MRI Contrast Agents DOI: 10.1002/ange.200905133 Gadolinium-Conjugated Dendrimer Nanoclusters as a Tumor-Targeted T 1 Magnetic Resonance Imaging Contrast Agent** Zhiliang Cheng, Daniel L. J. Thorek, and Andrew Tsourkas* Contrast agents are being used increasingly in diagnostic magnetic resonance (MR) imaging to help detect and characterize pathological abnormalities. In fact, it has been estimated that nearly 50 % of all MR examinations already involve the use of MR contrast agents, with chelated gadolinium compounds being by far the most widely used. [1, 2] Most clinically relevant gadolinium-based agents are small, non-targeted compounds that passively distribute into the intravascular and interstitial space. [3] However, there has recently been emerging interest in the development of paramagnetic contrast agents that are capable of probing the molecular profile of tissues by ligand targeting, enzymatic activity, and multiplexing. [4, 5] It is envisioned that these agents could be used to acquire a more specific clinical diagnosis and thus improve patient management. To compensate for the low signal enhancement generated by individual gadolinium ions, most targeted gadolinium compounds have relied on the development of nanoplatforms that can carry a high payload of gadolinium and enhance the longitudinal relaxivities (R 1 ) per gadolinium. A wide range of macromolecules and other nanoparticulate systems have already been tested as platforms for gadolinium labeling, including dendrimers, [6–12] polymers, [13] emulsions, [14] silica nanoparticles, [15–17] and vesicles. [18–21] Some of these agents have relaxivities on the order of 10 5 to 10 6 mm À1 s À1 per nanoparticle. [14, 17, 18] As the R 1 value for chelated gadolinium is typically only between 5 and 30 mm À1 s À1 when attached to these nanoparticulate carriers, these contrast agents clearly benefited most from their ability to carry a high gadolinium payload. Furthermore, as the theoretical maximum R 1 for gadolinium is estimated to be only about 80 mm À1 s À1 (1.5 T), [22] it can be argued that any major future improve- ments in the R 1 value per particle will be achieved through the development of nanoplatforms that support higher gadoli- nium payloads. Considering that most current nanoplatforms are only labeled with gadolinium chelates on their outer surface, to ensure high water accessibility, we hypothesized that higher gadolinium payloads could be achieved by the development of highly porous nanoparticles that contained a high gadolinium content throughout the intraparticular volume. Herein, we show that this could be accomplished by creating dendrimer nanoclusters (DNCs) composed of individual gadolinium-labeled PAMAM dendrimers that have been cross-linked to form larger nanoparticulate car- riers. We also demonstrate that these gadolinium-labeled DNCs can readily be functionalized with targeting ligands (e.g. folic acid) and used for in vivo molecular imaging. The synthesis of a folate-receptor targeted gadolinium-labeled DNC is shown in Figure 1. Paramagnetic DNCs were prepared by first crosslinking fifth-generation PAMAM dendrimers with the homobifunc- tional amine-reactive crosslinking agent NHS-PEG-NHS. The presence of a polyethylene glycol (PEG) spacer arm helped maintain the high water solubility of the formed dendrimer clusters. To control nanocluster size, the molar ratio between NH 2 -containing PAMAM dendrimer and NHS- containing BS(PEG) 5 cross-linker was varied. It was found that at a molar ratio of 50:1 [NH 2 ]:[NHS] it was possible to obtain DNCs with an average hydrodynamic diameter of 150 nm and a relatively narrow size distribution, as deter- mined by dynamic light scattering (DLS) measurements (see Supporting Information, Figure S1A). Non-crosslinked indi- vidual dendrimers, with an average diameter of 5.8 nm, were removed by repeated washes on a 100 nm centrifugal filter device. The purified DNCs were labeled with gadolinium by reacting the amine functional groups with the chelating agent diethylenetriaminopentaacetic acid (DTPA) dianhydride. The resulting paramagnetic DNCs were further functional- ized with the optical imaging dye fluorescein isothiocyanate (FITC) and the tumor-targeting ligand folic acid. Transmission electron microscopy (TEM) confirmed the labeling of the DNCs with gadolinium (see Supporting Information, Figure S1B). Owing to the presence of the electron-dense gadolinium ions, DNCs were directly placed on a carbon-coated copper grid and observed without using any additional staining agents that are often required to enhance the contrast of unmodified dendrimers. [23] The DNCs observed by TEM were approximately spherical in shape and 75–150 nm in diameter. These values are slightly smaller than the size measurements acquired by DLS and may reflect the difference between the hydrodynamic diameter measured by DLS and the physical diameter measured by TEM. The smaller average DNC size, based on TEM measurements, could also be a consequence of the limited number of DNCs analyzed in TEM micrographs (n = 20). To assess the paramagnetic properties of the gadolinium- conjugated DNCs, the amount of gadolinium within the [*] Dr. Z. Cheng, D. L.J. Thorek, Dr. A. Tsourkas Department of Bioengineering, University of Pennsylvania 210 South 33rd Street, 240 Skirkanich Hall Philadelphia, PA 19104 (USA) Fax: (+ 1) 215-573-2071 E-mail: atsourk@seas.upenn.edu [**] This work was supported in part by the National Institute of Health (NCI) R21 CA-132658 and the American Cancer Society RSG-07-005- 01. Supporting information for this article, including detailed exper- imental procedures, materials used, DLS/TEM measurements, cellular uptake, cell pellets, and cell viability, is available on the WWW under http://dx.doi.org/10.1002/anie.200905133. Zuschriften 356  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. 2010, 122, 356 –360