Effect of osmotic gradient on the physical properties of membrane lipids in liposomes

Osmotic swelling of multilamellar liposomes was shown to produce structural changes in liposomal membranes. Creation of transmembrane osmotic gradients results in an increased lateral mobility of membrane incorporated hydrophobic probe, pyrene. This is accompanied by some decrease in order parameter of membrane phospholipid polar head group regions. The perturbations are more marked in hydrophobic than in polar regions of the membranes. It is suggested that some functional changes in biomembranes arising upon osmotic swelling may be associated with structural alterations similar to those observed in liposomes.     

Physical separation of the aqueous phase and lipoidal lamellae from multilamellar liposomes: An analytical and preparative procedure

A method for the determination of the urinary melanocyte metabolite 5,6-dihydroxyindole-2-carboxylic acid is described. The catecholic indole is adsorbed onto alumina, chromatographed on a reverse-phase octadecyl silica column, and detected by fluorometry. The sensitivity of the method permits detection of 1 pmol injected.     

A coarse grained molecular dynamics study on the structure and stability of small-sized liposomes

The dependence of geometric structure and thermal stability of liposomes on their component phospholipid molecules and distribution of molecules in the inner and the outer layers of the liposome is investigated by conducting molecular simulations in explicit water for the eight types of liposomes constructed from different phospholipids. Using molecular mechanics structure-relaxation based on the coarse grained (CG) model, stable structures of the solvated liposomes are obtained. In addition, the molecular dynamics (MD) simulations based on the CG model are carried out at 310 and 360 K for elucidating the change in structure of the solvated liposomes. The MD simulations reveal that liposomes having the same number of lipids (SNL) in both the inner and the outer layers keep their spherical structures even at 360 K. In particular, the SNLs composed of palmitoyloleoyl-phosphatidyl-ethanolamine1 or dimyristoylglycero-phosphatidyl-choline lipid exhibit a compact spherical shape. In contrast, liposomes having the same density of lipids in the inner and the outer layers cannot keep their spherical shapes at 360 K. The obtained results contribute toward developing novel liposomes with enhanced thermal stability.     

Effect of glycerol on the interfacial properties of dipalmitoylphosphatidylcholine liposomes as measured with merocyanine 540.

Liposomes of dipalmitoylphosphatidylcholine (DPPC) prepared in increasing glycerol/glucose ratios show an increase in the absorbance at 570 nm of merocyanine spectra at temperatures below the phase transition. Since this effect is not observed when liposomes are prepared in solutions containing solely glucose, it is attributed to specific interactions of glycerol with the membrane phase. The increase in the 570 nm absorbance is ascribed to a partial fluidification of the membrane interface and is dependent on the distribution of the dye between the inner and the outer compartments of the liposomes and on their osmotic state. The greatest differences in the absorbance ratio are obtained when merocyanine is added to the external media. In consequence, the changes in the spectra of MC are dependent on the surface state of the liposomes which can be modified by an increase of glycerol or glucose in the external media. The present results are examined in the light of the perturbations that glycerol can induce on the barrier properties of the bilayer.     

Effect of phospholipid composition on pharmacokinetics and biodistribution of epirubicin liposomes

The effects of phospholipid composition on the pharmacokinetics (PK) and biodistribution of epirubicin (EPI) liposomes, as well as the in vitro macrophage uptake of various liposome formulations, were investigated. Three liposome formulations were investigated: HSPC:Chol (L-EPI; 5:4 molar ratio), HSPC:Chol:DSPG (D-EPI; 5:4:1 molar ratio), and HSPC:Chol:DSPG:DSPE-mPEG₂₀₀₀ (S-EPI; 5:4:1:0.3 molar ratio). Small unilamellar liposomes were prepared by the modified thin-film hydration method with extrusion through polycarbonate filters, and EPI was remote loaded into liposomes by the transmembrane ammonium sulfate gradient method. Macrophages were used to evaluate in vitro the cellular uptake of EPI-loaded liposomes. The following decreasing order of uptake amount was observed: L-EPI>D-EPI>S-EPI. D-EPI showed a relatively low level of uptake, probably because of the steric hindrance provided by the glycerol head group on DSPG, protecting it from the direct recognization by cell-membrane receptors. With the presence of serum, uptake values for all liposome formulations were increased for the activation of the complement system. In the PK study, S-EPI showed significantly prolonged circulating time and reduced clearance. The following increasing order of area under the concentration versus time curve was observed among the various liposome formulations: L-EPI<D-EPI<S-EPI. The biodistribution study indicated that S-EPI decreased drug disposition in the liver, spleen, lung, and heart and increased that in the kidney with respect to the other liposomes. The encouraging property of S-EPI, in terms of prolonging circulating time and reducing heart toxicity, might describe a promising perspective toward clinical application, and all the results would support further research into liposome-based drug carriers.     

Determination of the size distribution of liposomes by SEC fractionation,and PCS analysis and enzymatic assay of lipid content

In this study, small liposomes obtained by high-pressure homogenization were fractionated according to their particle sizes by size exclusion chromatography (SEC). The subfractions were analyzed by photon correlation spectroscopy (PCS) as well as enzymatic phosphatidylcholine (PC) assay for their particle sizes and lipid contents, respectively. For small egg PC-liposomes, a size range of 15 nm to 60 nm was found, with 80% of the vesicles being smaller than 30 nm in size. This is in contradiction to a mean size of 85±32 nm as indicated by PCS without fractionation. The PCS technique appears to underestimate very small particles below 30 nm if (few) bigger particles are present. The PCS particle size analysis of unfractionated hydrogenated egg PC/cholesterol-liposomes (2:1, mole/mole) by PCS did not yield any significant results. On fractionation, however, a particle size range of 40 nm to 120 nm was determined in a reproducible manner. Our results indicate that the combination of size exclusion fractionation with subsequent photon correlation spectroscopic particle size analysis and enzymatic PC assay can give both more detailed and more reliable insight into the particle size distribution of small liposomes than PCS alone. Published: May 15, 2002.     

Effect of carbohydrates on the survival of Lactobacillus helveticus during vacuum drying

AIMS: To assess four carbohydrates for the protective effect against Lactobacillus helveticus cells inactivation during vacuum drying, and to study the effect of selected carbohydrate on changes of inactivation kinetics. METHODS AND RESULTS: Early stationary phase L. helveticus cells grown in MRS media were recovered from fermentation broth, washed with PBS buffer (pH 7.0), and then mixed with different concentrations of four carbohydrates, namely lactose, sorbitol, inulin, and xanthan gum. Cells were dried in a vacuum drier at 100 mbar, 43 degrees C for 12 h. Only cells with 1% sorbitol addition showed higher survival (18%) over cells without added carbohydrate (8%). Using in situ microbalance technique whereby cell weight during vacuum drying was continuously monitored via precision balances integrated into the vacuum chamber, drying and inactivation kinetics of cells and cells mixed with sorbitol were established. CONCLUSION: Survival of L. helveticus during the vacuum drying could be improved by the addition of optimal concentration of 1% sorbitol. Addition of sorbitol did not cause drastic changes in drying rate, water content and water activity of samples. The protection mechanisms of sorbitol seemed not to be due to a direct physical effect, which could be related to drying rate. SIGNIFICANCE AND IMPACT OF THE STUDY: The increase in survival of cells after vacuum drying by the addition of a protective carbohydrate may provide an alternative mean to preserve starter cultures at a higher level of activity.     

Synergistic Effect of Phospholipid-Based Liposomes and Propylthiouracil on U-937 Cell Growth

Scientific evidence indicates that exogenous phospholipids in the form of liposomes can affect cell growth. Effects of liposomes on cell growth depend on several factors including composition of liposomes, lipid concentration, and type of cells studied. Because phagocytic cells such as monocytes and macrophages are natural targets of liposomes, intracellular delivery of drugs to modulate cellular activity of these cells is of interest. We explored the effects of phospholipid-based liposomes composed of soy bean phosphatidylcholine (PC) as the main lipid component on U-937 cell growth. Effects of charge-imposing lipids and cholesterol were also studied. In addition, we investigated whether phospholipid-based liposomes would exert any interaction on cell growth with propylthiouracil, a drug with known antiproliferative activity. We found that PC in the form of extruded liposomes had intrinsic antiproliferative activity on U‐937 cells at concentrations of 200 μM and up without any appreciable cytotoxiciy. Phosphatidylserine and phosphtidylglycerol, but not dicetlylphosphate, at 10 mol% increased growth retardation activity of PC liposomes. Cholesterol at 30 mol% did not have any effect on cell growth, except for liposomes composed of PC and phosphatidylserine, where growth retardation was negated in the presence of cholesterol. Synergistic effect on cell growth was seen with certain liposome compositions when 5.5 μg/mL of propylthiouracil was coincubated. The results of this study suggest that the effects of exogenous lipids on cell growth should be taken into consideration when PC-based liposomes are to be used as drug delivery systems, especially when the targets are cells with phagocytic activity.     

Mechanisms of attack and defence at the cell surface: The use of phospholipid bilayers as models for cell membrane

Electrical conductivity across phospholipid bilayers induced by various cytotoxic proteins has been used to analyse the damaging action of such proteins on cells; the protective effect of divalent cations and protons against such attack has also been investigated. The predominant effect of divalent cations and protons is to promote the closed state of membrane pores, i.e. to gate protein-induced lesions.     

The influence of polyalcohols and carbohydrates on the thermostability of alpha-amylase

The influence of polyhydric alcohols and carbohydrates on the thermostability, i.e., the heat inactivation kinetics, of Bacillus licheniformis alpha-amylase was studied in the temperature range 96 degrees to 130 degrees C. High concentrations (from 9 to 60 weight percent) of glycerol, sorbitol, mannitol, sucrose, or starch can markedly decrease the inactivation rate constant, k, and in the studied cases, this stabilizing effect grows stronger with increasing additive concentration. Statements about stabilization should, however, be specified carefully with respect to temperature, because E(A) is mostly altered likewise. For dissolved enzyme E(A) was almost always decreased in the presence of polyol or carbohydrate, whereas for immobilized enzyme it was augmented in each studied instance. The inactivation of dissolved enzyme can, in all the studied cases, be adequately described as a first-order process. Immobilized enzyme, however, shows biphasic then first-order inactivation kinetics, depending on the additive concentration and temperature.     

Influence of glycerol on the structure and redox properties of horse heart cytochrome c. A circular dichroism and electrochemical study

The effect of glycerol on the structure and redox properties of horse heart cytochrome c was investigated by absorption spectroscopy, circular dichroism, and dc cyclic voltammetry techniques. The results show that the organic solvent increases the -helix structure of the protein and induces slight changes at the active-site environment; however, the overall tertiary structure does not appear to be significantly perturbed. Glycerol stabilizes cytochrome c, the free energy of denaturation (G ₀) being approximately 0.7 kcal/mol larger than that determined in phosphate buffer under the same conditions, and influences the heterogeneous electron transfer kinetics at a chemically modified gold electrode; on the other hand, the redox potential of the protein is unaltered. On the whole, the results obtained indicate that glycerol acts as a suitable stabilizing agent of cytochrome c, which is of interest for application in biotechnology; the organic solvent does not alter the tertiary structure significantly or the redox properties of the protein. This has to be interpreted not only in terms of the glycerol-induced solvent ordering around the protein surface, but also as due to the specific features of the protein matrix.     

Effect of trehalose on the phase properties of hydrated and lyophilized dipalmitoylphosphatidylcholine multilayers

The structure and thermal behavior of hydrated and lyophilized dipalmitoylphosphatidylcholine (DPPC) multilayers in the presence of trehalose were investigated by differential scanning calorimetry and X-ray diffraction methods. Trehalose enters the aqueous space between hydrated bilayers and increases the interbilayer separation (from 0.36 to 1.37 nm in the different DPPC phases at 1 M trehalose). It does not affect the lipid chain packing and also the slow isothermal conversion at 4 degrees C of the metastable L beta' phase into the equilibrium crystalline Lc phase. Addition of trehalose leads to a slight upward shift (about 1 degrees C at 1 M trehalose) of the three phase transitions (sub-, pre-, and main transition) in fully hydrated DPPC while their other properties (enthalpy, excess specific heat, and transition width) remain unchanged. The effect of trehalose on the thermal behavior of DPPC multilayers freeze-dried from an initially completely hydrated state is qualitatively similar to that of water. These data support the "water replacement" hypothesis about trehalose action. It is suggested that trehalose prevents the formation of direct interbilayer hydrogen bonds in states of low hydration.     

Effect of onion-type multilamellar liposomes on Trametes versicolor laccase activity and stability

Trametes versicolor laccase was encapsulated into onion-type, lipid-based multilamellar vesicles (MLVs). When encapsulated, laccase was isolated from the assay medium but was still active once freed from its capsule. The encapsulation efficiency was larger than 65% at 25 °C and 37 °C and decreased to 55% by introducing 140 mM NaCl into the buffered medium (pH = 4.5). MLVs were shown to drastically improve both laccase stability and activity. At 25 °C, laccase activity was doubled in the presence of MLVs. At 37 °C in the salt-free medium, the half-life time of laccase was increased from 2hr 30-65 h without and with MLVs, respectively. This effect was even more pronounced in the salted medium where laccase activity was unchanged for 6 days in the presence of MLVs. These beneficial effects were attributed to the immobilization of laccase onto MLV surface. Laccase activity as well as stability was notably shown to be directly correlated to MLV stability.     

Effect of berberine on the antioxidant status, ultrastructural modifications and protein bound carbohydrates in azoxymethane-induced colon cancer in rats

Chemically induced carcinogenesis models in the rat are widely used for studying the biology of cancer and for developing and evaluating cancer prevention strategies. The azoxymethane(AOM)-induced rat colon tumor is a valuable tool for studying the interaction between tumor development and exogenous factors. Malignant conditions are characterized by enhanced levels of lipid peroxidative products, protein bound carbohydrates indicative of membrane damage and an ineffective antioxidant scavenging system. In the present study, AOM-induced rat showed lipid peroxidative damage, alterations in the membrane glycoprotein component and antioxidant defense system, along with ultrastructural changes like disruption in goblet cell and its membrane, swollen mitochondria with cristae dispersed, elongated endoplasmic reticulum and other features of neoplastic invasion. Berberine significantly attenuated the increases in lipid peroxidation, protein bound carbohydrates and enhanced the antioxidative status. The present study suggests that berberine prevents the appearance of malignant morphology and ultrastructural changes of AOM induced cancer by producing apoptosis like changes. Thus berberine inhibits neoplastic transformation by the induction of antioxidant defence system and ability to induce apoptotic like changes—thus elucidating its anti cancer role.     

Quantitative estimation of the surface carbohydrates on the infection structures of rust fungi with enzymes and lectins

Lectins of Triticum vulgaris (WGA), Concanavalia ensiformis (ConA), Phaseolus vulgaris (PHA), Lotus tetragonolobus (LTA), Arachis hypogaea (PNA), Ricinus communis (RCA I), Griffonia simplicifolia (GSA II) and the enzymes endo-(13)--D-glucanase, exo-(13)--D-glucanase and laminarinase were tested for binding to the infection structures of Puccinia coronata and Uromyces appendiculatus. The enzymes and lectins were labeled with fluorescein and the fluorescence was measured with a microscope photometer. GSA II and ConA bound to all parts of the two rust fungi to a certain extent. The germ tubes of P. coronata bound at least two times more WGA than did the germ tubes of U. appendiculatus. The appressoria of both rust fungi additionally bound exo-(13)--glucanase, endo-(13)--glucanase and laminarinase. The substomatal vesicle and the infection hypha of both rust fungi mainly bound the glucanases. Furthermore, the substomatal vesicle of U. appendiculatus bound PHA. No obvious binding with LTA, RCA I and PNA was observed. Binding generally could be inhibited by appropriate haptens. Binding to uredospores generally appeared unspecific. The results indicate that the germ tubes have chitin on their outer surfaces, the appressoria chitin and glucans and the substomatal vesicles and infection hyphae mainly glucans. Compared to P. coronata, U. appendiculatus has more terminal linked glucose residues or the glucan has more (13)--linkages. Also, U. appendiculatus has N-acetylgalactosamine or a similar sugar on the surface of the substomatal vesicle.Abbreviations ConA Concanavalia ensiformis agglutinin - FITC fluorescein isothiocyanate - GSA II Griffonia simplicifolic agglutimin II - LTA Lotus tetragonolobus agglutinin - PBS phosphate buffered saline - PNA Peanut agglutinin - RCA I Ricinus communis agglutinin I - PHA Phaseolus vulgaris agglutinin - WGA Wheat germ agglutinin     

Effects of glycerol on the in vitro stability and regulatory activation/inactivation of pyruvate,orthophosphate dikinase of Zea mays L.

Glycerol stabilizes the activity of pyruvate, orthophosphate dikinase extracted from darkened or illuminated maize leaves. It serves as a better protectant of activity than dithiothreitol for the active day-form and the glycerol concentration needed for full protection is inversely related to the level of protein. The night-form of the enzyme is also protected by glycerol not only against inactivation, but also against partial reactivation in storage. Glycerol does not prevent the P_i-dependent activation nor the ADP-dependent inactivation of pyruvate, orthophosphate dikinase, but the rates of both processes are substantially decreased. The ability of the inactive night-form for P_i-dependent activation is also sustained by glycerol for at least 2 h at 20°C, apparently through stabilization of the labile regulatory protein.Abbreviations BSA bovine serum albumin - G-6-P glucose-6-phosphate - MDH malate dehydrogenase - PCMB p-chloromercuribenzoate - PEP phosphoenolpyruvate - PEPCase phosphoenol-pyruvate carboxylase - PPDK pyruvate, orthophosphate dikinase - PVP polyvinylpyrrolidone     

The effect of soil drought on the composition of carbohydrates in yellow lupin seeds and triticale kernels

Carbohydrate analysis was made of yellow lupin seeds (cv. Juno) and triticale kernels (cv. Dagro), produced by plants exposed to drought stress for 21 days after the initial flowering of the first node of lupin and initial earing of triticale. The seeds of all experimental variants were harvest at full maturity, dried and stored in linen bags at 18–20 °C. Soluble carbohydrates were extracted and analysed as described by Horbowicz and Obendorf (1994). Gas chromatographic separation of carbohydrates showed that raffinose family oligosaccharides (RFO) were dominant in lupin seeds. The other carbohydrates present were sucrose (10 %), cyclitols and galactosyl cyclitols (12–13 %). Soil drought resulted in higher levels of verbascose, but decreased the quantities of the other carbohydrates in lupine seeds. In triticale kernels, over 50 % of soluble sugars were composed of sucrose and maltose, while 17.7 % were raffinose and stachyose. In response to drought the content of mono- and oligosaccharides declined. The decrease of soluble carbohydrates content in seeds of lupin and triticale kernels has no effect on the seed germination and vigour. It is assumed that the changes in the concentration of soluble sugars observed under drought may impair the storability of triticale kernels, but improve it for lupine seeds.     

Effects of phosphate on the activity, stability and regulatory properties of phosphoenolpyruvate carboxylase from the C4 plant Cynodon dactylon

The extraction of phosphoenolpyruvate carboxylase, PEPC (EC 4.1.1.31) from leaves of Cynodon dactylon (L.) Pers. with phosphate buffer (pH 7.4, 105 mM) was advantageous in comparison to the usual extraction with Tris-HCl buffer (pH 7.4, 100 mM); a higher activity was obtained, which was most evident at low substrate (phosphoenolpyruvate) concentrations. The PEPC activity was stable under dilution or in storage for at least 48 h at room temperature. The effects of phosphate buffer were not due to inhibition of phosphatase(s) action during the extraction, since they were also observed when the phosphates were added after the extraction with Tris-HCl. The phosphate-extracted enzyme was less responsive to both L-malate inhibition and activation by glucose-6-phosphate. The effects of phosphates might be due to preferential exclusion from the enzymic protein domain and, therefore, to a confinement of the enzyme to a fraction of the total volume. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of Ca2+ on the retention of glycerol and amino acids in Dunaliella tertiolecta

When D. tertiolecta cells, previously incubated in a 0.5 kmol m⁻³ NaCl medium with 1mol m⁻³ Ca²⁺, were transferred to an isotonic NaCl medium without Ca²⁺, the intracellular glycerol, as well as intracellular amino acids, was transiently lost to the medium within 30 min. The transient leakage of glycerol and amino acids was enhanced by the addition of EGTA (1 mol m⁻³), while the addition of SrCl₂ (1 mol m ⁻³) or polyamines such as spermidine (5 mol m⁻³) and spermine (5 mol m⁻³) restrained the leakage caused by the lack of external Ca²⁺ of intracellular glycerol and amino acids.