M. De Cuyper

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.

Synthesis of magnetic Fe3O4 particles covered with a modifiable phospholipid coat

This work reports the synthesis of iron oxide cores by coprecipitation of Fe2+ and Fe3+ ions with NaHCO3 or NH3. Depending on the experimental conditions, particles of two different sizes (13 or 130 nm diameter) were produced. X-ray diffractometry revealed Fe3O4 (magnetite) to be the main constituent. The smaller particles, which, in contrast to the larger ones, are superparamagnetic, were stabilized with a phospholipid bilayer consisting of a 9:1 molar ratio of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol, thereby creating so-called magnetoliposomes. In a subsequent step, poly(ethylene glycol)-(PEG-) derivatized dipalmitoylphosphatidylethanolamine was introduced into the lipid envelope by incubating the magnetoliposomes with pre-formed sonicated vesicles containing the PEGylated lipid. The mechanism by which lipid transfer occurred was determined from the kinetic profiles. The relevance of these observations to a wide range of biomedical applications is briefly discussed.

A successful strategy for the production of cationic magnetoliposomes

Summary The present work describes a strategy and the mechanistic background for synthesizing magnetoliposomes (MLs) in which the bi-layered coating is partly composed of positively charged lipids. In a first step, neutral MLs are prepared from zwitterionic phosphatidylcholine vesicles and magnetite nanocores stabilized with an anionic surfactant, and then incubated with cationic vesicles formed from phosphatidylcholine and dioleoyltrimethylammoniumpropane. During this latter step, spontaneous intermembrane lipid transfer occurs. After reaching an equilibrium state, the desired cationic MLs are captured from the mixture in a high-gradient magnetophoresis setup.

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

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‐CCK9, produced a significantly longer anorectic effect than unmodified CCK9. The present study assessed the dose–dependency of this response and the effect of two selective CCK1 receptor antagonists, with different abilities to cross the blood‐brain barrier (BBB), on PEG‐CCK9‐induced anorexia.

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.

Biodistribution and biocompatibility investigation in magnetoliposome treated mice

Magnetoliposomes (MLs) may be successfully applied for several purposes. A dimyristoylphosphatidyl-choline- based ML (ML-2) sample was developed as a precursor of more complex thermal cancer therapy systems. The present study reports on morphology and magnetic resonance (MR) investigations carried out with the magnetite-based ML-2 sample. For the experiments, adult female Swiss mice were endovenously treated with a bolus dose of 100 µl of ML-2. Morphology and room-temperature MR studies (X-band experiments) were performed in several organs collected from 1 hour to 28 days after ML administration. Histological data showed magnetic nanoparticle (MNP) clusters up to the 28th day in the liver and spleen tissues. In spite of the presence of MNP clusters, no morphological alterations were observed, supporting the biocompatibility of the ML-2. MR signal was detected only in the liver and spleen tissues and showed that the MNPs concentration was not altered from 48 hours to 28 days after ML injection. Using MR data, important pharmacokinetic parameters, such as the effective clearance (half-life) and peak concentration, were obtained for the liver and spleen.

Raman spectroscopy of magnetoliposomes

In this study Raman spectroscopy was used to investigate monolayer and bilayer magnetite-based magnetoliposomes (MLs). The Raman probe is the hydroxyl (OH) group chemisorbed at the magnetite nanoparticle surface. Measurements were performed at room temperature in the typical OH stretching region. The data gathered for both samples are compared to each other and with those obtained for pure water. In comparison to liquid water (2.74 kcal/ mol), it was found that the hydrogen bond strength between the chemisorbed OH-group and the polar headgroup of the inner phospholipid layer was reduced in both the monolayer (2.22 kcal/mol) and the bilayer (1.83 kcal/mol) ML samples.

Design and Development of Magnetoliposome-Based Theranostics

Magnetoliposomes (MLs) consist of nanometre-sized magnetite cores, enwrapped by a bilayer of phospholipid molecules. In the past we showed that these nanocolloids can be exploited as powerful biocompatible magnetic resonance imaging (MRI) contrast agents. In the present work, we report on the partitioning of the amphiphilic drug, (R/S)-propranolol, within the lipidic envelope of MLs, built up of the zwitterionic dimyristoylphosphatidylcholine. Furthermore, it is also shown that MLs are easily internalized by 3T3 fibroblasts, used in this study as a representative cell model, without damaging cell viability. Overall, the results deliver the proof-of-concept that drug-loaded MLs have great potential as unique intracellular theranostics, i.e., as a nanoscale delivery system with combinatory therapeutic-diagnostic imaging modalities.

Phase transitions in phospholipid dispersions studied with an intramolecular excimer forming fluorescent probe, 1,3-bis (β-naphthyl)propane

Abstract We introduce the use of an intramolecular excimer forming, non-conjugated bichromophoric molecule: 1,3-bis (β-naphthyl)propane as a new probe for measuring thermal phase transitions in aqueous dispersions of phospholipids. Intermolecular excimer forming systems, such as pyrene, have been intensely studied as probes for the “microfluidity” of phospholipid dispersions. The probe we used has the added advantage that intramolecular excimer formation follows a pseudomonomolecular mechanism. This makes observations independent of probe concentration and allows for minute concentrations of the probe to be used, lowering the risk of perturbation of the phospholipid phase. Phase transition temperatures determined from 1,3-bis (β-naphthyl) fluorescence are in good agreement with differential scanning calorimetry and light scattering measurements.

Birefringence and transmission electron microscopy of monolayer and bilayer magnetoliposomes

In this study, static magnetic birefringence (SMB) and transmission electron microscopy (TEM) were used to investigate magnetite-based monolayer and bilayer magnetoliposomes (MLs). The SMB data were analyzed using the recent model proposed by Skeff Neto et al. (J. Appl. Phys. 89 (2001) 3362). The SMB data indicate that monolayer-based MLs internalize magnetic nanoparticles as dimers while bilayer-based MLs internalize both isolated nanoparticles and dimers. The higher content of dimers inside monolayer MLs has been confirmed by TEM data.