Photoinitiated destabilization of sterically stabilized liposomes

A considerable effort has been devoted to the development of liposomes for the transport and buffering of drugs in the body. Several research groups have reported the increased localization of sterically stabilized liposomes (PEG-liposomes) at tumor sites. If PEG-liposomes are to be effective carriers of therapeutic agents, their drug permeability must be sufficiently low that little passive release occurs during the circulation time of the PEG-liposomes. However, once PEG-liposomes reach tumor sites, it may be desirable to accelerate the release of the encapsulated drug. The use of light to stimulate the release of encapsulated compounds from liposomes is attractive, because it is possible to control the spatial and temporal delivery of the radiation. PEG-liposomes composed in part of the photosensitive lipid, bis-SorbPC, can be prepared in a manner that effectively encapsulates water soluble compounds, yet releases them upon exposure to ultraviolet light in the presence of oxygen. The observed increase in liposome permeability is about 200-fold at high photoconversion of the monomeric bis-SorbPC. The increase in permeability is dependent on the extent of photolysis, but independent of both the charge on the PEG-lipid and the mole fraction of PEG-lipid included in the liposome. Therefore the photoinitiated destabilization of these PEG-liposomes is not a consequence of micellization of the PEG-lipid, but probably due to the formation of defects in the bilayer during crosslinking of the bis-SorbPC. The photoinduced increase in liposome permeability is great enough to make it possible to release therapeutic agents from PEG-liposomes at specific sites in a manner of tens of minutes to hours.     

Spontaneous transfer of stearic acids between human serum albumin and PEG:2000-grafted DPPC membranes

Electron spin resonance (ESR) spectroscopy is used to study the transfer of stearic acids between human serum albumin (HSA) and sterically stabilized liposomes (SSL) composed of dipalmitoylphosphatidylcholine (DPPC) and of submicellar content of poly(ethylene glycol:2000)-dipalmitoylphosphatidylethanolamine (PEG:2000-DPPE). Protein/lipid dispersions are considered in which spin-labelled stearic acids at the 16th carbon atom along the acyl chain (16-SASL) are inserted either in the protein or in the SSL. Two component ESR spectra with different rotational mobility are obtained over a broad range of temperature and membrane composition. Indeed, superimposed to an anisotropic protein-signal, appears a more isotropic lipid-signal. Since in the samples only one matrix (protein or membranes) is spin-labelled, the other component accounts for the transfer of 16-SASL between albumin and membranes. The two components have been resolved and quantified by spectral subtractions, and the fraction, f _p (16-SASL), of spin labels bound non-covalently to the protein has been used to monitor the transfer. It is found that it depends on the type of donor and acceptor matrix, on the physical state of the membranes and on the grafting density of the polymer-lipids. Indeed, it is favoured from SSL to HSA and the fraction of stearic acids transferred increases with temperature in both directions of transfer. Moreover, in the presence of polymer-lipids, the transfer from HSA to SSL is slightly attenuated, especially in the brush regime of the polymer-chains. Instead, the transfer from SSL to HSA is favoured by the polymer-lipids much more in the mushroom than in the brush regime.     

Kinetics of fatty acid binding ability of glycated human serum albumin

Kinetics of fatty acid binding ability of glycated human serum albumin (HSA) were investigated by fluorescent displacement technique with 1-anilino-8-naphtharene sulphonic acid (ANS method), and photometric detection of nonesterified-fatty-acid (NEFA method). Changing of binding affinities of glycated HSA toward oleic acid, linoleic acid, lauric acid, and caproic acid, were not observed by the ANS method. However, decreases of binding capacities after 55 days glycation were confirmed by the NEFA method in comparison to control HSA. The decrease in binding affinities was: oleic acid (84%), linoleic acid (84%), lauric acid (87%), and caproic acid (90%), respectively. The decreases were consistent with decrease of the intact lysine residues in glycated HSA. The present observation indicates that HSA promptly loses its binding ability to fatty acid as soon as the lysine residues at fatty acid binding sites are glycated.     

Characterization of sterically stabilized cisplatin liposomes by nuclear magnetic resonance

Extensive scientific efforts are directed towards finding new and improved platinum anticancer agents. A promising approach is the encapsulation of cisplatin in sterically stabilized, long circulating, PEGylated 100 nm liposomes. This liposomal cisplatin (STEALTH cisplatin, formerly known as SPI-77) shows excellent stability in plasma and has a longer circulation time, greater efficacy and lower toxicity than much free cisplatin. However, so far, the physicochemical characterization of STEALTH cisplatin has been limited to size distribution, drug-to-lipid ratio and stability. Information on the physical state of the drug in the liposome aqueous phases and the drug's interaction with the liposome membrane has been lacking. This study was aimed at filling this gap. We report a multinuclear NMR study in which several techniques have been used to assess the physical nature of cisplatin in liposomal formulations and if and to what extent the drug affects the liposome phospholipids. Since NMR detects only the soluble cisplatin in the liposomes and not the insoluble drug, combining NMR and atomic absorption data enables one to determine how much of the encapsulated drug is soluble in the intraliposomal aqueous phase. Our results indicate that almost all of the cisplatin remains intact during the loading process, and that the entire liposomal drug is present in a soluble form in the internal aqueous phase of the liposomes.     

Kinetics and mechanism of the interaction between human serum albumin and monomeric haemin.

The interaction of human serum albumin with monomeric haemin has been investigated by detailed kinetic analysis in dimethyl sulphoxide/water (3:5, v/v). The results obtained under conditions of albumin saturation of haemin and under pseudo-single turnover conditions indicate that methaemalbumin is formed in a two-stage, single-intermediate process. The initial association between the haemin and human serum albumin is a chemically controlled process (k1 = 1.7 X 10(5) mol-1 . s-1 . dm3 at 24 degrees C); the variation of K1 with pH exhibited a well defined pK of 5.9. The overall equilibrium constant, calculated by using microscopic rate constants, is 1.1 (+/- 0.5) X 10(8) mol-1 at 24 degrees C. The data and conclusions are consistent with a general binding mechanism for albumin in which intermediate formation is followed by an entropy-controlled internalization of the ligand.     

Sulfenic acid in human serum albumin

Summary. Sulfenic acid (RSOH) is a central intermediate in both the reversible and irreversible redox modulation by reactive species of an increasing number of proteins involved in signal transduction and enzymatic pathways. In this paper we focus on human serum albumin (HSA), the most abundant plasma protein, proposed to serve antioxidant functions in the vascular compartment. Sulfenic acid in HSA has been previously detected using different methods after oxidation of its single free thiol Cys34 through one- or two-electron mechanisms. Since recent evidence suggests that sulfenic acid in HSA is stabilized within the protein environment, this derivative represents an appropriate model to examine protein sulfenic acid biochemistry, structure and reactivity. Sulfenic acid in HSA could be involved in mixed disufide formation, supporting a role of HSA-Cys34 as an important redox regulator in extracellular compartments.     

Kinetics and mechanism of bilirubin binding to human serum albumin

The kinetics of bilirubin binding to human serum albumin at pH 7.40, 4 degrees C, was studied by monitoring changes in bilirubin absorbance. The time course of the absorbance change at 380 nm was complex: at least three kinetic events were detected including the bimolecular association (k1 = 3.8 +/- 2.0 X 10(7) M-1 S-1) and two relaxation steps (52 = 40.2 +/- 9.4 s-1 and k3 = 3.8 +/- 0.5 s-1). The presence of the two slow relaxations was confirmed under pseudo-first order conditions with excess albumin. Curve-fitting procedures allowed the assignment of absorption coefficients to the intermediate species. When the bilirubin-albumin binding kinetics was observed at 420 nm, only the two relaxations were seen; apparently the second order association step was isosbestic at this wavelength. The rate of albumin-bound bilirubin dissociation was measured by mixing the pre-equilibrated human albumin-bilirubin complex with bovine albumin. The rate constant for bilirubin dissociation measured at 485 nm was k-3 = 0.01 s-1 at 4 degrees C. A minimum value of the equilibrium constant for bilirubin binding to human albumin determined from the ratio k1/k-3 is therefore approximately 4 X 10(9) M-1.     

Characterization of the PEG layer of sterically stabilized liposomes: a SAXS study

Synchrotron small-angle X-ray scattering analysis of the bilayer structure of a pharmacologically relevant sterically stabilized liposome system is presented. Describing the electron density profile of the bilayer with the superposition of Gaussian functions, the contribution of the poly(ethylene glycol) (PEG) layers to the total electron density was identified. The changes in the thickness of the PEG layer as well as the distribution of the PEG chains among the outer and inner leaflets of the bilayers were followed by changing the molar ratio of the PEG-lipid and the molar weight of the PEG molecule.     

Kinetics of transfer in aqueous solution of ferriprotoporphyrin IX from human serum albumin to sperm whale apomyoglobin

Kinetic aspects of the transfer reaction of ferriprotoporphyrin IX (FP) from its complex with human serum albumin (HSA) to sperm whale apomyoglobin were investigated by spectrophotometry in aqueous solution. At molar ratios of 2:1 of both HSA and apomyoglobin to FP (1 × 10^?5M), the initial rate of transfer at pH 7.4 (0.025M Tris buffer) and 25°C was virtually independent of the concentration of apomyoglobin up to a sixfold excess of the latter. The rate-limiting step in the transfer reaction is considered to be the dissociation of FP from the FP-HSA complex with an apparent rate constant of the order of 10^?3 s^?1 under the above conditions. The initial rate decreased with increasing concentrations of HSA, indicating competition between HSA and apomyoglobin for free FP in the recombination reactions. A steady-state concentration of the order of 10^?9M is estimated for free FP under the conditions given. The initial rate of transfer decreased markedly with pH in the range of pH 5–9 and was also dependent on the type and concentration of the buffer used. Also, various electrolytes at different concentrations showed very different and specific effects on the initial rate of reaction. Similarly, various drugs and analogous substances such as penicillins and salicylate affected the rates of dissociation in different concentration ranges. From the temperature dependence of the initial rate at pH 7.4 in the range of 5–25°C, an overall energy of activation of about 8 kcal/mol and an entropy of activation of about ?50 e.u. are evaluated, indicating a much higher order of molecular groups around FP or other steric constraints in the transition state. It appears that kinetic parameters of the biopolymer–small molecule system are more sensitive to environmental changes than corresponding equilibrium data as measured by light absorption.     

A growth factor antagonist as a targeting agent for sterically stabilized liposomes in human small cell lung cancer

The ability of a growth factor antagonist, [D-Arg(6),D-Trp(7,9)-N(me)Phe(8)]-substance P(6-11), named antagonist G, to selectively target polyethylene glycol-grafted liposomes (known as sterically stabilized liposomes) to a human classical small cell lung cancer (SCLC) cell line, H69, was examined. Our results showed that radiolabeled antagonist G-targeted sterically stabilized liposomes (SLG) bound to H69 cells with higher avidity than free antagonist G and were internalized (reaching a maximum of 13000 SLG/cell), mainly through a receptor-mediated process, likely involving clathrin-coated pits. This interaction was confirmed by confocal microscopy to be peptide- and cell-specific. Moreover, it was shown that SLG significantly improved the nuclear delivery of encapsulated doxorubicin to the target cells, increasing the cytotoxic activity of the drug over non-targeted liposomes. In mice, [(125)I]tyraminylinulin-containing SLG were long circulating, with a half-life of 13 h. Use of peptides like antagonist G to promote binding and internalization of sterically stabilized liposomes, with their accompanying drug loads, i.e., anticancer drugs, genes or antisense oligonucleotides, into target cells has the potential to improve therapy of SCLC.     

Interactions of human serum albumin with chlorogenic acid and ferulic acid

The interactions of chlorogenic acid and ferulic acid with human serum albumin (HSA) have been investigated by fluorescence and Fourier transformed infrared (FT-IR) spectrometry. Fluorescence results showed that one molecule of protein combined with one molecule of drugs at the molar ratio of drug to HSA ranging from 1 to 10, and their binding affinities (KA) are 4.37 x 10(4) M(-1) and 2.23 x 10(4) M(-1) for chlorogenic acid and ferulic acid, respectively. The primary binding site for chlorogenic acid is most likely located on IIA and that for ferulic acid in IIIA. The main mechanism of protein fluorescence quenching was static quenching process. Combining the curve-fitting results of infrared amide I and amide III bands, the alterations of protein secondary structure after drug complexation were estimated. With increasing the drug concentration, the protein alpha-helix structure decreased gradually and the reduction of protein alpha-helix structure reached about 7% and 5% for protein binding with chlorogenic acid and ferulic acid individually at the drug to protein molar ratio of 30. This indicated a partial unfolding of HSA in the presence of the two acids. From the fluorescence and FT-IR results, the binding mode was discussed.     

Reactivity of sulfenic acid in human serum albumin

Sulfenic acid is formed upon oxidation of thiols and is a central intermediate in the redox modulation of an increasing number of proteins. Methods for quantifying or even detecting sulfenic acid are scarce. Herein, the reagent 7-chloro-4-nitrobenz-2-oxa-1,3-diazole was determined not to be suitable as a chromophoric probe for sulfenic acid in human serum albumin (HSA-SOH) because of lack of specificity. Thionitrobenzoate (TNB) reacted with HSA exposed to hydrogen peroxide, but not control or thiol-blocked HSA. The reaction was biphasic. The first phase was approximately 20-fold faster than the second phase and first order in HSA-SOH and TNB (105 +/- 11 M-1 s-1, 25 degrees C, pH 7.4), allowing quantitative data on HSA-SOH formation and reactivity to be obtained. Exposure of reduced HSA (0.5 mM) to hydrogen peroxide (4 mM, 37 degrees C, 4 min) yielded 0.18 +/- 0.02 mol of HSA-SOH per mol of HSA. HSA-SH reacted with hydrogen peroxide at 2.7 +/- 0.7 M-1 s-1 (37 degrees C, pH 7.4), while HSA-SOH reacted at 0.4 +/- 0.2 M-1 s-1, yielding sulfinic acid (HSA-SO2H), as detected by mass spectrometry. The rate constants of HSA-SOH with targets of analytical interest such as dimedone and sodium arsenite were determined. HSA-SOH did not react appreciably with the plasma reductants ascorbate or urate, nor with free basic amino acids. In contrast, HSA-SOH reacted rapidly with the plasma thiols cysteine, glutathione, homocysteine, and cysteinylglycine at 21.6 +/- 0.2, 2.9 +/- 0.5, 9.3 +/- 0.9, and 55 +/- 3 M-1 s-1 (25 degrees C, pH 7.4), respectively, supporting a role for HSA-SOH in the formation of mixed disulfides.     

Interaction of perfluorooctanoic acid with human serum albumin

Background  

Recently, perfluorooctanoic acid (PFOA) has become a significant issue in many aspects of environmental ecology, toxicology, pathology and life sciences because it may have serious effects on the endocrine, immune and nervous systems and can lead to embryonic deformities and other diseases. Human serum albumin (HSA) is the major protein component of blood plasma and is called a multifunctional plasma carrier protein because of its ability to bind an unusually broad spectrum of ligands.     

The interaction between cholesterol and human serum albumin

The interaction between cholesterol and Human Serum Albumin (HSA) was studied by fluorescence technique. Addition of cholesterol causes decreasing of the fluorescence intensity of HSA and the mechanism can be attributed to static quenching. Both negative enthalpy and entropy change indicate this binding was an "enthalpy-driven" reaction. The number of binding site and distance between residues and ligands were also calculated: n = 0.98, r = 3.84 nm. UV-vis spectra showed HSA molecules unfolded to some extent and the hydrophobicity was decreased in the presence of cholesterol.     

Fluorescence resonance energy transfer between bovine serum albumin and fluoresceinamine

Physical binding‐mediated organic dye direct‐labelling of proteins could be a promising technology for bio‐nanomedical applications. Upon binding, it was found that fluorescence resonance energy transfer (FRET) occurred between donor bovine serum albumin (BSA; an amphiphilic protein) and acceptor fluoresceinamine (FA; a hydrophobic fluorophore), which could explain fluorescence quenching found for BSA. FRET efficiency and the distance between FA and BSA tryptophan residues were determined to 17% and 2.29 nm, respectively. Using a spectroscopic superimposition method, the saturated number of FAs that bound to BSA was determined as eight to give a complex formula of FA₈–BSA. Finally, molecular docking between BSA and FA was conducted, and conformational change that occurred in BSA upon binding to FA molecules was also studied by three‐dimensional fluorescence microscopy. Copyright © 2015 John Wiley & Sons, Ltd.     

An apolipoprotein E-derived peptide mediates uptake of sterically stabilized liposomes into brain capillary endothelial cells

A promising strategy to solve the problems of insufficient membrane penetration of drugs and low target specificity is the localization of targeting and uptake-facilitating ligands on the surface of drug-carrier systems. This study investigated the role of a peptide derived from the LDL receptor (LDLr)-binding domain of apolipoprotein E (apoE) in initiating endocytosis in brain capillary endothelial cells. The highly cationic tandem dimer of apoE residues (141-150) was coupled covalently onto poly(ethylene glycol)-derivatized liposomes. Membrane binding and cellular uptake was monitored qualitatively by confocal-laser-scanning microscopy as well as quantitatively using a fluorescence assay. The peptide mediated an efficient, energy-dependent translocation of liposomes across the membrane of brain capillary endothelial cells. Liposomes without surface-located peptides displayed neither membrane accumulation nor cellular uptake. Low peptide affinity to LDLr and internalization of the complex into fibroblasts with up- and down-regulated receptor expression levels, as well as complex translocation into cells incubated with an antibody against the LDLr, pointed to a dominating role of an LDLr-independent transport route. Enzymatic digestion of heparan sulfate proteoglycan (HSPG) with heparinase I and addition of heparin and poly-l-lysin as competitors of HSPG and HSPG ligands, respectively, resulted in a significant loss in liposome internalization. The results suggested that HSPG played a major role in the apoE-peptide-mediated uptake of liposomes into endothelial cells of brain microvessels.