Currently, an important task in the field consists of providing the CAs with targeting capabilities. After the implementation of extracellular and blood pool systems, the new frontier in MRI is represented by the possibility of imaging molecular targets that act as signatures of a given pathology. This will allow early diagnosis of diseases as the detection of an altered biochemical process largely anticipates the anatomical changes that are at the basis of current diagnostic modalities.
Several modalities have contributed to the early stages of this innovative approach, namely positron emission tomography (PET), single photon emission computed tomography (SPECT), MRI and Optical Imaging. MRI-Gd(III)-based agents are much less sensitive than radionuclear and optical imaging probes. Therefore, molecular imaging based on MRI, invariantly involves the need for accumulating a high number of contrast enhancing units at the site of interest. The basis for the design of a Gd-containing imaging probe is first dictated from the concentration and localization (vascular, extracellular matrix, on the cellular membrane, intracellular) of the target molecule.36 Of course, the most accessible targets are those present on the surface of endothe-lial vessels. In principle they can be visualized by a number of macromolecular conjugates containing many Gd(III) complexes functionalized with the proper vector recognizing the given target. A nice example of targeting an endothelial site has been reported by Sipkins etal.37 in the targeting of a specific angiogen-esis marker, the endothelial integrin avp3, whose presence has been shown to correlate with the tumour growth. The imaging probe used in this work is a Gd-containing polymerized liposome. The target is first bound by a biotinilated antibody against avp3, which is successfully recognized by an avidin moiety on the surface of the liposome. Each liposome has a mean diameter of 300-350 nm,
which appears suitable to avoid the uptake by the reticulo-endothelial system (Figure 28.11). This approach provided an enhanced and detailed detection of a rabbit carcinoma through the imaging of the angiogenetic vasculature.
Recently, the same avp3 target has been addressed with lipidic nanoparticles containing a huge number of Gd-chelated units (94,400 Gd/particle characterized by r1 = 19.1 s^mM"1 (per Gd) r1 = 1,800,000 per particle). One of the lipidic components, integrin peptidomimetic antagonist, is covalently bound to the avp3.38
The large molecular size of these systems limits their delivery to targets on the endothelial walls. To target receptors in solid tissues other routes have to be followed. Bhujwalla and co-workers39 have recently developed and applied a two-component Gd-based avidin-biotin system for the visualization of HER-2/ scan receptors. The latter is a member of the epidermal growth factor family and is amplified in multiple cancers. Their approach consisted of addressing the extracellular domain of the receptors by means of a biotinilated mAb. After clearance of the unbound mAb, Gd-labelled avidin is administered and binds, with a high affinity, to the biotinilated mAb. The expression level of the receptor was estimated at 7 x 105 receptors/cell and the average number of Gd-DTPA units per avidin molecule was 12.5. The method has been successfully applied in an experimental mouse model of breast carcinoma.
For labelling cells in culture, the most straightforward route is represented by incubating the cells in culture media added with the CA. In the absence of any specific interaction, the internalization of the agent may occur through pino-cytosis, which is a process consisting of the invagination of the cell membrane to form small vescicles (<nm diameters) called endosomes. Therefore, incubation of cells, for a sufficiently long time, in a medium containing the imaging probe at relatively high concentration may lead to the internalization of the agent at amounts that may be sufficient for MRI visualization. Among a number of systems, the neutral, highly hydrophilic Gd-HPDO3A was considered as a good candidate for labelling stem cells by the pinocytotic route.40 The in vivo MR visualization of labelled stem cells will allow their monitoring after transplantation. In a typical experiment of uptake via pinocytosis, a few millions of stem cells are incubated in a culture medium containing Gd-HPDO3A in the mM concentration range (10-50 mM) for a few hours. Upon incubation no saturation effect is observed and the amount of up taken Gd is linearly proportional to the concentration of the paramagnetic agent in the incubation medium. Once the cell has been internalized, the Gd-HPDO3A molecules get entrapped in endosomic vesicles as it can be checked out by observing the cells incubated with Eu-HPDO3A in the confocal microscope. In fact, Gd and Eu chelates with the same ligands display the same chemical/ biological behaviour and the fluorescent response of Eu-HPDO3A acts as a histological reporter of the localization of Gd-HPDO3A in the cell. The potential of this approach has been proved by observing a mouse model of angiogen-esis, on which blood-derived endothelial progenitor cells (EPCs) had been implanted subcutaneously, within a matrigel plug. After a few days, the histo-logic examination showed large capillary structures transposing the gel plugs. MR images parallel histologic findings as hyperintense spots corresponding to the labelled cells were clearly detected.
Phagocytosis is the process of internalization of particles by cells endowed with phagocytic activity. In such a case, this route appears highly efficient for a singlestep internalization of a large amount of imaging probes. However, to be effective on MR images, Gd chelates must be water soluble. Therefore the particles must be biodegradable in order to release soluble Gd chelates once internalized into phagocytic cells. One may envisage several ways for the release of the Gd chelates. For instance, one may think of gel nano-particles of chitosan loaded with negatively charged Gd chelates. Such particles (200-400 nm diameter) are easily phagocyted and slowly degraded once internalized into the cells. Another approach to biodegradable Gd-containing particles has been pursued by designing particles whose insolubility is a property of the Gd chelates themselves. This goal is easily reached by introducing long aliphatic chains on the surface of the ligand. However, the fate of these systems can be controlled by means of the functionality used to link the insolubilizing moiety to the Gd chelate. In fact, by using ester or peptidic functionalities, the insolu-bilizing synthon can be displaced from the Gd chelates by the activity of the proper enzyme (Figure 28.12).
Another route for pursuing a high concentration of the imaging probe at the target of interest consists of its accumulation inside the cell. One way is offered by the 'cell internalization via receptors' route that is the procedure of choice in a number of nuclear medicine assays. For MRI, the design of the imaging probe requires the attachment of one or more Gd(III) chelates to the ligand molecule. Such structural modification may drastically affect the internalization process in respect to the mechanism occurring for the native ligand.
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