Preparation and Quality Control of Nano Sized Dendrimer Based Paramagnetic MRI Contrast Agents

The details of preparation methods for each agent have been previously published in Kobayashi et al. (2003b, 2001a,b,d). In order to load maximum gadolinium ions on a single dendrimer molecule, minor modification of reaction condition was made in each dendrimer. Other researchers used similar but different synthetic methods and chelates, and reported variable conjugation ratio of the chelate molecules and gadolinium ions to dendrimer molecules (Bryant et al., 1999; Wiener et al., 1994). All of the dendrimers investigated for MRL ranged from generation-2 (G2; 3 nm) to generation-10 (G10; 15 nm) for polyami-doamine (PAMAM) and from generation-2 to generation-4 for polypropy-lenimine diaminobutane (DAB). The dendrimer substrate was obtained from commercial sources (Dendritech, Inc., Midland, MI or Aldrich Chemical Co., Milwaukee, WI). These compounds are highly soluble in aqueous solution and both possess a spherical surface topology composed of primary amino groups that increase exponentially in number with each generation (Tomalia et al., 1990; Wu et al., 1994) (Figure 2.1). The well-defined structure, monodispersity of size, and large number of available reactive surface amino groups have led to the use of dendrimers as substrates for the attachment of large numbers of chelating agents for creating macromolecular MR contrast agents (Bryant et al., 1999; Kobayashi and Brechbiel, 2003, 2004, 2005; Wiener et al., 1994). During our synthesis, dendrimers were initially concentrated in phosphate buffer at mild basic pH, and thereafter reacted with molar equivalent amounts of DTPA-derivatives as chelating moieties (Kobayashi et al., 2001d,e; Wu et al., 1994), equal to the number of surface amine residues on the dendrimer molecule and

PAMAM-dendrimer

DAB(PPI)-dendrimer

H2lsf NH,

H2lsf NH,

- -Ca,CH2CONHCH,CH2-

Figure 2.1. Schema of dendrimer cores used for contrast agents.

repeated this reaction 2 to 4 times to conjugate sufficient numbers of chelates with each dendrimer molecules. As a quality control study, a radiolabeling assay that employed either 111In or 153Gd as a tracer demonstrated the number of 1B4M-DTPA molecules conjugated to each dendrimer (Kobayashi et al., 2001e). When the reaction was stopped, more than 90% of the exterior primary amine groups on these dendrimers theoretically reacted with the 1B4M-DTPA thereby providing consistent end products suitable for animal studies (Kobayashi et al., 2001e). However, this analysis and the calculation were based on the weight of totally dehydrated and desalted chelate molecules. As a result, this HPLC-based assay may overestimate the number of chelate molecules on a dendrimer because of the water and salt content in the lyophilized chelate molecules (Kobayashi and Brechbiel, 2005).

Dendrimer-1B4M-DTPA conjugates were mixed with Gd(III) citrate at pH 4.5 and, after formation of the complex, the excess Gd(III) in each preparation was removed by diafiltration with an appropriate molecular weight filtration membrane. As a quality control, a replacement assay that employed 153Gd as a tracer demonstrated that the number of 1B4M-DTPA molecules of the dendrimer-1B4M-DTPA conjugates chelating Gd(III) atoms ranged from 75% to 90% (Kobayashi et al., 2001e). The larger agents generally were able to hold a smaller proportion of Gd ions. The purified contrast agents were separated by size-exclusion high performance liquid chromatography (SE-HPLC) using Sephadex series of size-exclusion columns and analyzed by the 280 nm UV absorption and 153Gd radioactivity. The number of Gd(III) ions per a dendrimer-based contrast agent was determined by the replacement assay and independently validated by the inductively coupled plasma mass spectrometry (ICP-MS) assay. The number of Gd(III) ions per a contrast agent was consistent on both assays (Kobayashi and Brechbiel, 2003).

The size of the dendrimer-based contrast agents were analyzed by SE-HPLC, mass spectroscopy, and light scattering methods. Although the size of the agents varied with technique, variations in size were generally small (~15%) (Kobayashi et al., 2001a; Yordanov et al., 2003).

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