Christian Bergemann

Enhanced Leukemia Cell Detection Using a Novel Magnetic Needle and Nanoparticles

Acute leukemia is a hematopoietic malignancy for which the accurate measurement of minimal residual disease is critical to determining prognosis and treatment. Although bone marrow aspiration and light microscopy remain the current standard of care for detecting residual disease, these approaches cannot reliably discriminate less than 5% lymphoblast cells. To improve the detection of leukemia cells in the marrow, we developed a novel apparatus that utilizes antibodies conjugated to superparamagnetic iron oxide nanoparticles (SPION) and directed against the acute leukemia antigen CD34, coupled with a magnetic needle biopsy. Leukemia cell lines expressing high or minimal CD34 were incubated with anti-CD34-conjugated SPIONs. Three separate approaches including microscopy, superconducting quantum interference device magnetometry, and in vitro magnetic needle extraction were then used to assess cell sampling. We found that CD34-conjugated nanoparticles preferentially bind high CD34-expressing cell lines. Furthermore, the magnetic needle enabled identification of both cell line and patient leukemia cells diluted into normal blood at concentrations below those normally found in remission marrow samples. Finally, the magnetic needle enhanced the percentage of lymphoblasts detectable by light microscopy by 10-fold in samples of fresh bone marrow aspirate approximating minimal residual disease. These data suggest that bone marrow biopsy using antigen-targeted magnetic nanoparticles and a magnetic needle for the evaluation of minimal residual disease in CD34-positive acute leukemias can significantly enhance sensitivity compared with the current standard of care.

Intentional formation of a protein corona on nanoparticles: Serum concentration affects protein corona mass, surface charge, and nanoparticle–cell interaction

The protein corona, which immediately is formed after contact of nanoparticles and biological systems, plays a crucial role for the biological fate of nanoparticles. In the here presented study we describe a strategy to control the amount of corona proteins which bind on particle surface and the impact of such a protein corona on particle-cell interactions. For corona formation, polyethyleneimine (PEI) coated magnetic nanoparticles (MNP) were incubated in a medium consisting of fetal calf serum (FCS) and cell culture medium. To modulate the amount of proteins bind to particles, the composition of the incubation medium was varied with regard to the FCS content. The protein corona mass was estimated and the size distribution of the participating proteins was determined by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Additionally, the zeta potential of incubated particles was measured. Human blood-brain barrier-representing cell line HBMEC was used for in vitro incubation experiments. To investigate the consequences of the FCS dependent protein corona formation on the interaction of MNP and cells flow cytometry and laser scanning microscopy were used. Zeta potential as well as SDS-PAGE clearly reveal an increase in the amount of corona proteins on MNP with increasing amount of FCS in incubation medium. For MNP incubated with lower FCS concentrations especially medium-sized proteins of molecular weights between 30kDa and 100kDa could be found within the protein corona, whereas for MNP incubated within higher FCS concentrations the fraction of corona proteins of 30kDa and less increased. The presence of the protein corona reduces the interaction of PEI-coated MNP with HBMEC cells within a 30min-incubation.

Suitability of Viability Assays for Testing Biological Effects of Coated Superparamagnetic Nanoparticles

The aim of this study was to establish an in vitro model test system for the analysis of cellular effects of nanoparticles on endothelial cells using the human brain microvascular endothelial cell line HBMEC, a representative of the blood-brain barrier. At first we defined a toxic end point by using superparamagnetic iron oxide nanoparticles coated with cationic substances (polyethylenimine, chitosan, diethylamine ethyl). To study the toxic effects of nanoparticles we evaluated three different viability assays and one cytotoxicity assay for their suitability. We could show that a luminescence-based assay is most promising for an accurate testing, because it is reproducible and unaffected by assay conditions and reagents.

Comprehensive analysis of the in vitro and ex ovo hemocompatibility of surface engineered iron oxide nanoparticles for biomedical applications

A set of biomedically relevant iron oxide nanoparticles with systematically modified polymer surfaces was investigated regarding their interaction with the first contact partners after systemic administration such as blood cells, blood proteins, and the endothelial blood vessels, to establish structure–activity relationships. All nanoparticles were intensively characterized regarding their physicochemical parameters. Cyto- and hemocompatibility tests showed that (1) the properties of the core material itself were not relevant in short-term incubation studies, and (2) toxicities increased with higher polymer mass, neutralu2009=u2009anionicu2009<u2009cationic surface charge and charge density, as well as agglomeration. Based on this, it was possible to classify the nanoparticles in three groups,xa0to establish structure–activity relationships and to predict nanosafety. While the results between cyto- and hemotoxicity tests correlated well for the polymers, data were not fully transferable for the nanoparticles, especially in case of cationic low molar mass polymer coatings. To evaluate the prediction efficacy of the static in vitro models, the results were compared to those obtained in an ex ovo shell-less hen’s egg test after microinjection under dynamic flow conditions. While the polymers demonstrated hemotoxicity profiles comparable to the in vitro tests, the size-dependent risks of nanoparticles could be more efficiently simulated in the more complex ex ovo environment, making the shell-less egg model an efficient alternative to animal studies according to the 3R concept.

Magnetic particle spectroscopy allows precise quantification of nanoparticles after passage through human brain microvascular endothelial cells.

Crossing the blood-brain barrier is an urgent requirement for the treatment of brain disorders. Superparamagnetic iron oxide nanoparticles (SPIONs) are a promising tool as carriers for therapeutics because of their physical properties, biocompatibility, and their biodegradability. In order to investigate the interaction of nanoparticles with endothelial cell layers in detail, in vitro systems are of great importance. Human brain microvascular endothelial cells are a well-suited blood-brain barrier model. Apart from generating optimal conditions for the barrier-forming cell units, the accurate detection and quantification of SPIONs is a major challenge. For that purpose we use magnetic particle spectroscopy to sensitively and directly quantify the SPION-specific iron content. We could show that SPION concentration depends on incubation time, nanoparticle concentration and location. This model system allows for further investigations on particle uptake and transport at cellular barriers with regard to parameters including particles shape, material, size, and coating.