NANO LETTERS
A Highly Effective, Nontoxic T1 MR Contrast Agent Based on Ultrasmall PEGylated Iron Oxide Nanoparticles
2009 Vol. 9, No. 12 4434-4440
Ulrich I. Tromsdorf,*,† Oliver T. Bruns,‡ Sunhild C. Salmen,† Ulrike Beisiegel,‡ and Horst Weller*,† Institute of Physical Chemistry, UniVersity of Hamburg, Grindelallee 117, 20146 Hamburg, Germany, and IBM II: Molecular Cell Biology, UniVersity Medical Center HamburgsEppendorf, Martinistrasse 52, D-20246 Hamburg, Germany Received August 20, 2009
ABSTRACT In this study we systematically developed a potential MR T1 contrast agent based on very small PEGylated iron oxide nanoparticles. We adjusted the size of the crystalline core providing suitable relaxometric properties. In addition, a dense and optimized PEG coating provides high stability under physiological conditions together with low cytotoxicity and low nonspecific phagocytosis into macrophage cells as a part of the reticulo endothelial system at biologically relevant concentrations. The as developed contrast agent has the lowest r2/r1 ratio (2.4) at 1.41 T reported so far for PEGylated iron oxide nanoparticles as well as a r1 relaxivity (7.3 mM-1 s-1) that is two times higher compared to that of Magnevist as a typical T1 contrast agent based on gadolinium as a clinical standard.
Introduction. Superparamagnetic nanoparticles are of special interest for various applications in biotechnology and biomedicine. Their unique magnetic properties which might be fine-tuned on the nanometer scale make them particularly promising in both diagnosis and therapy. Currently, one of the most important and rapidly growing fields is the use of iron oxide particles as contrast agents for magnetic resonance imaging (MRI).1-3 The main task of contrast agent application in MRI is a shortening of the relaxation times T1 and T2 which characterize the two independent processes of proton relaxation. T1 describes the spin-lattice or longitudinal relaxation whereas T2 specifies the spin-spin or transverse relaxation of the excited protons. The efficiency of a contrast agent is usually expressed as its relaxivity r1 or r2, respectively, that is, the ability to shorten the relaxation time per millimole of the contrast agent. For a first classification, contrast agents can be divided into two major types. Positive contrast agents act to shorten mainly the relaxation time T1 and at the same time provide moderate impact on T2, thus generating a bright image. Negative contrast agents on the other hand mainly shorten the transverse relaxation time T2 and lead to signal reduction, that is, a dark image. * Corresponding authors,
[email protected] and
[email protected]. † Institute of Physical Chemistry, University of Hamburg. ‡ IBM II: Molecular Cell Biology, University Medical Center Hamburgs Eppendorf. 10.1021/nl902715v CCC: $40.75 Published on Web 10/02/2009
2009 American Chemical Society
Positive contrast agents commonly consist of paramagnetic chelates such as Gd-DTPA.4,5 Their relaxivity ratio r2/r1 commonly is in the range of 1-2. Recently, MnO nanoparticles have also been used although they exhibited low relaxivities.6 Negative contrast agents predominantly consist of iron oxide particles which can be roughly classified according to their hydrodynamic sizes. They show high r2/ r1 ratios of at least 10. In this sense one important group are iron oxides with hydrodynamic sizes of 40-100 nm which are applied to image cells of the reticulo endothelial system (RES), i.e., macrophages in the liver or the spleen. Smaller particles of approximately 20 nm size can also be used for MR lymphography. The use of iron oxide particles as negative contrast agent arises from the large hydrodynamic diameter of many clinically applied products or controlled clustering7,8 of individual particles. Even single particles with smaller hydrodynamic diameter are preferentially suitable for T2 weighted MRI due to their strong magnetization at common field strengths used for MRI which is associated with their superparamagnetic behavior.9,10 Recently, we systematically investigated the impact of surface modification and compartmentalization of superparamagnetic nanoparticles on negative contrast enhancement and developed a negative contrast agent that allows direct imaging of metabolic processes.11,12 Other results confirmed the importance of surface chemistry on proton relaxivity.13 The use of iron oxide particles in T1 weighted imaging is in most cases limited due to the large r2/r1 ratio although
the impact on T1 is significant and often higher compared to paramagnetic chelates. Therefore, only few examples are published so far where iron oxide particles are applied as T1 contrast agent.14 An important example are so-called blood pool contrast agents that are applied to image particular vessel structures in MR angiography15 (MRA) and provide longer blood half-life compared to the classes described above. Iron oxide based MRA consists of very small iron oxide particles and are coated with small molecules such as citrate.16 As a main advantage over conventional Gd based T1 contrast agents, iron oxide particles provide low long-term toxicity. Gd-based contrast agents have been shown recently to be associated with the development of nephrogenic systemic fibrosis in patients with impaired kidney function, a common disease with increasing incidence in the elderly.17 This severe side effect of Gd-based contrast agents might render these patients wheel-chair dependent and led to new recommendations for the application of these contrast agents. A strategy to form T1 contrast agents suitable for MRA out of iron oxide should involve the following aspects. The size of the crystal core must be suitably synthesized for T1 shortening while the impact on T2 has to be limited. This is in particular the case for ultrasmall iron oxide nanoparticles of core sizes around 5 nm. Second, the organic shell surrounding the core must be designed carefully with respect to stability under physiological conditions as well as a complete prevention of aggregation of individual particles which would result in T2 contrast enhancement again.18-20 Third, these particles should exhibit a low degree of nonspecific uptake by phagocytic cells to display a prolonged circulation time. This paper presents the development of a T1 blood pool contrast agent consisting of very small iron oxide nanoparticles that are coated with poly(ethylene glycol) (PEG) based ligands. Core size and length of the PEG chain were optimized according to stability, relaxometric properties, cytotoxicity, and unspecified cell uptake. So far, a lot of work has been done on the use of PEG as ligand for iron oxide nanocrystals.21-24 However, the coating of nanoparticles with PEG often results in large hydrodynamic diameters and the formation of at least small amounts of aggregates25,26 which in turn enables these systems to act as T2 but not as T1 contrast agent. We synthesized monodisperse (less than 10% standard deviation) iron oxide nanoparticles with core sizes of 4 and 6 nm, and therefore optimized the relaxometric properties. We then used phosphate-functionalized PEG for phase transfer to aqueous solution and adjusted the PEG chain length in order to completely prevent aggregation of particles under physiological conditions and to minimize cytotoxicity and unspecific cell uptake into macrophages. Therefore, we were able to synthesize an iron oxide based T1 contrast agent with a robust PEG coating providing the smallest r2/r1 ratio of 2.4 at clinical relevant fields (1.41 T) reported so far for PEG coated superparamagnetic nanoparticles. The r1 relaxivity of 7.3 mM-1s-1 (calculated from the analytical iron concentration) is approximately two times higher than Nano Lett., Vol. 9, No. 12, 2009
conventional Magnevist (Gd-DTPA). However, our contrast agent should provide low long-term toxicity. Results and Discussion. Oleic acid stabilized superparamagnetic iron oxide nanoparticles (4 and 6 nm mean core diameter) were synthesized as reported previously.27-29 The particles show a narrow size distribution (standard deviation
