PEGylated lipids impede the lateral diffusion of adsorbed proteins at the surface of (magneto)liposomes.

Protein binding to nanoparticles is a crucial issue in biomedicine, as it triggers their clearance from the bloodstream after intravenous injection. Many techniques are available for measuring strong protein binding interactions, but weak dynamic interactions are more difficult to assess. To tackle the latter problem, in the present work, cytochrome c was chosen as a representative model of a water-soluble protein and the adsorbing particulates were either small unilamellar phospholipid vesicles or 14 nm diameter solid superparamagnetic iron oxide cores onto which a phospholipid bilayer was strongly chemisorbed (so-called magnetoliposomes). Incorporation of cytochrome c oxidase into the phospholipid bilayer allowed the association of cytochrome c with the surface of the particles to be measured with high sensitivity by VIS-spectrophotometry. The impact of enzyme density as well as some of the physical features of the PEG corona (degree of PEGylation and PEG chain length) adjacent to the surface of the lipid structures on the overall kinetics was also investigated.

Evaluation of peri-tumoral vessels surrounding colorectal liver metastases after intravenous injection of extruded magnetoliposomes in rats: correlation with 3T MRI and histopathology.

BACKGROUND: Magnetoliposomes have pronounced signal-enhancing effect on T1-weighted (T1w) images of the liver using qualitative analysis which may be benefical for demonstrating peritumoral vasculature. PURPOSE: To correlate peri-tumoral vasculature (ring-enhancement) surrounding colorectal liver metastases after injection of magnetoliposomes using T1-weighted (T1w) imaging with histopathology in a rat model. MATERIAL AND METHODS: All experiments were approved by the responsible Animal Care Committee. Three rats injected with CC531 coloncarcinoma cells in the portal vein were imaged at 3T using a small diameter four channel coil. The presence of liver metastases, signal intensity changes within intrahepatic vessels, peri-tumoral vasculature (ring-enhancement) surrounding liver metastases on T1w imaging and histopathology, and the histopathological distribution of iron particles were evaluated. SS SE-EPI and T1w GE sequences were used. Images were evaluated qualitatively and MRI findings were correlated with histopathology. RESULTS: Fifteen liver metastases were present which were all detected at MRI (mean diameter 2.4 mm (SD 0.8 mm, range 1.5-4.7 mm)). Ring-enhancement surrounding liver metastases at contrast-enhanced T1w GE sequences was present in all liver metastases. Correlation with histopathology showed the corresponding presence of dilated sinusoids filled with iron particles surrounding the liver metastases. CONCLUSION: Blood-pooling of iron oxide particles within magnetoliposomes was demonstrated with increased and hyperintensity of vessels after injection of magnetoliposomes. Qualitatively, ring-enhancement surrounding the liver metastases was seen on T1w imaging and corresponded histopathologically with the presence of iron particles (magnetoliposomes) within the dilated sinusoids surrounding the liver metastases.

Partitioning of propranolol in the phospholipid bilayer coat of anionic magnetoliposomes.

This work deals with the partitioning of the cationic amphiphilic drug, propranolol, in the coating of so-called magnetoliposomes (MLs), which consist of nanometre-sized, magnetizable iron oxide cores covered with a phospholipid bilayer. MLs of two types were used: either the ML coat consisted entirely of anionic dimyristoylphosphatidylglycerol, or it was mixed with zwitterionic dimyristoylphosphatidylcholine in a 5/95 molar ratio. To separate sorbed from non-sorbed propranolol, high-gradient magnetophoresis was used. The sorption profiles clearly show that electrostatic interactions play a key role in the sorption process as drug incorporation in the ML coat was favoured by increasing the anionic character of the ML envelope and by reducing the salt concentration of the medium. Also, upon drug binding some phospholipid molecules were expelled from the ML coat. The observations may be of relevance in the biomedical field, i.e. in the development of ML-based, intracellular theranostics.

Specific heating power of fatty acid and phospholipid stabilized magnetic fluids in an alternating magnetic field.

Magnetic fluids (MFs) with a similar narrow size distribution of the iron oxide core were stabilized with lauric acid (MF 1), oleate (MF 2) or, after dialysis in the presence of liposomes, with phospholipid molecules (MF 3 and MF 4, respectively). The hydrodynamic sizes of the MF 1 and MF 3 were half those found for MF 2 and MF 4. The MFs were exposed to inductive heating in an alternating magnetic field at a frequency of 200 kHz and a maximum magnetic field strength of 3.8 kA m(-1). Specific absorption rates (SAR) of 294 ± 42 (MF 1), 214 ± 16 (MF 2), 297 ± 13 (MF 3) and 213  ± 6 W g(-1) Fe (MF 4) were obtained. The data for MF 2 and MF 4 were identical to those found for the commercially available ferucarbotran. The biomedical relevance of the phospholipid-coated MFs is briefly discussed.

PEGylated cholecystokinin prolongs satiation in rats: dose dependency and receptor involvement.

BACKGROUND AND PURPOSE: Acute intraperitoneal (i.p.) administration of cholecystokinin (CCK) is known to induce a significant, but short-lasting, reduction in food intake, followed by recovery within hours. Therefore, we had covalently coupled CCK to a 10 kDa polyethylene glycol and showed that this conjugate, PEG-CCK(9), produced a significantly longer anorectic effect than unmodified CCK(9). The present study assessed the dose-dependency of this response and the effect of two selective CCK(1) receptor antagonists, with different abilities to cross the blood-brain barrier (BBB), on PEG-CCK(9)-induced anorexia. EXPERIMENTAL APPROACH: Food intake was measured, for up to 23 h, after i.p. administration of different doses (2, 4, 8, 16 and 32 microg kg(-1)) of CCK(9) or PEG-CCK(9) in male Wistar rats. Devazepide (100 microg kg(-1)), which penetrates the BBB or 2-NAP (3 mg kg(-1)), which does not cross the BBB, were coadministered i.p. with PEG-CCK(9) (6 microg kg(-1)) and food intake was monitored. KEY RESULTS: In PEG-CCK(9)-treated rats, a clear dose-dependency was seen for both the duration and initial intensity of the anorexia whereas, for CCK(9), only the initial intensity was dose-dependent. Intraperitoneal administration of devazepide or 2-NAP, injected immediately prior to PEG-CCK(9), completely abolished the anorectic effect of PEG-CCK(9). CONCLUSIONS AND IMPLICATIONS: The duration of the anorexia for PEG-CCK(9) was dose-dependent, suggesting that PEGylation of CCK(9) increases its circulation time. Both devazepide and 2-NAP completely abolished the anorectic effect of i.p. PEG-CCK(9) indicating that its anorectic effect was solely due to stimulation of peripheral CCK(1) receptors.

Preparation and Characterization of a Phospholipid Membrane-Bound Tetrapeptide That Corresponds to the C-Terminus of the Gastrin/Cholecystokinin Hormone Family.

The present work deals with the synthesis of a hydrophobized peptide and its localization at the membrane surface, after its incorporation into phospholipid vesicles. The tetrapeptide, Trp-Met-Asp-Phe-NH(2), which corresponds to the C-terminus of the cholecystokinin/gastrin hormone family, is conjugated to N-glutaryldioleoylphosphatidylethanolamine using a carbodiimide-catalyzed reaction method. Sonication of the lipophilized hormone in the presence of dimyristoylphosphatidylcholine results in a strong sequestration of the conjugate in the artificial membrane structures that are formed. More detailed information on the localization of the peptide moiety with respect to the membrane surface is gathered from fluorescence measurements. Both the observed blue shift in the fluorescence spectra and the quenching of Trp emission in the presence of potassium iodide point to a partial screening of the hormone moiety from the surrounding aqueous phase. The different parameters that may influence the physicochemical behavior of a hydrophobized peptide in a membrane structure are briefly discussed Copyright 2000 Academic Press.

Short- vs. long-circulating magnetoliposomes as bone marrow-seeking MR contrast agents.

We evaluated the relaxation enhancement and biodistribution of short- vs. long-circulating magnetoliposomes as a new contrast agent for magnetic resonance (MR) imaging of bone marrow. Magnetoliposomes with (ML-PEG) and without (ML) incorporation of polyethylene glycol (PEG, Mw 2000) were prepared, measuring 40 nm in diameter with 1-6 iron oxide crystals/vesicle. PEGylation selectively enhanced the T2 relaxivity of magnetoliposomes by 10% to 15%, with R1 and R2 values of 3 and 240 s(-1)/mM at 1.5 T and 37 degrees C. ML (n = 6) and ML-PEG (n = 6) preparations were administered IV into young (6-8 weeks old) and adult (>1 year old) Sprague-Dawley rats at 100 pmol Fe/kg. PEGylation increased blood half-life (P<0.05 for t > 30 minutes), following a biexponential clearance with a long half-life of 53.2+/-13.2 minutes. The clearance of ML was monoexponential, with a half-life 7.4+/-0.4 minutes. MR imaging revealed a pronounced uptake in bone marrow, including the iliac bone, femur, tibia, and upper and lower vertebrae. The bone marrow uptake of ML-PEG was comparable to that of ML, with both reaching a plateau within 30 minutes following injection. Fast spin-echo T2-weighted imaging was found to provide optimal contrast enhancement and allowed a clear depiction of red to yellow marrow conversion due to normal aging. While the use of magnetoliposomes can provide the added benefit of therapeutic drug or gene delivery, further investigation is warranted to assess their usefulness in differentiating normal vs. abnormal marrow conditions.

A simplified purification procedure of alpha-lactalbumin from milk using Ca(2+)-dependent adsorption in hydrophobic expanded bed chromatography.

The technique of expanded bed adsorption is originally designed for a direct recovery of proteins from fermentor feedstocks. In this article we describe the use of expanded bed adsorption for the recovery of alpha-lactalbumins from defatted milk using the hydrophobic Streamline Phenyl gel. alpha-Lactalbumins are Ca(2+)-binding proteins. Upon Ca2+ removal, they undergo a significant conformation change rendering them more hydrophobic. Based on this unique property we develop a protocol for fast and efficient purification of alpha-lactalbumin from milk. The use of this technique results in a reduction of the number of chromatographic purification steps.

Catalytic durability of magnetoproteoliposomes captured by high-gradient magnetic forces in a miniature fixed-bed reactor.

Cytochrome c-oxidase, used as a model membrane-bound enzyme, was embedded in phospholipid bilayer membranes, attached to nanometer-sized Fe(3)O(4) colloids (so-called magnetoliposomes). In comparison with the lipid-depleted free enzyme, both the activity and the enzymatic stability of the complexes, stored at 4 degrees C, were considerably enhanced. These beneficial properties of magnetoproteoliposomes have been successfully exploited in a magnetically controlled fixed-bed bioreactor, operating in a continuous flow regime. (c) 1996 John Wiley & Sons, Inc.

Simple two-step procedure for the preparation of highly active pure equine milk lysozyme.

A fast, simple two-step purification scheme is presented for the isolation of lysozyme at a high yield from equine milk. In the first step, fluidized bed technology, using the Streamline system, was exploited. In the following step, advantage was taken of Ca(2+)-induced conformational changes to obtain a pure, high specific activity, enzyme fraction by hydrophobic interaction chromatography.

Binding characteristics and thermal behaviour of cytochrome-C oxidase, inserted into phospholipid-coated, magnetic nanoparticles.

A strategy for the immobilization of cytochrome-c oxidase, used as a representative membrane-bound enzyme, into so-called magnetoliposomes has been developed. The latter structures consist of a phospholipid bilayer which covers nanometer-sized Fe3O4 colloids. Incorporation of the enzyme into the phospholipid envelope is facilitated by a short sonication step. Upon adsorption, the reaction characteristics of the lipid-depleted enzyme are drastically changed. With double-layered phosphatidylcholine (PC) magnetoliposomes the activity increases by a factor of approximately 5. After a first magnetic fractionation step, approximately 67% of the activity remains with the magnetoliposome retentate. Subsequent magnetophoresis cycles show that the adsorbed enzyme is firmly fixed into the phospholipid coat. Upon immobilization, the thermal behavior is also profoundly affected. The heating inactivation curves show two sigmoidal transition zones. Irrespective of the PC type used, a first inflection point is located near 39 degrees C, whereas a second one, which is located at higher temperatures, clearly depends on the acyl chain length (56 degrees C with dimyristoyl-PC and 60 degrees C for dioleoyl-PC and Ovothin-200). An identical behavior is observed with classical proteoliposomes with an equal phospholipid composition. By contrast, monolayer-coated dimyristoyl-PC magnetic structures are inferior with respect to both their reactivation potency and their ability to strongly affix cytochrome-c oxidase and to improve the thermal stability of the enzyme.

Potentialities of magnetoliposomes in studying symmetric and asymmetric phospholipid transfer processes.

Using high-gradient magnetophoresis, the non-protein-mediated transfer and exchange of phosphatidylglycerol (PG) molecules between sonicated phospholipid dispersions and magnetoliposomes is studied. The latter structures consist of nanometer-sized magnetite (Fe3O4) cores which are enwrapped by a phospholipid bilayer. Their dimensions are similar to those of small unilamellar vesicles (De Cuyper and Joniau (1988) Eur. J. Biophys. 15, 311-319). Using these particles, spontaneous lipid movements were studied in three different cases. In a first setup, symmetric exchange between dimyristoylphosphatidylglycerol (DMPG) magnetoliposomes, labelled with [3H]DMPG, and DMPG vesicles was followed. Within the time scale of the experiment (1 day) both the lipid molecules residing in the inner and outer leaflet of the magnetoliposomes participate in the exchange process, although 'flip-flop' movements have a retarding effect. In the second approach a unidirectional flux of DMPG from DMPG magnetoliposomes to distearoylphosphatidylglycerol (DSPG) acceptors is noted. In this case, the outer phospholipid leaflet of the magnetoliposomes (in contrast to the inner one) can be largely stripped off; the extent of depletion is determined by the relative amount of the DSPG receiving structures. Furthermore, it is found that with a 15-fold molar excess of receptors, the whole depletion course can be described by a single first-order rate expression. The reluctancy of the inner shell phospholipids to migrate is further illustrated by the virtual lack of transfer, observed with monolayer-coated Fe3O4 colloids. In the third case, asymmetric bidirectional PG transfer is followed between equimolar amounts of DMPG magnetoliposomes and dipentadecanoylphosphatidylglycerol vesicles. In the initial stage of the incubation period, the mmol PG/g Fe3O4 ratio decreases, but progressively restores later on. By quantitatively measuring the transfer rate of each of the individual components, this complex behavior could be unravelled.

Reaction of alpha-tubulin with iodotyrosines catalyzed by tubulin:tyrosine ligase: carboxy-terminal labeling of tubulin with [125I]monoiodotyrosine.

We have studied the capacity of different iodinated derivatives of phenylalanine and tyrosine to inhibit the incorporation of [3H]tyrosine into tubulin catalyzed by tubulin:tyrosine ligase. In contrast to thyronine and its iodinated derivatives, iodotyrosines were efficient inhibitors. That they also functioned as substrates of the enzyme was shown by the effective incorporation of [125I]mono- and diiodotyrosine into tubulin. The label was shown to be located at the carboxy terminus. Labeling by this method conserves the polymerization capacity of tubulin in contrast with classical radioiodination methods involving oxidation.

Magnetoliposomes. Formation and structural characterization.

The adsorption of different types of phosphatidylglycerols onto magnetizable solid particles is studied. The super-paramagnetic magnetite spheres used have an average diameter of only 14 nm and are stabilized by lauric acid to keep them in solution. During incubation and dialysis of this water-based magnetic fluid in the presence of preformed sonicated phospholipid vesicles, magnetoliposomes are formed which are captured from solution with high efficiency by high-gradient magnetophoresis. Support for the bilayer character of the phospholipid coat is derived from both theoretical calculations and experimental data. Phospholipids which form the inner monolayer are adsorbed very quickly with their charged head-group orientated towards the iron oxide surface. The high-affinity character of the binding is reflected in the adsorption isotherms and is further illustrated by their non-extractability with high concentrations of Tween 20. The outer layer assembles through interaction with the exposed hydrocarbon chains. As compared to the inner layer, the phospholipids adsorb at a much slower rate and are displaced by Tween 20 concentrations which usually disrupt conventional membranes. The adsorption isotherms for this layer obey the Langmuir expression. The affinity constants, derived from them, progressively increase as the hydrophobic nature of the phosphatidylglycerols is more pronounced.

An azide-insensitive low-affinity ATPase stimulated by Ca2+ or Mg2+ in basal-lateral and brush border membranes of kidney cortex.

Basal-lateral and brush border membranes from pig kidney cortex were prepared by differential centrifugation followed by free-flow electrophoresis. In each type of membrane, azide-insensitive, low-affinity Ca2+-ATPase and Mg2+-ATPase activities are demonstrated. A comparative study for both membranes further reveals the following analogies between these ATPases: (a) they show maximal activity between pH 8 and 8.5; (b) they exhibit Km values for Ca-ATP or Mg-ATP in the millimolar range and have a comparable low substrate specificity; (c) they are insensitive to 10 microM of vanadate, N,N'-dicyclohexylcarbodiimide, e diethylstilbestrol, quercetin, harmaline and amiloride. The partial inhibition by 1 mM of the various compounds is rather aspecific. In view of these similarities it is concluded that only one enzyme entity is responsible for the activity which is measured in both membrane types. The HCO3-stimulated Mg2+-ATPase activity in pig kidney cortex was also studied. This enzyme, however, is clearly of mitochondrial origin since the HCO3-stimulation coincides with the distribution profile of succinate dehydrogenase, a mitochondrial marker; and since it is inhibited by azide.

An intramolecular excimer forming probe used to study the interaction of alpha-lactalbumin with model membranes.

The nonconjugated bichromophoric molecule 1,3-di(1-pyrenyl)propane shows, besides the pyrene monomer fluorescence, a structureless emission due to an intramolecular excited dimer (excimer). In the case of intramolecular excimer forming systems, the ratio of the emission intensities of excimer vs. monomer (IE/IM) is sensitive to changes in membrane structure as will be illustrated here. The present molecule is a useful probe to report on lipid-protein interactions, at least in our model system. We have introduced it into the hydrocarbon layer of dimyristoylphosphatidylcholine vesicles in order to study their interaction with alpha-lactalbumin (alpha-LA) as a function of pH and temperature. On the basis of steady-state fluorescence, kinetic, and energy transfer studies, we have found that, at pH 4, alpha-LA strongly interacts with the lipid bilayer. In steady-state fluorescence experiments, changes of the ratio IE/IM and shifts of the transition temperature have been observed, reflecting changes of membrane structure caused by interaction with alpha-LA. Kinetic studies of the rate of interaction of alpha-lactalbumin and measurements of energy transfer from excited tryptophan(s) to the fluorescent probe confirm the steady-state experiments. Our results agree with previously reported microcalorimetric, gel chromatographic, and fluorescence polarization studies.

Behavior of beef-heart cytochrome c oxidase in reconstituted proteovesicles. A systemtic evaluation of the influence of phospholipid polar headgroup and fatty-acyl side chains.

1. Phospholipid-depleted cytochrome c oxidase is incorporated in vesicles, built up of phospholipids of known polar headgroup and fatty-acyl side chains. 2. Maximal reactivation is obtained only when the fatty-acyl side chains provide a fluid environment. 3. Fluid zwitterionic phospholipids are found to be more efficient reactivators than fluid anionic ones. 4. Irrespective of the polar headgroup type, two narrow ranges of activation energies for the enzymatic reaction are calculated from the Arrhenius plots: 81--92 kJ/mol in solid and 51--61 kJ/mol in fluid conditions. 5. Cytochrome c oxidase is also incorporated in a series of vesicles, each built up of an equimolar amount of two phospholipids which differ in their polar headgroup type and/or their fatty-acyl side chain characteristics. From the localization of the enzyme activity profiles, obtained with these mixtures, tentative deductions are made about the preference of cytochrome c oxidase for different phospholipid molecules.