Intracellular Distribution of the Imaging Probe

When the imaging probe is internalized into the cell, its effect may depend upon its location. To improve our insight into this issue, we have compared the relaxation enhancement measured for Gd-HPDO3A internalized in HTC (post-hepatocarcinoma cell line) by pynocytosis and by electroporation, respectively, [84]. Whereas the former route causes the entrapment of the contrast agent into endosomic vesicles, the latter one leads to its dispersion into the cytoplasm. Electroporation consists of the formation of transient hydrophilic pores on the cell membrane upon the application of suitable electric pulses between the two electrodes placed in the cell suspension [85]. As shown in Figure 4.9, the relaxation rate of the cells labeled by pynocytosis shows a saturation effect upon increasing the amount of the internalized Gd-HPDO3A with limiting Rj values of approximately 3 s"Conversely, the Rj values for the cellular pellets labeled by electroporation are markedly higher, and even more importantly, they are linearly dependent upon the amount of the internalized complex. A cellular pellet (and a portion of tissue as well) can be considered as a multisite system where the water molecules are distributed in the extra- and in the intracellular (or cytosolic) compartments. Such compartments are separated by the cellular membrane, whose water permeability is crucial for determining the relaxometric behavior of the whole pellet. The behavior observed in Figure 4.9 may be explained in terms of a three-site water exchange model when the imaging probe is entrapped into endosomes (extracellular/cytoplasm/endosome compartments) and in terms of a two-site exchange model when the paramagnetic agent is only dispersed into the cytoplasm. On this basis, the quenching effect of the exchange on the relaxation rate of cytosolic water protons is the responsible factor for the saturation of the relaxation rate observed at high concentrations of the internalized probe for the HTC pellets labeled by pynocytosis. The results obtained note the importance of the procedure used for labeling cells and demonstrate that the cytosol confinement of the probe yields higher relaxing efficiency, in turn allowing the MRI detection of a smaller number of cells with respect to the entrapment into endosomes.

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