Specific Targeting of Tumor Angiogenesis by RGD-Conjugated Ultrasmall Superparamagnetic Iron Oxide Particles Using a Clinical 1.5-T Magnetic Resonance Scanner Chunfu Zhang, 1,2 Manfred Jugold, 1,2 Eva C. Woenne, 1,2 Twan Lammers, 3 Bernd Morgenstern, 1,2 Margareta M. Mueller, 4 Hanswalter Zentgraf, 5 Michael Bock, 2 Michael Eisenhut, 6 Wolfhard Semmler, 2 and Fabian Kiessling 1,2 1 Junior Group Molecular Imaging, 2 Department of Medical Physics in Radiology, 3 Clinical Cooperation Unit Radiotherapeutic Oncology, 4 Tumor and Microenvironment, 5 Applied Tumor Virology, and 6 Radiopharmaceutical Chemistry, German Cancer Research Center, Heidelberg, Germany Abstract 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, novel A v B 3 integrin–targeted ultrasmall superpara- magnetic 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-aminopropyltrime- thoxysilane (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 compet- itively 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 A v B 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 A v B 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 A v B 3 integrins expressed on endothelial cells. Further- more, these molecular MR imaging probes are capable of distinguishing tumors differing in the degree of A v B 3 integrin expression and in their angiogenesis profile even when using a clinical 1.5-T MR scanner. [Cancer Res 2007;67(4):1555–62] Introduction Angiogenesis is an essential step for the growth and spread of malignant tumors (1, 2) and its extension correlates with the malignant potential of several cancers, including breast cancer, malignant melanoma, and skin squamous cell carcinoma (3–5). The cell adhesion molecule a v h 3 integrin is a specific marker of angiogenesis, which is overexpressed in activated and proliferating endothelial cells (6). Clinical studies showed that the expression of a v h 3 integrin correlates with tumor grade (7, 8) and thus suggested a v h 3 integrin as marker of malignancy. Therefore, the ability to noninvasively detect a v h 3 expression in living subjects would allow a better characterization of tumors and help to identify tumor regions with higher aggressiveness. This might be valuable to improve radiotherapy planning and the monitoring of antiangio- genic and other noninvasive antitumor therapies (9). For targeting angiogenic vessels, a v h 3 integrin antibodies were created and are currently evaluated in clinical trials as antiangio- genic therapeutics (10, 11). Additionally, a v h 3 integrins can be targeted by small peptides. A suitable short amino acid sequence that binds to the a v h 3 integrin receptor is Arg-Gly-Asp (RGD). Linear (12, 13) and cyclic RGD peptides (14) have been tested to target a v h 3 integrins for different purposes. Cyclic RGD peptides thatconsistofaringsystemflankedbyunrelatedaminoacidswere shown to better resist proteolysis and to have a higher affinity to the target than their linear counterparts. Several diagnostic compounds based on the above-mentioned targeting vectors have been developed and used in positron emission tomography (PET; refs. 15–17), single-photon emission computed tomography (18, 19), optical imaging (20, 21), and ultrasound (22, 23). Using [ 18 F]galacto-RGD, it was shown by PET that the tracer uptake by melanoma xenografts correlated with its a v h 3 integrin expression level. Furthermore, these specific probes were found to be capable of detecting angiogenesis in squamous cell carcinoma xenografts in mice (A431), which expressed a v h 3 integrins only on angiogenic vessels but not on tumor cells (15). Magnetic resonance (MR) imaging (MRI) is also a highly desirable modality for molecular imaging because it provides not only high spatial resolution but also excellent soft tissue contrast. However, the low sensitivity of MRI to contrast agents often reduces the success of imaging approaches with targeted contrast agents. Thus, particles, polymers, or liposomes loaded with high amounts of gadolinium or superparamagnetic iron oxide particles are usually conjugated to the specific ligands to generate a sufficiently high tissue contrast. Addressing extravascular targets with these conjugates, however, is often problematic due to their limited extravasation and high uptake by the reticuloendothelial system. On the other hand, imaging of angiogenic targets is promising because the targets are presented on the surface of the vessels and thus can directly be addressed from the blood. In this context, T 1 contrast agents have been developed to target a v h 3 integrins by MRI. Sipkins et al. (24) showed that paramagnetic Requests for reprints: Fabian Kiessling, Junior Group Molecular Imaging, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. Phone: 49-6221-422533; Fax: 49-6221-422572; E-mail: f.kiessling@dkfz-heidelberg.de. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-1668 www.aacrjournals.org 1555 Cancer Res 2007; 67: (4). 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