The Use of Fluorescence Resonance Energy Transfer to Study the Disintegration Kinetics of Liposomes Containing Lysolecithin and Oleic Acid in Rat Plasma (original) (raw)
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Organic solvents used in various pharmaceutical preparations may be associated with chronic health effects, with special emphasis on halogenated solvents. Liposomes, lipid bilayer membrane carriers, have potential applications in targeted drug delivery systems. The non-halogenated solvents, acetonitrile and ethanol, were used in comparison to commonly used chloroform. The effect of solvents and dispersion medium was demonstrated using physicochemical properties, stability studies and hemolytic activity. Increased sonication time showed decreased particle size in phosphate buffer saline and water medium. Vesicles prepared from all solvents exhibited better stability in phosphate saline buffer than water when evaluated by particle size and zeta potentials. Liposomes showed a positive zeta potential in buffer solution whereas liposomes in water showed negative zeta potential. In vitro hemolytic activity of liposomes was done with fresh human red blood cells. Results in buffer solution were in the range of 1-4 % which further proved this medium superior to pure water. The findings of this study are helpful in suggesting the formulation of thin films by less hazardous solvents in terms of the environmental integrity and human health.
A Stability Test of Liposome Preparations Using Steady-State Fluorescent Measurements
Drug Delivery, 2001
The stability of liposome preparations under the action of the nonionic detergent Triton X-100 was measured using the fluorescent molecular probe octadecylrhodamine B (R18). The probe inserted in the lipid bilayer shows a self-quenched fluorescence and the degree of quenching depends both on the probe concentration and the phase state of the lipid membrane. The addition of detergent to the liposomes produces a steep decrease in self-quenching caused by dilution of the probe in the bilayer. The curves of steady-state fluorescence intensity show an abrupt change in slope that corresponds to the point at which liposomes break down into lipid-detergent mixed entities that are different from the earlier liposome-monodisperse population. The lytic process was followed in parallel by dynamic light scattering (DLS), and the analysis of the DLS results agree with the interpretation of the fluorescence measurements. The probe R18 therefore is a useful marker to test the stability of liposome ...
Colloids and Surfaces B: Biointerfaces, 2007
In order to study mechanisms involved in liposome-cell interaction, this work attempted to assess the influence of vesicle composition on the delivery of liposomal content to Hela cells. In particular, to evaluate pH-sensitive properties and cell interaction of the prepared liposomes, the lipid formulations contained cholesterol (Chol) and they were varied by using phosphatidylcholines with different purity degree: soy lecithin (SL; 80% phosphatidylcholine), a commercial mixture of soy phosphatidylcholine (P90; 90% phosphatidylcholine) or dipalmitoylphosphatidylcholine (DPPC; 99% of purity). A second series of liposomes also contained stearylamine (SA). Dehydration-rehydration vesicles (DRV) were prepared and then sonicated to decrease vesicle size. Vesicle-cell interactions and liposomal uptake were examined by fluorescence microscopy using carboxyfluorescein (CF) and phosphatidylethanolamine-dioleoyl-sulforhodamine B (Rho-PE) as fluorescent markers. Fluorescence dequenching assay was used to study the influence of pH on CF release from the liposomal formulations. Liposome adhesion on the cell surface and internalization were strongly dependent on vesicle bilayer composition. SA vesicles were not endocytosed. DPPC/Chol liposomes were endocytosed but did not release their fluorescent content into the cytosol. SL/Chol and P90/Chol formulations displayed a diffuse cytoplasmic fluorescence of liposomal marker. (A.M. Fadda).
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1990
The interaction of liposomes with macrophage cells was monitored by a new fluorescence method (Hong, K., Straubinger, R.M. and Papahadjopoulos, D., J. Cell Biol. 103 (1986) 56a) that allows for the simultaneous monitoring of binding, endocytosis, acidification and leakage. Profound differences in uptake, cell surface-induced leakage and leakage subsequent to endocytosis were measured in liposomes of varying composition. Pyranine (1-hydroxypyrene-3,6,8-trisulfonic acid, I-IllS), a highly fluorescent, water-soluble, pH sensitive dye, was encapsulated at high concentration into the lumen of large unilamellar vesicles. HPTS exhibits two major fluorescence excitation maxima (403 and 450 nm) which have a complementary pH dependence in the range 5-9: the peak at 403 nm is maximal at low pH values while the peak at 450 nm is maximal at high pH values. The intra-and extracellular distribution of liposomes and their approximate pH was observed by fluorescence microscopy using appropriate excitation and barrier filters. The uptake of liposomal contents by cells and their subsequent exposure to acidified endosomes or secondary lysosomes was monitored by spectrofluorometry via alterations in the fluorescence excitation maxima. The concentration of dye associated with cells was determined by measuring fluorescence at a pH independent point (413 nm). The average pH of ceil-associated dye was determined by normalizing peak fluorescene intensities (403 nm and 450 nm) to fluorescence at 413 nm and comparing these ratios to a standard curve. HPTS-containing liposomes bound to and were acidified by a cultured murine macrophage cell line (J774) with att/ 2 of 15-20 min. The acidification of liposomes exhibited biphasic kinetics and 50-80% of the liposomes reached an average pH lower than 6 within 2 h. A liposomal lipid marker exhibited a rate of uptake similar to HPTS, however the lipid component selectively accumulated in the cell; after an initial rapid release of liposome contents, 2.5-fold more lipid marker than liposomal contents remained associated with the cells after 5 h. Coating haptenated iiposomes with antibody protected liposomes from the initial release. The leakage of iiposomal contents was monitored by co-encapsulating I-IPTS and p-xylene-bis-pyridinium bromide, a fluorescence quencher, into liposomes. The time course of dilution of liposome contents, detected as an increase in HPTS fluorescence, was coincident with the acidification of HPTS. The rate and extent of uptake of neutral and negatively charged liposomes was similar; however, liposomes opsonized with antibody were incorporated at a higher rate (2.9-fold) and to a greater extent (3.4-fold). In addition, the rate and extent of incorporation of liposome encapsulated HPTS was dependent on temperature and the metabolic state of the cell, consistent with uptake of liposomes by endocytosis. The use of HlYFS allowed accurate and simultaneous quantitation of iiposome uptake, acidification, cell-induced leakage of liposomes, and regurgitation of liposome contents. In addition to cell-surface induced leakage, liposomes leaked extensively during endocytosis coincident with acidification; half of the cell-induced dilution of liposome contents was accounted for by leakage at the cell surface, while the remainder occurred coincident with acidification. Liposome contents labeled the aqueous space of endosomes and lysosomes and were regurgitated rapidly as liposomal lipid accumulated selectively. Opsonization of iiposomes, to induce Fc-mediated endocytosis, afforded protection to the initial dilution of liposome contents, but not the rate of leakage, after endocytosis. Implications of these studies for the use of liposomes as drug delivery vehicles are discussed.
Stability of anionic liposomes in serum and plasma
African Journal of Pharmacy and Pharmacology, 2011
It is well-known that serum components destabilize liposomal membranes. Therefore, most in-vitro transfection protocols avoid serum, which make the extrapolation to in-vivo situations difficult. In this study, we investigated the stability of different anionic liposomal formulations including artificial viral envelopes (AVEs) in 100% fetal calf serum (FCS), human serum (HS) and human plasma (HP) by measuring the release of entrapped carboxyfluorescein (CF). We observed that FCS and HP induce leakage of CF from vesicles, while HS did not induce a pronounced leakage from the liposomes. In addition, we studied the effect of the phosphatidylethanolamine (PE) moiety, negatively charged lipid components and cholesterol (CHOL) on the stability of AVE liposomes. We found that the liposomes composed of DMPE/DPPG/CHOL (1:2:1) were the most stable liposomes in FCS and HP, among the examined liposomal formulations. Liposomes having a lipid composition similar to viral envelopes (AVE) were more stable in serum than pH-sensitive liposomes also used in gene therapy.
Journal of Biomedical Optics, 2005
Liposomes are known to be taken up by the liver cells after intravenous injection. Among the few techniques available to follow this process in vivo are perturbed angular correlation spectroscopy, nuclear magnetic resonance spectroscopy, and scintigraphy. The study of the intracellular pathways and liposomal localization in the different liver cells requires sacrifice of the animals, cells separation, and electronic microscopy. In the acidic intracellular compartments, the in situ rate of release of liposomes remains poorly understood. We present a new method to follow the in situ and in vivo uptake of liposomes using a fluorescent pH-sensitive probe 5,6carboxyfluorescein (5,6-CF). 5,6-CF is encapsulated in liposomes at high concentration (100 mM) to quench its fluorescence. After laparotomy, liposomes are injected into the penile vein of Wistar rats. Fluorescence images of the liver and the skin are recorded during 90 min and the fluorescence intensity ratio is calculated. Ratio kinetics show different profiles depending on the liposomal formulation. The calculated intracellular liver pH values are, respectively, 4.5 to 5.0 and 6.0 to 6.5 for DSPC/chol and DMPC liposomes. After sacrifice and flush with a cold saline solution, the pH of the intracellular site of the liver (ex vivo) is found to be 4.5 to 5.0. This value can be explained by an uptake of liposomes by the liver cells and subsequent localization into the acidic compartment. An intracellular event such as dye release of a drug carrier (liposomes loaded with a fluorescent dye) can be monitored by pH fluorescence imaging and spectroscopy in vivo and in situ.
Liposome disposition in vivo. III. Dose and vesicle-size effects
Biochimica et biophysica acta, 1981
The effect of lipid dose (4,3-512.8 mumol total lipid/kg body weight), administered intravenously as liposomes encapsulating radioactive inulin, upon the ability of mouse organs to bind and/or take-up the radioactive label has been studied in vivo. Three different liposome diameters were investigated: 0.46 micrometers (L), 0.16 micrometers (M) and 0.058 micrometers(S). All liposomes were negatively charged with lipid composition of phosphatidylcholine/phosphatidic acid/cholesterol/alpha-tocopherol in the molar ration 4 : 1 : 5 : 0.1 or 4 : 1 : 1 : 0.05. Overall radioactive label disposition after 2 h was consistent with localization predominantly in the reticuloendothelial system. A saturation of liver with increasing lipid dose was demonstrated for all three sizes, together with a corresponding increase in blood levels. Spleen radioactivity increased with increasing dose of L- and M-liposomes, but decreased for increasing doses of S-liposomes. Levels in residual carcass exhibited n...
Journal of Pharmaceutical Sciences, 1992
Small unilamellar liposomes containing fluorinated steroids (flumethasone and dexamethasone) were obtained. A physicochemical evaluation was conducted based on photon correlation spectroscopy (PCS) and fluorine-19 (IgF) nuclear magnetic resonance (NMR) spectroscopy compared with standard biochemical methods (HPLC). The PCS method provides a fast and adequate evaluation of some critical features of liposomes (size, physical stability). In addition, ''F NMR spectroscopy gives substantial information, in a nondestructive manner, on steroid behavior in the membrane upon encapsulation and also when the temperature of liposomes is increased. The combined spectroscopic approach proposed here might prove useful in (1) the management of liposomal formulation, especially in the documentation of physicochemical properties, (2) pharmaceutical control in the industrial production line, and (3) control preceding injection at the clinical site. Spectroscopic techniques might offer a complementary approach to classical biochemical methods in the evaluation of the properties of a liposomal formulation and could be usefully integrated into quality control procedures. Liposomes constitute a particular pharmaceutical formulation allowing significant modifications in the pharmacokinetic properties of a drug, such as its parenchymal delivery. Preparations of liposomes containing a fluorinated steroid (dexamethasone) have been recently proposed in topical1 and ocular2.3 drug administration. Therapeutic applications of liposomes require a rigorous quality control specific to this pharmaceutical form: vesicle size, encapsulation ratio, and kinetics of release constitute the main parameters which define a liposomal preparation. The usual biochemical evaluation of liposomes generally requires separation or destruction of vesicles, which are then analyzed in a sequential manner by chromatography, centrifugation, and radioactive or fluorescent labeling techniques.
Pharmaceutical Methods, 2016
Objective: The objective of the study was to assess the effect of liposomal concentration on the characterization of liposome using dynamic light scattering technique. Method: Liposome of a water soluble active, Niacinamide, was taken for the study. Phospholipid combination (INCI name lecithin and Propane diol) was used to form liposomes of Niacinamide. Concentrated liposomal suspension was prepared using various ratios of phospholipid. The concentrated liposomal suspension was diluted five times using distilled water. Concentrated liposomal suspension and diluted suspension were characterized for particle size, PDI, conductivity and Zeta potential. Their surface morphology was studied with SEM images. Results: The data on conductivity, PDI, zeta potential and particle size were significantly different between concentrated and diluted liposomal samples. The use of various phospholipid ratios appears to significantly affect PDI, zeta potential and particle size in concentrated sample. The data on diluted samples indicated that the phosholipid level has no significant effect on these parameters. Conclusion: In conclusion, characterizing liposomes using techniques which involves dynamic light scattering, that is widely used, liposome concentration during measurement is important to get reliable results.