DOI: 10.1002/adma.200702770 Dendrimer-Functionalized Shell-crosslinked Iron Oxide Nanoparticles for In-Vivo Magnetic Resonance Imaging of Tumors** By Xiangyang Shi, * Su He Wang, * Scott D. Swanson, Song Ge, Zhengyi Cao, Mary E. Van Antwerp, Kevin J. Landmark, and James R. Baker, Jr.* Non-invasive diagnosis and detection of early-stage tumors is regarded as one of the current challenges in the biomedical sciences. Magnetic resonance (MR) imaging is a powerful, non-invasive imaging technique because of its high spatial resolution and tomographic capabilities. However, the signal sensitivity of MR imaging for specific biological targets is largely dependent on the specificity and selectivity of the ligand used to target magnetic nanoparticles (NPs) to specific tissues. Development of tumor-targeted magnetic NPs is necessary to enhance the MR signal sensitivity for in-vivo tumor detection. Various proteins such as transferrin, [1,2] anti-carcinoembryonic antigen monoclonal antibody rch 24, [3] herceptin, [4–6] and chlorotoxin [7] have been conjugated onto iron oxide NP surfaces. Unfortunately, these protein ligands tend to display immunogenecity and the biological macromolecules used are very expensive and not available for many types of cancer, which thereby limits their applications. One of the most widely used cancer-targeting ligands is folic acid (FA), which targets FA receptors (FAR) that are overexpressed in several human carcinomas including breast, ovary, endometrium, kidney, lung, head and neck, brain, and myeloid cancers. [8–10] Several groups have investigated the conjugation of folic acid (FA) onto iron oxide NPs for targeting tumor cells. [11–17] However, many of these reports are limited to in-vitro studies. This is largely a result of difficulties related to the in-vivo stability and macrophage uptake of many FA-modified magnetic NPs. It implies that a biocompatible and robust polymer coating onto iron oxide NP surfaces may be essential for a successful in-vivo MR imaging of a tumor. The authors have recently embarked on the surface modification of iron oxide NPs with dendrimers for biomedical imaging applications. Dendrimers are a new class of highly branched, monodispersed, and synthetic macromolecules with well-defined structure, composition, and architecture. Den- drimers, especially poly(amidoamine) (PAMAM) dendrimers, have been shown to be capable of conjugating targeting ligands, imaging agents, and drug molecules for targeted cancer therapeutics. [18–21] It is expected that appropriately manip- ulating the iron oxide NP surfaces with dendrimer chemistry may offer possibilities for sensing of various biological systems. Early work has shown that carboxy-terminated PAMAM dendrimers can be successfully assembled onto Fe 3 O 4 NPs for intracellular uptake studies. [22] However, because of the large amount of carboxy groups on the dendrimer surface, the Fe 3 O 4 NPs modified with FA do not show specific binding to the FAR-expressing cells in vitro. In a previous work, it has been shown that Fe 3 O 4 NPs modified through an approach that combines a layer-by-layer (LbL) self-assembly technique [23–34] and dendrimer chemistry [18–20] can specifically target tumor cells overexpressing FAR in vitro. [35] In these studies, a bilayer composed of polystyrene sulfonate sodium salt (PSS) and FA- and FI (fluorescein isothiocyanate)-functionalized PAMAM dendrimers of generation 5 (G5.NH 2 -FI-FA) were assembled onto Fe 3 O 4 NPs through electrostatic LbL assembly, followed by acetylation of the remaining surface amine groups of the assembled G5 dendrimers. Unfortunately, in-vivo data show that most of these bilayer-modified Fe 3 O 4 NPs accumulate in the liver of mice, which suggests that the particles lack in-vivo stability (unpublished results). Development of a robust polymer shell coating onto Fe 3 O 4 is necessary to achieve a successful in-vivo MR image of a tumor. Approaches to accomplish this involve increasing the polymer layer thickness and/or chemically crosslinking the polymer shells. [36–41] Literature reports show that poly(glutamic acid) (PGA) and poly(L-lysine) (PLL) multilayers can be successfully self- assembled on planar substrates [42,43] and display very good biocompatibility for implant coatings. [44] In this present study, iron oxide NPs are assembled with multilayers of PGA and PLL, followed by assembly with G5.NH 2 -FI-FA dendrimers. The interlayers are then crosslinked through EDC COMMUNICATION [*] Dr. X. Shi, Dr. S. H. Wang, Z. Cao, M. E. Van Antwerp Prof. J. R. Baker, Jr. Michigan Nanotechnology Institute for Medicine and Biological Sciences University of Michigan, Ann Arbor, MI 48109 (USA) E-mail: xshi@umich.edu; shidasui@umich.edu; jbakerjr@umich.edu Dr. S. D. Swanson Department of Radiology University of Michigan, Ann Arbor, MI 48109 (USA) S. Ge, K. J. Landmark Department of Physics University of Michigan, Ann Arbor, MI 48109 (USA) [**] X. Shi and S. H. Wang contributed equally to this work. This project has been funded in whole or in part by the National Institutes of Health (NIH) (under the contract # NIH 1 RO1 EB002657, NOI-CO-97111, and NIH 1 RO1 CA119409) and the Michigan Economic Development Corporation-Life Sciences Corridor Fund (under award GR-472). The authors thank Sasha Meshinchi for his assistance with the TEM experiments and valuable discussions. Supporting Information is available online from Wiley InterScience or from the author. Adv. Mater. 2008, 20, 1671–1678 ß 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 1671