Hanswalter Zentgraf

Publications of Hanswalter Zentgraf

• RGD-labeled USPIO Inhibits Adhesion and Endocytotic Activity of {alpha}v{beta}3-Integrin-expressing Glioma Cells and Only Accumulates in the Vascular Tumor Compartment.

  Authors: Fabian Kiessling, Jochen Huppert, Chunfu Zhang, Jabadurai Jayapaul, Stefan Zwick, Eva C Woenne, Margareta M Mueller, Hanswalter Zentgraf, Michael Eisenhut, Yoseph Addadi, Michal Neeman, Wolfhard Semmler

  Radiology. 09/2009;

  Purpose: To investigate the biologic effect of arginine-glycine-aspartic acid (RGD)-labeled ultrasmall superparamagnetic iron oxide (USPIO) (referred to as RGD-USPIO) on human umbilical veinPurpose: To investigate the biologic effect of arginine-glycine-aspartic acid (RGD)-labeled ultrasmall superparamagnetic iron oxide (USPIO) (referred to as RGD-USPIO) on human umbilical vein endothelial cells (HUVECs), ovarian carcinoma (MLS) cells, and glioblastoma (U87MG) cells and on U87MG xenografts in vivo. Materials and Methods: All experiments were approved by the governmental review committee on animal care.USPIOs were coated with integrin-specific (RGD) or unspecific (arginine-alanine-aspartic acid [RAD]) peptides. USPIO uptake in HUVECs, MLS cells, and U87MG cells and in U87MG tumor xenografts was determined with T2 magnetic resonance (MR) relaxometry in 16 nude mice. Cells and tumors were characterized by using immunofluorescence microscopy. Trypan blue staining and lactate dehydrogenase assay were used to assess cytotoxicity. Statistical evaluation was performed by using a Mann-Whitney test or a linear mixed model with random intercept for the comparison of data from different experiments. Post hoc pairwise comparisons were adjusted according to a Tukey test. Results: HUVECs and MLS cells internalized RGD-USPIOs significantly more than unspecific probes. Controversially, U87MG cells accumulated RGD-USPIOs to a lesser extent than USPIO. Furthermore, only in U87MG cells, free RGD and alpha(v)beta(3) integrin-blocking antibodies strongly reduced endocytosis of nonspecific USPIOs. This was accompanied by a loss of cadherin-dependent intercellular contacts, which could not be attributed to cell damage. In U87MG tumors, RGD-USPIO accumulated exclusively at the neovasculature but not within tumor cells. The vascular accumulation of RGD-USPIO caused significantly higher changes of the R2 relaxation rate of tumors than observed for USPIO. Conclusion: In glioma cells with unstable intercellular contacts, inhibition of alpha(v)beta(3) integrins by antibodies and RGD and RGD-USPIO disintegrated intercellular contacts and reduced endocytotic activity, illustrating the risk of inducing biologic effects by using molecular MR probes. Supplemental material: http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.2532081815/-/DC1 (c) RSNA, 2009.

• Specific targeting of tumor angiogenesis by RGD-conjugated ultrasmall superparamagnetic iron oxide particles using a clinical 1.5-T magnetic resonance scanner.

  Authors: Chunfu Zhang, Manfred Jugold, Eva C Woenne, Twan Lammers, Bernd Morgenstern, Margareta M Mueller, Hanswalter Zentgraf, Michael Bock, Michael Eisenhut, Wolfhard Semmler, Fabian Kiessling

  Cancer research. 03/2007; 67(4):1555-62.

  Angiogenesis is essential for the development of malignant tumors and provides important targets for tumor diagnosis and therapy. To noninvasively assess the angiogenic profile of tumors, novelAngiogenesis is essential for the development of malignant tumors and provides important targets for tumor diagnosis and therapy. To noninvasively assess the angiogenic profile of tumors, novel alpha(v)beta(3) integrin-targeted ultrasmall superparamagnetic iron oxide particles (USPIOs) were designed and their specific uptake by endothelial cells was evaluated in vitro and in vivo. USPIOs were coated with 3-aminopropyltrimethoxysilane (APTMS) and conjugated with Arg-Gly-Asp (RGD) peptides. Accumulation in human umbilical vein endothelial cells (HUVECs) was evaluated using Prussian blue staining, transmission electron microscopy, magnetic resonance (MR) imaging, and inductively coupled plasma mass spectrometry. Uptake of RGD-USPIO by HUVECs was significantly increased when compared with unlabeled USPIO and could be competitively inhibited by addition of unbound RGD. The ability of the RGD-USPIO to noninvasively distinguish tumors with high (HaCaT-ras-A-5RT3) and lower (A431) area fractions of alpha(v)beta(3) integrin-positive vessels was evaluated using a 1.5-T MR scanner. Indeed, after RGD-USPIO injection, there was a more pronounced decrease in T(2) relaxation times in HaCaT-ras-A-5RT3 tumors than in A431 tumors. Furthermore, T(2)*-weighted images clearly identified the heterogeneous arrangement of vessels with alpha(v)beta(3) integrins in HaCaT-ras-A-5RT3 tumors by an irregular signal intensity decrease. In contrast, in A431 tumors with predominantly small and uniformly distributed vessels, the signal intensity decreased more homogeneously. In summary, RGD-coupled, APTMS-coated USPIOs efficiently label alpha(v)beta(3) integrins expressed on endothelial cells. Furthermore, these molecular MR imaging probes are capable of distinguishing tumors differing in the degree of alpha(v)beta(3) integrin expression and in their angiogenesis profile even when using a clinical 1.5-T MR scanner.

• Silica- and alkoxysilane-coated ultrasmall superparamagnetic iron oxide particles: a promising tool to label cells for magnetic resonance imaging.

  Authors: Chunfu Zhang, Björn Wängler, Bernd Morgenstern, Hanswalter Zentgraf, Michael Eisenhut, Harald Untenecker, Ralf Krüger, Ralf Huss, Christian Seliger, Wolfhard Semmler, Fabian Kiessling

  Langmuir : the ACS journal of surfaces and colloids. 02/2007; 23(3):1427-34.

  In this study silica- and alkoxysilane-coated ultrasmall superparamagnetic iron oxide (USPIO) particles were synthesized, and their ability to label immortalized progenitor cells for magneticIn this study silica- and alkoxysilane-coated ultrasmall superparamagnetic iron oxide (USPIO) particles were synthesized, and their ability to label immortalized progenitor cells for magnetic resonance imaging (MRI) was compared. USPIO particles were synthesized by coprecipitation of ferric and ferrous salts. Subsequently, the particles were coated with silica, (3-aminopropyl)trimethoxysilane (APTMS), and [N-(2-aminoethyl)-3-aminopropyl]trimethoxysilane (AEAPTMS). The size of the USPIO particles was about 10 nm without a significant increase in diameter after coating. The highest T2 relaxivity was achieved for silica-coated USPIO particles, 339.80 +/- 0.22 s-1 mM-1, as compared with APTMS- and AEAPTMS-coated ones, reaching 134.40 +/- 0.01 and 84.79 +/- 0.02 s-1 mM-1, respectively. No toxic effects on the cells could be detected by trypan blue, TUNEL, and MTS assays. Uptake of USPIO particles was evaluated by Prussian blue staining, transmission electron microscopy, T2-MR relaxometry, and mass spectrometry. It was found that cell uptake of the different USPIO particles increased for longer incubation times and higher doses. Maximum cellular iron concentrations of 42.1 +/- 4.0 pg/cell (silica-coated USPIO particles), 37.1 +/- 3.5 pg/cell (APTMS-coated USPIO particles), and 32.7 +/- 4.0 pg/cell (AEAPTMS-coated USPIO particles) were achieved after incubation of the cells with USPIO particles at a dose of 3 micromol/mL for 6 h. The decrease of the T2 relaxation time of the cell pellets was most pronounced for cells incubated with silica-coated USPIO particles followed by APTMS- and AEAPTMS-coated particles, respectively. In gelatin gels even small clusters of labeled cells were detected by 1.5 T MRI, and significant changes in the T2 relaxation times of the gels were determined for 10000 labeled cells/mL for all particles. In summary, as compared with APTMS- and AEAPTMS-coated particles, silica-coated USPIO particles provide the highest T2 relaxivity and most effectively reduce the T2 relaxation time of immortalized progenitor cells after internalization. This suggests silica-coated USPIO particles are most suited for cell labeling approaches in MRI.