Evaluation of tumoral enhancement by superparamagnetic iron oxide particles: comparative studies with ferumoxtran and anionic iron oxide nanoparticles.

This study was designed to compare tumor enhancement by superparamagnetic iron oxide particles, using anionic iron oxide nanoparticles (AP) and ferumoxtran. In vitro, relaxometry and media with increasing complexity were used to assess the changes in r2 relaxivity due to cellular internalization. In vivo, 26 mice with subcutaneously implanted tumors were imaged for 24 h after injection of particles to describe kinetics of enhancement using T1 spin echo, T2 spin echo, and T2 fast spin echo sequences. In vitro, the r2 relaxivity decreased over time (0-4 h) when AP were uptaken by cells. The loss of r2 relaxivity was less pronounced with long (Hahn Echo) than short (Carr-Purcell-Meiboom-Gill) echo time sequences. In vivo, our results with ferumoxtran showed an early T2 peak (1 h), suggesting intravascular particles and a second peak in T1 (12 h), suggesting intrainterstitial accumulation of particles. With AP, the late peak (24 h) suggested an intracellular accumulation of particles. In vitro, anionic iron oxide nanoparticles are suitable for cellular labeling due to a high cellular uptake. Conversely, in vivo, ferumoxtran is suitable for passive targeting of tumors due to a favorable biodistribution.

Intracellular uptake of anionic superparamagnetic nanoparticles as a function of their surface coating.

A new class of superparamagnetic nanoparticles bearing negative surface charges is presented. These anionic nanoparticles show a high affinity for the cell membrane and, as a consequence, are captured by cells with an efficiency three orders of magnitude higher than the widely used dextran-coated iron oxide nanoparticles. The surface coating of anionic particle with albumin strongly reduces the non specific interactions with the plasma membrane as well as the overall cell uptake and at the same time restores the ability to induce specific interactions with targeted cells by the coadsorption on the particle surface of a specific ligand. Kinetics of cellular particle uptake for different cell lines are quantitated using two new complementary assays (Magnetophoresis and Electron Spin Resonance).

Binding of biological effectors on magnetic nanoparticles measured by a magnetically induced transient birefringence experiment.

We have investigated the relaxation of the magnetically induced birefringence in a suspension of magnetic nanoparticles in order to detect the binding reaction of polyclonal antibodies on the particle surface. The birefringence relaxation is driven by the rotational diffusion of the complex formed by the magnetic nanoparticles bound to the antibody and thus is directly related to the hydrodynamic size of this complex. Birefringence relaxations are well described by stretched exponential laws revealing a polydisperse distribution of hydrodynamic diameters. Comparing the size distribution of samples with different initial ratios of immunoglobuline added per magnetic nanoparticles, we evidence the graft of an antibody on particle and eventually the onset of particles aggregation. Measurements on samples separated in size by gel filtration demonstrate the robustness of our experiment for the determination of size distribution and its modification due to the adsorption of a macromolecule. The immunoglobuline binding assay is performed comparatively for ionic magnetic nanoparticles with different coatings.

Detection of phosphatidylserine surface exposure on human erythrocytes using annexin V-ferrofluid.

The asymmetric transbilayer distribution of phospholipids in the plasma membrane and the regulation of phosphatidylserine (PS) exposure at the cell surface of animal cells are of high physiological significance. It has been shown previously that annexin V is one of the most sensitive tools with which the presence of small amounts of PS on the outer surface of eukaryotic cells can be detected. We present here the covalent coupling of annexin V molecules to magnetic nanoparticles of maghemite. The resulting annexin V-ferrofluid is used in the magnetic separation of PS exposing cells, as illustrated for human erythrocytes modified in their phospholipid transbilayer asymmetry by the use of a calcium ionophore. Results on stored human erythrocytes and comparison with results obtained using iodinated and fluorescein-labeled annexin V are also presented.

Biomedical applications of maghemite ferrofluid.

The use of cell-targeted ferrofluid in the characterization of modifications of cell membranes is reviewed. Maghemite ferrofluid was synthesized by the Massart method, complexed with dimercaptosuccinic acid (FF). Cell targeting by FF was developed by coupling FF to various biological effectors such as antibodies, lectins, etc, which enabled magnetic cell sorting. Modifications in erythrocyte membranes were studied using FF bound to recombinant human annexin V (AnxFF) which is very sensitive, compared to other Anx-based reagents, in the early detection of phosphatidylserine (PS) exposition on the outer leaflet of the plasma membrane. Thus PS exposition on mouse RBC was detected already after a 24-h storage at 4 degrees C and, transiently, 24 h after their infection by Plasmodium parasites, at which time the parasites are still confined to the liver, thus leading to the recruitment of young RBC and the accumulation of a species, intermediate between reticulocytes and erythrocytes, and the actual RBC target of plasmodial invasion. AnxFF revealed PS exposition on RBC from sickle cell anemia patients, following various inflammations and already after 20 days of human blood storage under blood bank conditions. Such a sensitive detection should be similar to that of macrophages which recognize exposed PS on cells and bring about the latter's elimination from the circulation. AnxFF binding determination was combined with that of cell electrophoretic mobility, glycerol resistance and filterability to characterize RBC membrane modifications in Alzheimer's disease patients which suggested a continuous damage and regeneration in RBC of these patients. A logistic analysis suggested that several three-parameter combinations could permit diagnosis of Alzheimer's disease with up to 95% accuracy. THP1 cells and macrophages, derived themselves by incubation with retinoic acid, were bound to FF and placed in a radio frequency alternating magnetic field. Magnetocytolysis was associated with FF attachment to the cells without damage to non-bound cells and without heating of the surrounding solution.

Surface modification of superparamagnetic nanoparticles (Ferrofluid) studied with particle electrophoresis: application to the specific targeting of cells.

Colloidal aqueous suspension of superparamagnetic nanoparticles (9 nm in diameter) composed of maghemite (gamma Fe2O3) and forming an ionic ferrofluid in aqueous solution are covalently coupled with lectins, enzymes or antibodies, using specific thiol chemistry. The surface charge modifications of nanoparticles, caused by ligand coupling, were monitored by measuring their electrophoretic mobilities using laser-Doppler velocimetry. Particle electrophoretic mobility (PEM) changes are shown to correlate well with the amount of ligand fixed on the particles, as probed by its biological activity. The PEM method provides a useful tool to optimize ligand immobilization at the surface of nanoparticles, and may be advantageous when biological activity measurements are not convenient.

Membrane modifications of red blood cells in Alzheimer's disease.

Red blood cells (RBC) from 24 Alzheimer's disease (AD) patients, 18 age- and sex-matched nondemented (ND) patients, hospitalized in the same facility for orthopedic problems, and 18 healthy volunteers aged 30-52 years were studied in order to gain insight into the nature of RBC membrane modifications in AD. Significant differences were found between RBC from AD and ND patients or young controls respectively for annexin V-binding (45.5 +/- 18.0% vs 27.1 +/- 14.7 and 2.7 +/- 1.9, p = .003), fraction of glycerol resistant cells (30.8 +/- 11.1% vs 19.6 +/- 6.4 and 10.2 +/- 3.1, p = .026), cell electrophoretic mobility in polymer (1.028 +/- 0.022 microns sec-1 V-1 cm vs 1.046 +/- 0.022 and 1.053 +/- 0.021, p = .02) and only limited significance for the filterability (1.46 +/- 0.12 msec vs 1.58 +/- 0.11 and 1.54 +/- 0.11, p = 0.1). A logistic analysis, using simultaneously several features as independent variables, suggested the combined use of annexinV- binding, glycerol resistance, and cell filterability which allowed the assignment of 95% of patients from this cohort to the right group. A prospective analysis of a larger cohort is required for the estimation of the diagnostic value of this test battery. In addition, the high level of annexin binding is characteristic of a disruption of the phospholipid asymmetry in aged or damaged cells, while the high glycerol resistance combined with low electrophoretic mobility an rigidity characterize young RBC, thus indicating an enhanced turnover of RBC in Alzheimer's disease.

Use of annexin V-ferrofluid to enumerate erythrocytes damaged in various pathologies or during storage in vitro.

Recombinant human annexin V was bound covalently to 9 nm maghemite (gamma Fe2O3) nanoparticles, yielding annexin-ferrofluid (AnxFF), and used to separate annexin-bound red blood cells (RBC) in a magnetic field and estimate their percentage in various bloods. Annexin binding in normal human RBC increased proportionately with storage from 8% on day 2 to 42% on day 100. Enhanced AnxFF binding was associated with various pathologies. Thus, normal blood contained 10.7 +/- 5.9% AnxFF binding RBC; bloods with normal sedimentation rates (albeit with some disease necessitating analysis) contained 23.5 +/- 6.2%; those with high sedimentation rates contained 51.5 +/- 12.3%; sickle cell anaemia patients' blood contained 50.0 +/- 9.3%, and bloods from patients with other pathologies (deforming rheumatic disease, cancer necessitating chemotherapy, etc.) contained 58.6 +/- 7.6% AnxFF binding RBC. Enhanced Ca+2-dependent annexin binding reflects a loss of the asymmetric distribution of anionic phospholipids in plasma membranes which may constitute a signal for the destruction of the modified cells by the reticuloendothelial system. Once these preliminary results are confirmed, the determination of the fraction of AnxFF bound erythrocytes, following their magnetic separation, could prove a simple and rapid quality test for example in the context of blood transfusion.