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.

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.

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.