Comparison of protein adsorption patterns onto differently charged hydrophilic superparamagnetic iron oxide particles obtained in vitro and ex vivo.

Protein adsorption patterns of superparamagnetic iron oxides (SPIO) were evaluated by two-dimensional electrophoresis (2-DE) after in vitro incubation of the particles in plasma or serum. SPIO particles having positive (MKK 1211), negative (MKA 1211), or neutral (MKG 1411) charge were used. Protein adsorption patterns of different charged SPIO particles acquired in vitro and recollected 5 min after intravenous injection into rats (ex vivo) were compared. For the uncharged MKG 1411 particles, the differences of protein adsorption patterns were negligible and only minor differences were found for the negatively charged MKA 1211 and positively charged MKK 1211 particles. A good correlation between in vitro and ex vivo data could be shown. For the evaluation of protein adsorption patterns of SPIO particles determining organ distribution and allowing estimation of site-specific delivery (drug targeting), the currently used protocol for 2-DE analysis could be confirmed.

USPIO-enhanced direct MR imaging of thrombus: preclinical evaluation in rabbits.

PURPOSE: To test the hypothesis that ultrasmall superparamagnetic iron oxide (USPIO) particles may diffuse into nonendothelialized fresh thrombi and thus allow for direct magnetic resonance (MR) imaging of a thrombus. MATERIALS AND METHODS: Stagnation thrombi of different thrombus ages (1, 3, 5, 7, and 9 days) were induced in the external jugular veins of 25 rabbits. Direct MR imaging of thrombi was performed by using a fat-saturated T1-weighted gradient-echo sequence (three-dimensional [3D] magnetization prepared rapid acquisition gradient echo) before and 24 hours after intravenous administration of USPIO (particle size, 25 nm; 200 micromol per kilogram of body weight). Thrombus length on 3D reconstruction images was compared with that depicted on a radiographic venogram and with histologic findings (joint reference standard). In addition, T2*-weighted gradient-echo images were acquired and scored semiquantitatively. RESULTS: The hyperintensity of the thrombus segment depicted on T1-weighted images (thrombus length determined with 3D reconstruction images divided by true thrombus length) increased significantly after administration of contrast medium at a thrombus age of 3 days (0.6 +/- 0.4 [SD] to 0.8 +/- 0.4; P =.02), 5 days (0.1 +/- 0.1 to 1.0 +/- 0.1; P <.001), and 7 days (0 to 0.6 +/- 0.4; P =.02), but not at an age of 1 and 9 days. No significant change in the thrombus signal intensity was observed on T2*-weighted images. CONCLUSION: The animal model showed that direct MR imaging of the thrombus improved 24 hours after USPIO administration with a T1-weighted sequence. No improvement was seen with the T2*-weighted sequence.

Evaluation of desorption of proteins adsorbed to hydrophilic surfaces by two-dimensional electrophoresis.

The evaluation of the plasma protein adsorption patterns of superparamagnetic iron oxide (SPIO) particles is of high interest concerning their in vivo fate and is carried out by two-dimensional electrophoresis (2-DE). The sample preparation is of great importance, especially the removal of the adsorbed proteins (desorption) from the particle surface for subsequent analysis by 2-DE. The removal is carried out by a desorption solution. In this study, negatively and positively charged SPIO model particles were under investigation concerning the desorption of proteins adsorbed on their surfaces. Firstly, the desorption process was determined quantitatively using the Bradford protein assay. Secondly, the removable or nonremovable protein species, from particles surface were under investigation by 2-DE. Looking at the desorption in a quantitative manner with the Bradford assay, the desorption efficacy from negatively charged particles was about 90%. In the case of the positively charged particles, the desorption efficacy seemed to be reduced, approximately 34% of the proteins remained on the surface. Comparing the protein patterns of the particles evaluated by 2-DE in the desorption solution and the proteins remaining on the particles, they confirmed the results from the protein quantification. After desorption, the IgG gamma-chains were found to be the dominant protein fraction remaining on the negatively charged particles. On the positively charged particles, many more protein species were found after desorption. The more basic the protein fragments, the more ineffective was the desorption from the positively charged model particle, and vice versa. Nevertheless, all protein spots were found qualitatively in the desorption solution, especially when the desorption solutions still containing the particles were used for the 2-DE analysis. In conclusion, 2-DE could be confirmed as the "gold standard" for determining the plasma protein adsorption patterns of nanoparticulate systems.

Two-time window and multiangle photon correlation spectroscopy size and zeta potential analysis--highly sensitive rapid assay for dispersion stability.

Polysaccharide-stabilized iron oxide particles are highly potent contrast agents in magnetic resonance imaging. After intravenous injection, the size of these small or ultrasmall particles strongly influences their distribution in the body. Knowledge about the uniformity of particle size distribution within this particle size range is not accessible by laser diffraction (lower edge of detection range), and photon correlation spectroscopy (PCS) data only yield insufficient information, the so-called polydispersity index. A combination of two-time window and multiangle analysis makes detailed characterization of particle size distribution and particle aggregation feasible, which was shown using five different iron oxide dispersions. Additional particle charge characterization yielded conclusions about the type of stabilization present in the dispersion - electrostatic or steric stabilization. Thus, this thorough particle size and charge analysis is a tool for quick detection of broad particle distributions or aggregates.

Superparamagnetic iron oxide-enhanced MRI of atherosclerotic plaques in Watanabe hereditable hyperlipidemic rabbits.

RATIONALE AND OBJECTIVES: Inflammatory atherosclerotic plaques are characterized by increased endothelial permeability and multiple macrophages. Blood-pool MRI contrast agents like superparamagnetic iron oxide (SPIO) have an affinity for the monocyte-macrophage system and thus, may label inflammatory plaques. The objective was to demonstrate SPIO uptake in plaques of atherosclerotic rabbits by MRI and histology. METHODS: Aortas of anesthetized Watanabe hereditable hyperlipidemic rabbits were studied with a moderately T2*-weighted gradient-echo sequence at 1.5 T. Four groups of five animals each were studied: (1) without ultrasmall SPIO (carboxydextran coating; particle size, 25 nm; estimated plasma half-life, 6 hours) or with imaging after intravenous injection of SPIO at a dose (micromol Fe/kg) and postcontrast time delay (hours) of 50/8 (2), 50/24 (3), or 200/48 (4). In vivo MRI was compared with corresponding ex vivo histological iron stains. RESULTS: Animals receiving 200 micromol Fe/kg demonstrated areas of focal signal loss clearly confined to the aortic wall on a mean of 24 +/- 9 (31% +/- 11%) of 76 +/- 5 images compared with 0 +/- 0 of 76 +/- 5 images in controls (P = 0.009). The number of images with this finding in groups 2 and 3 was not significantly different compared with controls. By microscopy, SPIO-iron was seen in the endothelial cells and subendothelial intimal macrophages of atherosclerosis-prone aortic wall segments. Atherosclerotic lesions demonstrating iron uptake also showed a high macrophage content. CONCLUSIONS: SPIO accumulates in aortic plaques of atherosclerotic rabbits, producing a characteristic MRI finding. As SPIO accumulates in plaques with increased endothelial permeability and a high macrophage content, two established features of plaque inflammation, it may have a potential for noninvasive assessment of inflammatory atherosclerotic plaques.

Interactions of nanoparticles with body proteins--improvement of 2D-PAGE-analysis by internal standard.

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is the method of choice to investigate protein adsorption of blood proteins (opsonization) onto nanoparticular drug carriers. In general the reproducibility of the obtained adsorption patterns is satisfying. However, direct comparison between the amounts of single protein spots from gels obtained in different runs is difficult, because 2D-PAGE is a multistep procedure. A possible solution of the problem is to establish a protein as internal standard. Therefore, selected proteins (Bio-rad) were under investigation. Due to its molecular weight and isoelectric point, soybean trypsin inhibitor (TI) does not interfere with plasma components. Therefore, a method was established to use TI as an internal standard protein to improve comparability between the 2D-PAGE gels obtained in different analytical runs.

The influence of the sample preparation on plasma protein adsorption patterns on polysaccharide-stabilized iron oxide particles and N-terminal microsequencing of unknown proteins.

The in vivo organ distribution of i.v. injected drug carriers is strongly influenced by the adsorption of plasma proteins after i.v. injection, e.g. uptake by the mononuclear phagocytic system (MPS). 2-D PAGE could be established to analyze plasma protein adsorption patterns on polysaccharide-stabilized aqueous iron oxide dispersions used as contrast agents in Magnetic Resonance Imaging (MRI). After incubation in human plasma, centrifugation, a washing procedure and a solubilization step were carried out to obtain the proteins adsorbed onto these ultrasmall particles (65 nm in diameter). Patterns of adsorbed proteins were analyzed in dependence on the washing medium used, i.e. highly purified water, phosphate buffered saline and Krebs buffer pH 7.4. Conductivity and composition of the washing medium influenced the adsorption of IgG onto the particles, but had little effect on the other proteins present. IgG was strongly reduced when using the relatively high conductive buffers. The more stabilizing polysaccharide was desorbed the larger was the total amount of adsorbed proteins. Appearance of two unknown chains of spots in the range of appr. 92 kDa, accounting for appr. 10% and 2% of the overall detected protein amount, was observed only when using Krebs buffer during the washing process. Performing N-terminal microsequencing one unknown chain of spots could be identified as a dimer of fibrinogen gamma chains.

Targeting of ultrasmall superparamagnetic iron oxide (USPIO) particles to tumor cells in vivo by using transferrin receptor pathways.

Human transferrin was covalently coupled to ultrasmall superparamagnetic iron oxide (USPIO) particles, and the transferrin-USPIO obtained was investigated in vivo in experimental SMT/2A tumor-bearing rats (rat mammary carcinoma). Physicochemical characterization showed an overall size of 36 nm (DLS) with a core size of 5 nm (TEM). Relaxivities were R1 = 23.6 and R2 = 52.1 liter/mmol.s (0.47 T). Bound transferrin was 280 micrograms/mg of iron. Pharmacokinetic investigations revealed a half-life of 17 min in normal rats. The MR evaluation of tumor signal intensity over time showed a 40% (range 25-55%) signal reduction 150 min after injection with the reduction persisting for at least 8 h. Control experiments using the parent USPIO compound or USPIO labeled with a nonspecific human serum albumin (HSA-USPIO) showed a change of only 10% (range 5-15%) in tumor signal intensity over time. The results demonstrate that a combination of the USPIO relaxivity properties with the specificity of transferrin-mediated endocytosis allows in vivo detection of tumors by MR imaging.

Determination of plasma protein adsorption on magnetic iron oxides: sample preparation.

PURPOSE: The purpose of this study was to investigate the influence of the sample preparation on the plasma protein adsorption pattern of polysaccharide-stabilized iron oxide particles by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). METHODS: The iron oxide particles were incubated in vitro in human plasma for five minutes. Thereafter, four different methods for particle recovery, including adsorbed proteins from surplus plasma, were investigated: centrifugation, magnetic separation, gel filtration and membrane-based static microfiltration. Adsorbed proteins were desorbed from the particle surfaces by surfactants and analyzed by 2-D PAGE, as described elsewhere (1,2). RESULTS: All the techniques investigated were able to separate small-size iron oxides (approx. 110 nm) and adsorbed proteins from excess plasma. The gels obtained by the different separation procedures displayed almost identical adsorption patterns. Major proteins identified were: fibrinogen, IgG, albumin and an unclassified protein of about 70 kDa with a pI value of 6.5-7.5. CONCLUSIONS: Centrifugation was regarded as the most suitable separation method due to its speed and ease of use. In contrast to gel filtration, any washing media can be used. The magnetic separation process is restricted to particles with high inducible magnetic saturation, in particular, to iron oxides with overall sizes > 50 nm.

The influence of the sample preparation on plasma protein adsorption patterns on polysaccharide-stabilized iron oxide particles and N-terminal microsequencing of unknown proteins.

The in vivo organ distribution of i.v. injected drug carriers is strongly influenced by the adsorption of plasma proteins after i.v. injection, e.g. uptake by the mononuclear phagocytic system (MPS). 2-D PAGE could be established to analyze plasma protein adsorption patterns on polysaccharide-stabilized aqueous iron oxide dispersions used as contrast agents in Magnetic Resonance Imaging (MRI). After incubation in human plasma, centrifugation, a washing procedure and a solubilization step were carried out to obtain the proteins adsorbed onto these ultrasmall particles (65 nm in diameter). Patterns of adsorbed proteins were analyzed in dependence on the washing medium used, i.e. highly purified water, phosphate buffered saline and Krebs buffer pH 7.4. Conductivity and composition of the washing medium influenced the adsorption of IgG onto the particles, but had little effect on the other proteins present. IgG was strongly reduced when using the relatively high conductive buffers. The more stabilizing polysaccharide was desorbed the larger was the total amount of adsorbed proteins. Appearance of two unknown chains of spots in the range of appr. 92 kDa, accounting for appr. 10% and 2% of the overall detected protein amount, was observed only when using Krebs buffer during the washing process. Performing N-terminal microsequencing one unknown chain of spots could be identified as a dimer of fibrinogen gamma chains.

Identification of plasma proteins facilitated by enrichment on particulate surfaces: analysis by two-dimensional electrophoresis and N-terminal microsequencing.

Plasma protein adsorption on intravenously injectable drug carriers is regarded as an important factor for the fate of the particles in the body after their administration. Therefore, the plasma protein adsorption patterns on a number of different carrier systems were analyzed in vitro employing two-dimensional electrophoresis (2-DE). The particulate systems presented in this study were polystyrene (PS) model particles, PS nanoparticles surface-modified by adsorption of a surfactant, a commercial fat emulsion, and magnetic iron oxide particles used as contrast agents in magnetic resonance imaging. Most of the spots in the plasma protein adsorption patterns could be identified by matching the resulting 2-DE gels with a reference map of human plasma proteins. Several other proteins that indicated preferentially adsorbed proteins on the surface of the particles investigated have either not been identified on the reference map, or their identity was found to be ambiguous. The relevant proteins are all present in plasma in low abundance. Since these proteins were strongly enriched on the surface of the particles, the resulting spots on the 2-DE gels were successfully identified by N-terminal microsequencing. With this approach, two chains of spots, designated PLS:6 and PLS:8, were determined on a plasma reference map: inter-alpha-trypsin inhibitor family heavy chain-related protein (also named PK-120) and a dimer of fibrinogen gamma, respectively. Plasma gelsolin is presented in a 2-DE adsorption pattern of PS model particles. One of the main proteins adsorbed by droplets of a commercial fat emulsion was identified as apoliprotein H. Moreover, the positions of apolipoproteins apoC-II and apoC-III were also verified on the 2-DE protein map of human plasma. Thus, protein adsorption experiments of the kind presented in this study are increasing our insight into human plasma proteins.

Liver and spleen enhancement after intravenous injection of carboxydextran magnetite: effect of dose, delay of imaging, and field strength in an ex vivo model.

It has been predicted that liver and spleen enhancement after administration of superparamagnetic contrast agents may be different, depending on the strength of the main magnetic field. With the use of an ex vivo model, we investigated at 0.3, 0.5, and 1.5 T the effects on liver and spleen signal intensity of 5, 15, and 45 mumol/kg body weight of dextran magnetite (SHU 555A) in 54 rats. Nine rats served as controls. At different time delays since injection, the animals were killed, and after perfusion with saline, the liver, brain, and spleen were fixed in formalin. The specimens were embedded in an agar gel matrix and imaged with inversion recovery T1-weighted, proton density spin echo, and T2*-weighted gradient recalled echo (GRE) sequences. At each magnetic field strength, peak liver and spleen signal loss increased with increasing dose of the contrast medium. Signal loss was significantly more conspicuous after a dose of 15 than 5 mumol/kg body weight, but not after a dose of 45 compared with 15 mumol/kg. No signal change was observed in the brain. GRE images showed higher enhancement than proton density-weighted spin echo and inversion recovery images but were noisier. The enhancement showed a plateau between 30 min and 24 hours. Only the signal decrease of the liver after a low dose of contrast medium on GRE images was significantly higher (p < 0.01) at 1.5 than at 0.5 and 0.3 T. Other differences in respect to the field strength were less significant (p < 0.05) or nonsignificant. Differences in the spleen enhancement were nonsignificant. SHU 555A at a dose of 15 mumol/kg is an efficient intracellular contrast agent for liver and spleen at low, mid, and high field strength. Proton density spin echo images are probably the sequence of choice to exploit SHU 555A contrast effects and a wide time window for imaging after its intravenous injection does exist.

Magnetic resonance lymphography in rats: effects of muscular activity and hyperthermia on the lymph node uptake of intravenously injected superparamagnetic iron oxide particles.

RATIONALE AND OBJECTIVES: We investigated the influence of muscular activity and regional body temperature changes on the accumulation of intravenously (i.v.) administered, dextran-coated superparamagnetic iron oxide (SPIO) particles in the lymph nodes of rats. METHODS: Four groups of rats (N = 21) were used. Five rats were allowed to move freely after i.v. contrast administration (group 1). In another five rats, muscular inactivity (group 2) was induced during i.v. injection of SPIO particles and for up to 2 hr thereafter by anesthesia. In seven rats (likewise anesthetized), the contrast agent was administered while the extremities of one side of the body were warmed in a water bath for 2 hr (group 3). The rats in groups 1-3 received 100 mumol Fe/kg of the contrast agent. Four rats not given SPIO particles served as the control group (group 4). The lymph nodes of all animals were removed 24 hr after SPIO administration and were embedded in an agar matrix for magnetic resonance imaging at 1.5 T using a proton-density-weighted spin-echo (PD-SE) sequence and a T2*-weighted gradient-recalled echo (T2* GRE) sequence. RESULTS: Signal loss varied widely among the different lymph nodes in group 1. A pronounced signal reduction was observed in the mesenteric (PD-SE = 20 +/- 6%, T2* GRE = 55 +/- 19%), iliac (PD-SE = 13 +/- 13%, T2* GRE = 44 +/- 24%), and popliteal (PD-SE = 24 +/- 7%, T2* GRE = 70 +/- 11%) lymph nodes and only a moderate reduction in the mandibular (PD-SE = 4 +/- 7%, T2* GRE = 42 +/- 15%), axillary (PD-SE = 0 +/- 4%, T2* GRE = 8 +/- 7%), and inguinal (PD-SE = 5 +/- 5%, T2* GRE = 34 +/- 18%) lymph nodes. The least pronounced signal loss occurred in the peripheral lymph nodes of group 2, ranging from 0 +/- 3% for PD-SE sequences and 10 +/- 11% for T2* GRE sequences to 13 +/- 15% for PD-SE sequences and 41 +/- 19% for T2* GRE sequences. In group 3, the uptake of contrast material in the peripheral lymph nodes of the hyperthermal side was significantly more pronounced than on the contralateral side (p < .01), and the contrast agent was distributed more evenly to the different lymph node groups than in group 1. CONCLUSION: Muscular activity and regional hyperthermia markedly influence the accumulation of SPIO particles in different lymph node groups in rats. These findings must be considered in preclinical studies and in the clinical administration of MR lymphography.

[Toxic shock syndrome]. / Toxisches Schocksyndrom.

The entity of the variable pictures of an illness summed up by the description "toxic shock syndrome" has been focussed on by a great number of publications in Northern America since 1978, but has been described scarcely until yet in Germany. An etiological relationship with formerly described "toxic scarlet fever" and "Kawasaki syndrome" is to be discussed. This case in charge deals with a 25-year-old female patient, who developed acutely without any former disease during her menstruation an illness of severe clinical presentation. The findings were high fever, arterial hypotension, and reversible renal failure, watery diarrhea and vomitus , different cutaneous manifestations and signs of disseminated intravascular coagulation, and severe thrombocytopenia with bleeding mucosal ulcerations. Massive growth of Staphylococcus aureus was demonstrable as well as growth of Proteus mirabilis and E. coli in a culture of vaginal smear.

Reliable routine method for determination of perazine in serum by thin-layer chromatography with an internal standard.

The use of a high-performance thin-layer chromatography linear chamber and of thioridazine as internal standard increases the performance of thin-layer chromatography (TLC) with direct densitometric scanning, and allows the rapid determination of serum levels of the neuroleptic drug perazine under usual therapeutic conditions. TLC is superior to gas-liquid chromatography in so far as the main metabolite desmethylperazine can be easily separated and detected by the same procedure.

[Idiopathic orthostatic hypotension (Shy-Drager syndrome). Results of regional cerebral blood flow measurements (author's transl)]. / Idiopathische orthostatische Hypotension (Shy-Drager-Syndrom). Ergebnisse regionaler Hirndurchblutungsmessungen

Using a gamma camera and the stochastic method, regional cerebral blood flow was measured in a patient with the diagnosis of idiopathic orthostatic hypotension. Blood pressure was altered by tilting the patient in supine position head-up and head-down. A partial defect in the autoregulation and CO2 sensitivity was found. Sensitivity to Norepinephrine was checked by intravenous infusion of 0.02 mug/min/kg body weight. An immediate marked rise in blood pressure was observed. Clinical symptoms, therapeutic guidelines, and histopathological findings are discussed.

[Dichloroethane poisoning with myoclonic syndrome, seizures and irreversible cerebral defects (author's transl)]. / Dichloräthan-Vergiftung mit myoklonischem Syndrom, epileptischen Anfällen und bleibenden cerebralen Defekten

A 48-year-old man, who took by mistake a sip of ointment containing dichloroethane, survived, and showed a course of two phases of toxic symptoms. After an initial narcosis and an interval with few pathological symptoms seizures, myoclonia and somnolence occurred. Irreversible final disturbances were lasting mental defects, cerebellar dysarthria, ataxia, and hydrocephalus. Concomitant diseases were acute liver dystrophy, nephropathy, and anemia. The clinical picture of dichoroethane posoning is outlines, the pathogenesis of this particular cerebral lesion described, and the therapy discussed.