Figure 28.12 The insoluble system is represented by a Gd-DTPA-like complex functionalized with a long aliphatic chain which is bound to the chelate moiety through an ester bond; and the action of the esterase causes the cleavage of the bond between the insoluble moiety and the soluble Gd(III) chelate

Figure 28.13 Schematic representation of Gd-HPDO3A entrapped into the Apoferritin cavity (see Plate 6)

In order to deal with a system whose structural characteristics were unaltered by the loading with Gd(III) chelates, Apoferritin was chosen because it allows the imaging probes to be entrapped inside its inner cavity.41 (Figure 28.13) The exterior of such Gd(III)-loaded Apoferritin is exactly the same as in the parent Ferritin and then, once administered intravenously, it is quickly cleared up by the proper receptors on hepatocytes.42

In such a system, water can freely diffuse through the channels formed at the intersection of the protein subunits (10 channels), but the larger Gd-HPDO3A molecules cannot. The relaxivity shown by each Gd-HPDO3A entrapped in the Apoferritin cavity is very high (ca. 80s~1mMGd~1 at 20 MHz and 25 °C). It has been possible to assess that the Gd-loaded Apoferritin maintains its integrity upon the cell internalization process as the relaxivity observed for the cytoplasmatic extract corresponds to that of the intact system. Finally, the amount of cell-internalized Gd-loaded Apoferritin is similar to that reported for the native Ferritin (6.5 x 106 molecules per cell in 6h).

Probably, the most common route for the internalization of Gd(III) chelates is through a receptor-mediated endocytosis that is expected to occur any time a given ligand is modified by attaching one or more Gd-chelating units. Thus, the internalization process is no longer the one followed by the native ligand but the binding to the receptor stimulates an endocytotic process which starts with the folding of a portion of cellular membrane and ends up with the welding of its extremities. Through this process, a number of substances remain entrapped in endosomic vesicles, primarily the molecules bound to the membrane or in close proximity to the portion of membrane involved in the endosome formation.

Weiner etal.43 showed that the uptake of a folate-conjugate dendrimer into tumour cells over expressing high-affinity Folate Receptor (hFR) occurs through this type of endocytotic pathway. Another example of receptor mediated endocytosis of a large dendrimer has been recently reported.44 The macromolecular system comprising Avidin and a biotinilated dendrimer bearing 254 Gd-DTPA chelates (AV-G6Gd) has shown to accumulate 'in vitro' into SHIN3 cells (a cell line obtained originally from human ovarian cancer) 50-fold greater than Gd-DTPA. The internalization process is driven by an Avidin molecule, a glycoprotein that binds to ^-D-galactose receptors which are present on these tumour cells.

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