The major aim of this study was to prove or disprove the theories concerning the correlation between physicochemical properties of superparamagnetic iron oxide (SPIO) particles and their accumulation in the lymph nodes. New SPIO particles were produced using starch as stabilising polymer shell. The synthesis was done in a two-step procedure using conventional wet-chemical precipitation technique and subsequent coating of the iron oxide cores. The particles were physicochemically characterised and their lymphotrophy studied in rats using well described lymphotropic dextran-coated SPIO particles as reference. Despite the short blood half-lives of approximately 13 min and the relatively large sizes (approximately 60-90 nm), the starch SPIO particles proved at least as efficient in lymph node accumulation as the small 25 nm dextran SPIO particles having a half-life of 90 min. The currently accepted theories concerning the connection between particle properties and their uptake into lymph nodes are not generally valid, or have at least to be limited for dextran-coated SPIO particles. Lymph node targeting could be achieved despite the present theories consider small size (<30 nm) and long circulation times in the blood as prerequisites. Histological examination showed, that SPIO particles could only be found in lymph node areas where macrophages could be marked which enclosed the particles. Localisation in marginal areas of the lymph nodes indicates endothelial transcytosis as the major accumulation pathway.
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.
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.
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