RESUMO
A variety of monodisperse superparamagnetic iron oxide particles (SPIOs) was designed in which the surface was modified by PEGylation with mono- or bifunctional poly(ethylene oxide)amines (PEG). Using (125)I-labeled test proteins (transferrin, albumin), the binding and exchange of corona proteins was studied first in vitro. Incubation with (125)I-transferrin showed that with increasing grade of PEGylation the binding was substantially diminished without a difference between simply adsorbed and covalently bound protein. However, after incubation with excess albumin and subsequently whole plasma, transferrin from the preformed transferrin corona was more and more lost from SPIOs in the case of adsorbed proteins. If non-labeled transferrin was used as preformed corona and excess (125)I-labeled albumin was added to the reaction mixtures with different SPIOs, a substantial amount of label was bound to the particles with initially adsorbed transferrin but little or even zero with covalently bound transferrin. These in vitro experiments show a clear difference in the stability of a preformed hard corona with adsorbed or covalently bound protein. This difference seems, however, to be of minor importance in vivo when polymer-coated (59)Fe-SPIOs with adsorbed or covalently bound (125)I-labeled mouse transferrin were injected intravenously in mice. With both protein coronae the (59)Fe/(125)I-labelled particles were cleared from the blood stream within 30 min and appeared in the liver and spleen to a large extent (>90%). In addition, after 2 h already half of the (125)I-labeled transferrin from both nanodevices was recycled back into the plasma and into tissue. This study confirms that adsorbed transferrin from a preformed protein corona is efficiently taken up by cells. It is also highlighted that a radiolabelling technique described in this study may be of value to investigate the role of protein corona formation in vivo for the respective nanoparticle uptake.
RESUMO
Automated systems have been increasingly utilized for DNA extraction by many forensic laboratories to handle growing numbers of forensic casework samples while minimizing the risk of human errors and assuring high reproducibility. The step towards automation however is not easy: The automated extraction method has to be very versatile to reliably prepare high yields of pure genomic DNA from a broad variety of sample types on different carrier materials. To prevent possible cross-contamination of samples or the loss of DNA, the components of the kit have to be designed in a way that allows for the automated handling of the samples with no manual intervention necessary. DNA extraction using paramagnetic particles coated with a DNA-binding surface is predestined for an automated approach. For this study, we tested different DNA extraction kits using DNA-binding paramagnetic particles with regard to DNA yield and handling by a Freedom EVO(®)150 extraction robot (Tecan) equipped with a Te-MagS magnetic separator. Among others, the extraction kits tested were the ChargeSwitch(®)Forensic DNA Purification Kit (Invitrogen), the PrepFiler™Automated Forensic DNA Extraction Kit (Applied Biosystems) and NucleoMag™96 Trace (Macherey-Nagel). After an extensive test phase, we established a novel magnetic bead extraction method based upon the NucleoMag™ extraction kit (Macherey-Nagel). The new method is readily automatable and produces high yields of DNA from different sample types (blood, saliva, sperm, contact stains) on various substrates (filter paper, swabs, cigarette butts) with no evidence of a loss of magnetic beads or sample cross-contamination.
