共查询到20条相似文献,搜索用时 31 毫秒
1.
Casals E Galán AM Escolar G Gallardo M Estelrich J 《Chemistry and physics of lipids》2003,125(2):139-146
The physical stability of six liposome systems designed as platelet substitutes was determined on storage at 4 degrees C over a 3-month period under quiescent conditions. Liposomes used were large unilamellar vesicles. Correlation of the n-average mean diameter, polydispersity, zeta-potential and the presence of aminophospholipid on liposome surface (in those preparations which contain phosphatidylethanolamine (PE) and phosphatidylserine (PS)) led to the conclusion that liposomes that mimicked the composition of platelets were the most stable. When a net charge was present in the vesicles (liposomes with PS), the likelihood of aggregation was extremely low. In the period studied, a proportion of 25% of charged lipid (PS) conferred sufficient electrostatic stabilization to prevent vesicle fusion. An increase in this charge did not modify the stability characteristics. PE-containing liposomes behaved in a particular way: when PE content was 50%, the stability of the preparation was limited to 1 month; whereas if the content was 25%, the zeta-potential rose with time, as did the presence of PE in the liposome surface. 相似文献
2.
Fluorescence correlation spectroscopy (FCS) has been increasingly used to study the binding of fluorescently-labeled peptides and proteins to phospholipid vesicles. In this work, we present a new method to analyze partition data obtained by this technique based on the assumption that the number of fluorescently-labeled protein molecules bound per liposome follows a Poisson distribution. To not overestimate the recovered partition coefficients, we first show that the variation in liposome brightness caused by this statistical distribution must be considered explicitly in data analysis when the parameter used to establish the partition curves is the fractional instead of the absolute amplitudes associated with the slowest diffusing particles in the system (lipid vesicles), a choice frequently made in FCS partition studies. We further extend the theoretical model describing the membrane partition of a fluorescently-labeled protein by considering the presence of a trace amount of free fluorescent dye (non-binding component) in the system. We show that this situation can account for an apparent maximal binding level lower than 100% in the experimental partitioning curves obtained for Alexa 488 fluorescently-labeled lysozyme and liposomes prepared with variable anionic phospholipid content. The extreme sensitivity of the FCS technique allowed uncoupling lysozyme partition from the protein-induced liposome aggregation, confirming that lysozyme binding to negatively charged liposomes is dominantly driven by electrostatic interactions. 相似文献
3.
Timothy D. Heath David Robertson Michael S.C. Birbeck Anthony J.S. Davies 《生物化学与生物物理学报:生物膜》1980,599(1):42-62
We describe a method by which horseradish peroxidase may be attached covalently to the surface of liposomes under conditions which permit minimal non-covalent association of the enzyme with the lipids. The coupling method adopted does not allow the formation of homopolymers of liposomes or peroxidase. For phosphatidylethanolamine/phosphatidylcholine and stearylamine/phosphatidylcholine vesicles, minimal disruption of vesicular structure is observed, whilst for phosphatidylserine vesicles, the lipid-protein complex appears to form structures much smaller than 25 nm in diameter. Stearylamine/phosphatidylcholine vesicles have been shown to retain entrapped inulin, and activity measurements for the peroxidase suggest that it is located exclusively on the external surface of the liposome membrane. Peroxidase can be localized histochemically which has permitted the morphological study of the coated liposomes and their interactions with cells. 相似文献
4.
In order to facilitate the isolation of liposomes from blood components, we have developed a simple and rapid procedure combining chromatographic and centrifugal methods. This 'spin column' procedure was used to isolate liposomes from incubation mixtures with human serum or from the blood of CD1 mice after intravenous administration of liposomes. An advantage of this procedure is that processing times are fast (typically minutes) such that the isolation procedure can be done in the absence of chelators or other coagulation inhibitors which may affect protein/liposome interactions. Furthermore, several samples can be analyzed together and small sample volumes can be processed. In addition, we show that this spin column procedure can be employed to isolate large unilamellar vesicles averaging 100 nm in diameter from lipoproteins and plasma proteins. The applicability of this spin column procedure in studying protein/liposome interactions is demonstrated by quantitating the amount of human complement component C3 bound per liposome using a C3 competitive ELISA assay after incubation with human serum. The proteins associated with the recovered liposomes were further analyzed by conventional SDS-polyacrylamide gel electrophoresis. We show that egg phosphatidylcholine/cholesterol (55:45, mol/mol) or egg phosphatidylcholine/cholesterol/dioleoylphosphatidylserine (35:45:20, mol/mol) liposomes isolated from the circulation of CD1 mice within minutes of administration have distinct, complex profiles of associated proteins. By isolating circulating large unilamellar liposomes using the spin column method and characterizing the proteins associated with their membranes, this protein fingerprinting approach will expedite identifying protein interactions which affect liposome stability and clearance in vivo. 相似文献
5.
P Boquet 《European journal of biochemistry》1979,100(2):483-489
Diphtheria toxin, its fragments A, B and the protein serologically related to toxin, crm 45, have been studied for their hydrophobicity using the method of charge shift electrophoresis. These molecules were then assayed for liposome interaction. The results have shown that the diphtheria toxin B fragment behaves as an amphiphatic protein because it contains a hydrophobic domain located in that portion of the B chain which remains in protein crm 45. Toxin fragment A is hydrophilic. Incubation of protein crm 45 or toxin fragment B with preformed liposomes leads to association of these proteins with lipid vesicles. Fragment A does not interact with liposomes. Binding of protein crm 45 with lipid vesicles is dependent on time and temperature. Protein crm 45 is unidirectionally associated with liposomes, its enzymic fragment A directed outside the liposome. Fragment B or protein crm 45, upon binding with liposomes, does not affect the permeability of the vesicles. 相似文献
6.
We have compared drug transfer into target cells in vitro from liposomes of different sizes. Liposomes of mean diameter 800 Å, 2000 Å or 4000 Å, containing the folate analogue, methotrexate, and the fluorophore, carboxyfluorescein, were covalently coupled to Staphylococcus aureus protein A. Cells of the murine k haplotype were preincubated with an anti-H-2Kk monoclonal antibody. Excess antibody was removed and then cells were incubated with liposomes. The number of cell-bound liposomes was determined by fluorimetry. The drug effect was assayed by the methotrexate-mediated inhibition of radiolabeled deoxyuridine uptake. The drug effect was more important in the case of the 800 Å vesicles than for the larger liposomes, despite the fact that the quantity of drug bound to cells was several-fold greater for large liposomes than for small ones. Since fusion is excluded by the non-proportionality of drug binding and drug effect, the predominant manner of liposome entry seems to be endocytosis. At least for these in vitro studies, the endocytosis by target cells of small liposomes seems to be more efficient than that of large liposomes. 相似文献
7.
Reulen SW Brusselaars WW Langereis S Mulder WJ Breurken M Merkx M 《Bioconjugate chemistry》2007,18(2):590-596
Liposomes have become popular drug delivery vehicles and have more recently also been applied as contrast agents for molecular imaging. Most current methods for functionalization of liposomes with targeting proteins rely on reactions of amine or thiol groups at the protein exterior, which generally result in nonspecific conjugation at multiple sites on the protein. In this study, we present native chemical ligation (NCL) as a general method to covalently couple recombinant proteins in a highly specific and chemoselective way to liposomes containing cysteine-functionalized phospholipids. A cysteine-functionalized phospholipid (Cys-PEG-DSPE) was prepared and shown to readily react with the MESNA thioester of EYFP, which was used as a model protein. Characterization of the EYFP-liposomes using fluorescence spectroscopy showed full retention of the fluorescent properties of conjugated EYFP and provides a lower limit of 120 proteins per liposome. The general applicability of NCL was further tested using CNA35, a collagen-binding protein recently applied in fluorescent imaging of collagen. NCL of CNA35 thioester yielded liposomes containing approximately 100 copies of CNA35 per liposome. The CNA35-liposomes were shown to be fully functional and bind collagen with a 150-fold higher affinity compared to CNA35. Our results show that NCL is an attractive addition to existing conjugation methods that allows direct, covalent, and highly specific coupling of recombinant proteins to liposomes and other lipid-based assemblies. 相似文献
8.
《Journal of liposome research》2013,23(1):33-36
AbstractOur recent in vivo studies have investigated the surface adsorption property of various circulating liposomes to blood proteins, and have related this property to liposome clearance behavior. In particular, we have investigated liposomes composed of different charged or neutral lipids, fatty acyl chain length and saturation, and cholesterol content. From these studies an apparent inverse relationship between the amount of blood protein that associates with large unilamellar vesicles and the circulation half-lives of the liposomes is observed, indicating that protein-mediated liposome clearance mechanisms are dominant. Furthermore, by comparing the protein profiles of rapidly cleared liposomes with liposomes exhibiting enhanced circulation times, key blood proteins have been identified and implicated in the clearance process. 相似文献
9.
M.J. Hsu R.L. Juliano 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1982,720(4):411-419
Liposomes are taken up as intact vesicles by mouse peritoneal macrophages in a process which is temperature sensitive and is affected by inhibitors of glycolytic metabolism and of microfilament activity. Macrophages take up negatively charged vesicles more readily than positively charged vesicles (2-fold) or neutral vesicles (4-fold). Macrophages take up similar amounts of multilamellar liposomes, reversed phase liposomes and small unilamellar liposomes in terms of lipid, however this corresponds to vastly different numbers of particles and amounts of trapped volume. Coating the liposomes with macromolecular ligands capable of interacting with macrophage surface receptors can markedly promote liposome uptake. Thus, formation of an IgG-antigen complex on the liposome surface results in a 102-fold enhancement of liposome uptake, while coating the vesicles with fibronectin results in a 10-fold augmentation of uptake. Uptake via IgG-mediated and fibronectin-mediated processes seem to be independent since excess unlabelled, IgG-coated liposomes will inhibit the uptake of radioactively-labelled IgG-coated liposomes much more effectively than the uptake of radioactively-labelled fibronectin-coated liposomes. Cell-bound liposomes can readily be visualized on and inside of the macrophages using fluorescence microscopy techniques. 相似文献
10.
Using the electron paramagnetic resonance (EPR) method with spin-labeled fatty acids and gel-penetrating chromatography, the effect of phosphatidylglycerol on cytochrome P-450 incorporation into liposomes from dimyristoylphosphatidylcholine was investigated. An addition of phosphatidylglycerol caused an increase in the protein content of the proteoliposomes as well as their accelerated formation at temperatures below and above the liposome phase transition temperature (Ts). The dependence of the proteoliposome formation rate on the phosphatidylglycerol content in the liposome mixture is described by complex kinetics with a maximum in the presence of 10 mol.% of the negatively charged phospholipid. The mechanism of proteoliposome formation is discussed in terms of asymmetric distribution and phase state of the phospholipids in the original vesicles. 相似文献
11.
Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions3, monitor drug delivery4,5, and encapsulation4. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle.6 Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs)7. In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking8. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters <100 - 400 nm to study their behavior on cell functions3. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest9. Drug encapsulation can be achieved as reported during liposome formation9. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and (2) liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles.Nonetheless, the current liposome preparation technologies (sonication10, freeze-and-thaw10, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter <100 nm) with high consistency and efficiency10,5, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes.Manual extrusion using gas-tight syringes and polycarbonate membranes10,5 is a common practice but heterogeneity is often observed when using pore sizes <100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS)10, electron microscopy11 and nanoparticle tracking analysis (NTA)12 were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below. 相似文献
12.
The principle of a novel latex agglutination test based on combined results of electrophoretic mobility and particle aggregation measurements is described. Poly(styrene/alpha-tert-butoxy-omega-vinylbenzylpolyglycidol) (P(S/PGL)) microspheres were synthesized by a one step soap-free emulsion copolymerization of styrene and alpha-tert-butoxy-omega-vinylbenzylpolyglycidol macromonomer with number average molecular weight Mn = 2700 (polydispersity [Mw]/[Mn] = 1.10). Particles with monomodal size distribution (number average diameter Dn = 220 nm) and surface fraction of polyglycidol equal to f = 0.42 mol % were obtained. Human serum albumin (HSA) was covalently bound onto the surface of P(S/PGL) microspheres activated with 1,3,5-trichlorotriazine. In a model immunodiagnostic assay for anti-HSA, in which P(S/PGL) particles with covalently bound HSA have been used, the electrophoretic mobility and aggregation of microspheres were measured simultaneously. This approach allowed detection of anti-HSA in the serum in the range of anti-HSA concentrations from 0.1 to 150 microg/mL. The highest changes in electrophoretic mobility were registered for microspheres with surface concentration of immobilized HSA equal to Gamma = 9.2 x 10(-4) g/m2. 相似文献
13.
《生物化学与生物物理学报:生物膜》1985,814(1):151-155
A method is described for the preparation of liposomes carrying covalently attached protein at the outer surface. It is based upon the reaction between proteins, thiolated with the heterobifunctional reagent succinimidyl-S-acetylthioacetate (SATA), and liposomal maleimido-4-(p-phenylbutyryl)phosphatidyl-ethanolamine. Advantages of the procedure are that it is not restricted to proteins carrying native SH-groups and that it is substantially more convenient and less time-consuming than previously published methods. Applications for these liposomes are to be found in the fields of liposome targeting and the production of monoclonal antibodies. 相似文献
14.
B N Zimogliad V N Vasil'chenko N S Vasil'chenko V S Leont'ev A G Shkorbatov 《Biofizika》1984,29(2):259-263
To study the structural transformations and aggregation stability in liposomes, a new approach is proposed whose efficiency is demonstrated during the analysis of light scattering by lecithin liposome sols doped with low-molecular cryoprotectors . This approach is used to determine absolute dimensions, relative values of weight concentration, masses of vesicules and their surface densities as well as their polydispersity characteristics. The anisotropic scattering data suggest that cryoprotectors eliminate small scale defects which are partially restored at low temperatures. Low-molecular cryoprotectors are found to produce some favourable changes in dimensions and distribution of liposomes and to increase their aggregation and structural stability. 相似文献
15.
Carmo VA De Oliveira MC Reis EC Guimarães TM Vilela JM Andrade MS Michalick MS Cardoso VN 《Journal of liposome research》2008,18(1):59-70
Complement activation is an important step in the acceleration of liposome clearance. The anaphylatoxins released following complement activation may motivate a wide variety of physiologic changes. We performed physicochemical characterization and in vitro studies of the interaction of complement system with both noncirculating and long-circulating pH-sensitive and nonpH-sensitive liposomes. The liposomes were characterized by diameter, zeta potential, and atomic force microscopy (AFM). The study of liposome interactions with complement system was conducted using hemolytic assay in rat serum. All liposomes presented a similar mean diameter (between 99.8 and 124.3 nm). The zeta potential was negative in all liposome preparations, except in liposomes modified with aminopoly (ethyleneglycol) 2000-distearoylphosphatidylethanolamine (aPEG(2000)-DSPE), which presented positive zeta potential. Atomic force microscopy images showed that non-long-circulating pH-sensitive liposomes are prone to vesicles aggregation. Non-pH-sensitive liposomes complement system activates, while pH-sensitive liposomes showed to be poor complement activators in rat serum. 相似文献
16.
Lectin-mediated binding of liposomes to Hela cells was analyzed as a function of different parameters. We show that the amount of lectin covalently bound to liposomes can be accurately controlled. We chose to work with 500 - to 1 000 molecules of WGA bound per liposome of 1 micron diameter. These liposomes bound very efficiently to Hela cells as demonstrated by fluorescent microscopy, and fluorescent cell-sorting. We show that the number of liposomes bound is proportional to the input, over a wide range of concentrations. The liposomes bound very tightly to cells and could not be removed by trypsin or N-acetylglucosamine, which competes with WGA binding. 相似文献
17.
Grabielle-Madelmont C Lesieur S Ollivon M 《Journal of biochemical and biophysical methods》2003,56(1-3):189-217
This review focuses on the use of conventional (SEC) and high performance (HPSEC) size exclusion chromatography for the analysis of liposomes. The suitability of both techniques is examined regarding the field of liposome applications. The potentiality of conventional SEC is strongly improved by using a HPLC system associated to gel columns with a size selectivity range allowing liposome characterization in addition to particle fractionation. Practical aspects of size exclusion chromatography are described and a methodology based on HPSEC coupled to multidetection modes for on-line analysis of liposomes via label or substance encapsulation is presented. Examples of conventional SEC and HPSEC applications are described which concern polydispersity, size and encapsulation stability, bilayer permeabilization, liposome formation and reconstitution, incorporation of amphiphilic molecules. Size exclusion chromatography is a simple and powerful technique for investigation of encapsulation, insertion/interaction of substances from small solutes (ions, surfactants, drugs, etc.) up to large molecules (proteins, peptides and nucleic acids) in liposomes. 相似文献
18.
The nucleocapsid of Sindbis virus, a natural non-infectious complex of the viral RNA and protein molecules can be encapsulated in large, unilamellar vesicles and delivered efficiently to cells in an infectious form. It is shown that high infectivity of the vesicle entrapped nucleocapsids is partly due to the viral envelope proteins which enhance entrapment and liposome cell interaction.We believe that the efficiency of liposome mediated gene transfer of eukaryotic cells can be increased significantly by the insertion of fusogenic viral envelope proteins into the lipid bilayer of liposomes. 相似文献
19.
《Journal of liposome research》2013,23(4):303-320
AbstractCationic and anionic liposomes have been prepared by extrusion from dipalmitoylphosphatidylcholine (DPPC) and its mixtures with cholesterol and dimethyldioctadecyltrimethylammonium bromide (DDAB) and with phosphatidylinositol (PI) respectively covering a range of composition from 0 to 19 mole % DDAB and PI. The adsorption of liposomal lipid from the liposome dispersion onto particles of silica and titanium dioxide in suspension has been studied as a function of liposome composition and concentration. The adsorption isotherms have been fitted using a Langmuir equation from which the binding constants and maximum surface coverage were obtained. The Gibbs energies of adsorption for the cationic liposomes were on average -61.0 ± 2.1 kJ mol?1 (on silica) and -50.6 ± 2.9 kJ mol?1 (on titanium dioxide). On average saturation adsorption is equivalent to 3 to 10 lipid monolayers on silica and 3 to 7 on titanium dioxide. Using liposomes encapsulating D-glucose it is demonstrated that there is almost no release of glucose on adsorption of the lipid, indicating that the liposomes are adsorbed intact to form a liposome monolayer on the particle surfaces. Adsorption of intact liposomes to form a close-packed liposome monolayer of solid supported vesicles (SSV) is shown to be equivalent to on average 7.0 ± 0.2 phospholipid monolayers. The SSVs are shown to have increased stability to disruption by surfactants and when carrying the oil-soluble bactericide, Triclosan?, to be capable of inhibiting the growth of oral bacteria from immobilised biofilms. 相似文献
20.
Vildete A. S. Carmo Mônica C. De Oliveira Eduardo C. O. Reis Tânia M. P. D. Guimarães José M. C. Vilela Margareth S. Andrade 《Journal of liposome research》2013,23(1):59-70
Complement activation is an important step in the acceleration of liposome clearance. The anaphylatoxins released following complement activation may motivate a wide variety of physiologic changes. We performed physicochemical characterization and in vitro studies of the interaction of complement system with both noncirculating and long-circulating pH-sensitive and nonpH-sensitive liposomes. The liposomes were characterized by diameter, zeta potential, and atomic force microscopy (AFM). The study of liposome interactions with complement system was conducted using hemolytic assay in rat serum. All liposomes presented a similar mean diameter (between 99.8 and 124.3 nm). The zeta potential was negative in all liposome preparations, except in liposomes modified with aminopoly (ethyleneglycol) 2000-distearoylphosphatidylethanolamine (aPEG2000-DSPE), which presented positive zeta potential. Atomic force microscopy images showed that non–long-circulating pH-sensitive liposomes are prone to vesicles aggregation. Non–pH-sensitive liposomes complement system activates, while pH-sensitive liposomes showed to be poor complement activators in rat serum. 相似文献