Invited Review Insights into the Use of Paramagnetic Gd(III) Complexes in MR-Molecular Imaging Investigations Silvio Aime, PhD, 1 * Claudia Cabella, PhD, 2 Sebastiano Colombatto, PhD, 3 Simonetta Geninatti Crich, PhD, 1 Eliana Gianolio, PhD, 1 and Fabio Maggioni 2 Can gadolinium III [Gd(III)] complexes be considered good candidates for magnetic resonance (MR)-molecular imag- ing studies? In this review article, we examine the princi- pal issues that are the basis of successful use of Gd-based protocols in molecular imaging applications. High relax- ivity is the primary requisite. Therefore, the design of such paramagnetic probes has to be pursued keeping in mind the relationships between structure, dynamics, and the relevant parameters involved in paramagnetic relax- ation processes. Moreover, the limited number of target molecules on cellular membranes makes it necessary to define strategies aimed at delivering many Gd-containing moieties to the sites of interest. Examples are reported for the attainment of very high relaxivities for the design of new routes for pursuing the accumulation of small sized Gd(III) complexes at the targeting sites. An efficient cellular uptake of Gd-containing probes is the key step for attaining the visualization of targeted cells by MR imag- ing, and selected examples are reported. In this context, the problem of the assessment of the minimum amount of Gd(III) complexes necessary for the MR imaging-visualiza- tion of cells has been addressed by reporting the authors’ observations on the cell-internalization of Gd(III) com- plexes. A particularly efficient delivery system is repre- sented by Gd-loaded apoferritin, which allows the MR visualization of hepatocytes when the number of Gd-com- plexes per cell is 4  1  10 7 . Finally, the potential of responsive systems is considered by outlining the exploi- tation of the amplification effect brought about by the action of a specific enzymatic activity on the relaxivity of a suitably functionalized Gd(III) complex. Key Words: Molecular imaging; MRI; Gd(III) complexes; cel- lular uptake; relaxivity J. Magn. Reson. Imaging 2002;16:394 – 406. © 2002 Wiley-Liss, Inc. IT IS NOW WELL ESTABLISHED that magnetic reso- nance imaging (MRI) is the pre-eminent methodology among the various diagnostic modalities currently available, as it offers a powerful way to map structure and function in soft tissues by sampling the amount, flow, and environment of water protons in vivo. The intrinsic contrast can be augmented by the use of con- trast agents (CA) in both clinical and experimental set- tings. Thus, the use of CA adds highly relevant physiolog- ical information to the superb anatomical resolution obtained in MR images. Most of the work with CAs has dealt with the use of gadolinium (III) [Gd(III)] complexes, because this metal ion couples a large magnetic mo- ment with a long electron spin relaxation time. The new scenery of molecular imaging applications requires the development of a novel class of CAs char- acterized by a higher contrasting ability and improved targeting capabilities (1). In this survey, we intend to tackle some basic issues that are of fundamental im- portance for the use of Gd(III)-based systems in molec- ular imaging applications, namely: 1) actual under- standing of the determinants of T 1 -relaxivity of Gd(III) complexes, and how to proceed to attain very high re- laxivities; 2) how one may envisage efficient routes for the delivery of a high number of Gd(III) complexes at the site of interest; 3) at the currently available magnetic field strength, which is an estimate for the minimum number of Gd(III) complexes per cell necessary for MRI visualization; 4) the most practical ways to pursue the cell-internalization of a high number of Gd(III) com- plexes; and 5) how a Gd-based probe can act as a reporter of a specific parameter of the micro-environ- ment in which it distributes. RATIONAL DESIGN OF GD(III)-BASED SYSTEMS ENDOWED WITH HIGH RELAXIVITIES The relaxivity is usually measured in vitro and repre- sents the relaxation enhancement of water protons pro- moted by the paramagnetic complex at a 1 mM concen- tration. The theory of paramagnetic relaxation provides a basis for understanding the relationships between the structure and dynamic properties of a Gd(III) complex and its relaxivity (2,3). It has been shown that, at the imaging fields, high relaxivities can be obtained in the presence of long reorientational times ( R ) of the com- plex. The molecular motion of the complex can be slowed down by the formation of covalent or non-cova- lent conjugates between the paramagnetic complex and a slowly moving substrate, such as proteins (4), mi- 1 Dipartimento di Chimica I.F.M., Universita ` di Torino, Torino, Italy. 2 Bracco Imaging S.p.A, Milano, Italy. 3 Dipartimento di Medicina e Oncologia Sperimentale, Torino, Italy. Contract grant sponsor: MIUR (PRIN); Contract grant sponsor: CNR (Biotechnology PF, Oncology and L. 95/95); Contract grant sponsor: Bracco Imaging S.p.a (Milano Research Center). *Address reprint requests to: S.A., Dipartimento di Chimica Inorganica, Chimica Fisica e Chimica dei materiali, Universita ` di Torino, Via Pietro Giuria 7, I-10125 Torino, Italy. E-mail: silvio.aime@unito.it Received April 9, 2002; Accepted June 25, 2002. DOI 10.1002/jmri.10180 Published online in Wiley InterScience (www.interscience.wiley.com). JOURNAL OF MAGNETIC RESONANCE IMAGING 16:394 – 406 (2002) © 2002 Wiley-Liss, Inc. 394