The fate of a designed protein corona on nanoparticles in vitro and in vivo.

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.

The distribution and degradation of radiolabeled superparamagnetic iron oxide nanoparticles and quantum dots in mice.

(51)Cr-labeled, superparamagnetic, iron oxide nanoparticles ((51)Cr-SPIOs) and (65)Zn-labeled CdSe/CdS/ZnS-quantum dots ((65)Zn-Qdots) were prepared using an easy, on demand, exchange-labeling technique and their particokinetic parameters were studied in mice after intravenous injection. The results indicate that the application of these heterologous isotopes can be used to successfully mark the nanoparticles during initial distribution and organ uptake, although the (65)Zn-label appeared not to be fully stable. As the degradation of the nanoparticles takes place, the individual transport mechanisms for the different isotopes must be carefully taken into account. Although this variation in transport paths can bring new insights with regard to the respective trace element homeostasis, it can also limit the relevance of such trace material-based approaches in nanobioscience. By monitoring (51)Cr-SPIOs after oral gavage, the gastrointestinal non-absorption of intact SPIOs in a hydrophilic or lipophilic surrounding was measured in mice with such high sensitivity for the first time. After intravenous injection, polymer-coated, (65)Zn-Qdots were mainly taken up by the liver and spleen, which was different from that of ionic (65)ZnCl2. Following the label for 4 weeks, an indication of substantial degradation of the nanoparticles and the release of the label into the Zn pool was observed. Confocal microscopy of rat liver cryosections (prepared 2 h after intravenous injection of polymer-coated Qdots) revealed a colocalization with markers for Kupffer cells and liver sinusoidal endothelial cells (LSEC), but not with hepatocytes. In J774 macrophages, fluorescent Qdots were found colocalized with lysosomal markers. After 24 h, no signs of degradation could be detected. However, after 12 weeks, no fluorescent nanoparticles could be detected in the liver cryosections, which would confirm our (65)Zn data showing a substantial degradation of the polymer-coated CdSe/CdS/ZnS-Qdots in the liver.

The cell-type specific uptake of polymer-coated or micelle-embedded QDs and SPIOs does not provoke an acute pro-inflammatory response in the liver.

Semiconductor quantum dots (QD) and superparamagnetic iron oxide nanocrystals (SPIO) have exceptional physical properties that are well suited for biomedical applications in vitro and in vivo. For future applications, the direct injection of nanocrystals for imaging and therapy represents an important entry route into the human body. Therefore, it is crucial to investigate biological responses of the body to nanocrystals to avoid harmful side effects. In recent years, we established a system to embed nanocrystals with a hydrophobic oleic acid shell either by lipid micelles or by the amphiphilic polymer poly(maleic anhydride-alt-1-octadecene) (PMAOD). The goal of the current study is to investigate the uptake processes as well as pro-inflammatory responses in the liver after the injection of these encapsulated nanocrystals. By immunofluorescence and electron microscopy studies using wild type mice, we show that 30 min after injection polymer-coated nanocrystals are primarily taken up by liver sinusoidal endothelial cells. In contrast, by using wild type, Ldlr (-/-) as well as Apoe (-/-) mice we show that nanocrystals embedded within lipid micelles are internalized by Kupffer cells and, in a process that is dependent on the LDL receptor and apolipoprotein E, by hepatocytes. Gene expression analysis of pro-inflammatory markers such as tumor necrosis factor alpha (TNFα) or chemokine (C-X-C motif) ligand 10 (Cxcl10) indicated that 48 h after injection internalized nanocrystals did not provoke pro-inflammatory pathways. In conclusion, internalized nanocrystals at least in mouse liver cells, namely endothelial cells, Kupffer cells and hepatocytes are at least not acutely associated with potential adverse side effects, underlining their potential for biomedical applications.

Synthesis of radioactively labelled CdSe/CdS/ZnS quantum dots for in vivo experiments.

During the last decades of nanoparticles research, many nanomaterials have been developed for applications in the field of bio-labelling. For the visualization of transport processes in the body, organs and cells, luminescent quantum dots (QDs) make for highly useful diagnostic tools. However, intercellular routes, bio-distribution, metabolism during degradation or quantification of the excretion of nanoparticles, and the study of the biological response to the QDs themselves are areas which to date have not been fully investigated. In order to aid in addressing those issues, CdSe/CdS/ZnS QDs were radioactively labelled, which allows quantification of the QD concentration in the whole body or in ex vivo samples by γ-counting. However, the synthesis of radioactively labelled QDs is not trivial since the coating process must be completely adapted, and material availability, security and avoidance of radioactive waste must be considered. In this contribution, the coating of CdSe/CdS QDs with a radioactive (65)ZnS shell using a modified, operator-safe, SILAR procedure is presented. Under UV illumination, no difference in the photoluminescence of the radioactive and non-radioactive CdSe/CdS/ZnS colloidal solutions was observed. Furthermore, a down-scaled synthesis for the production of very small batches of 5 nmol QDs without loss in the fluorescence quality was developed. Subsequently, the radio-labelled QDs were phase transferred by encapsulation into an amphiphilic polymer. γ-counting of the radioactivity provided confirmation of the successful labelling and phase transfer of the QDs.