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Abstract:
introduction: Molecular imaging has an important role in diagnosis and thus the effective
treatment of diseases. Several strategies have been developed to specifically target/trace the early
biochemical, physiological and anatomical changes under pathological conditions. The development
of molecular probes selectively binding the biochemical targets has proved to be a boon in this area.
The most common strategy for designing these probes make use of the monoclonal antibody (mab)
fragments, high affinity mab, pretargeting strategies etc. The pretargeting techniques using the high
affinity of biotin to avidin has proved to be the most efficient technique for selective accumulation of
the probes at the target site 1.
Methods: The basic skeleton of the molecules is
do3a, functionalized and linked to biotin. The
linking is done in a way to increase the stability toward cleavage action of the biotinidase. Two
complexes,
gd-biotin1 and
gd-biotin2, have been synthesized and characterized by 1h
nMr, 13C
nMr, and
eSi-MS. The synthesized ligands were finally loaded with gadolinium and the exact
concentration of the complexes obtained was determined by bulk magnetic shift method. The
relaxivity in the absence and presence of avidin was measured at various magnetic fields (1.5T, 4.7T,
7T, and 9.4T). Shin-3 cells were measured with the complexes (+/- avidin) and relaxation times were
estimated in these cells at 3T in order to test if the magnetic properties are maintained in cellular
system.
results: The in vitro relaxivity measurement of
gd-biotin1 and -2 with avidin in buffer
demonstrated different longitudinal (r1) and transverse relaxation (r2) behaviour.
gd-biotin1
enhanced r2 by ~1000% with the addition of avidin at all four magnetic fields, while r1 showed field
dependent response.
at 9.4 T 47% decrease, at 7T 31% decrease; at 3T 82% increase and at 1.5 T an
increase of 266% was observed.
on the otherhand, r1 of
gd-biotin2 decreased by ~ 200% at 9.4T and
7T, 40% at 3T and increased by 40% at 1.5T. The r2 however, showed an increase of 240% at 7T. The
measurement of r2 at other magnetic field is under progress.
labelling of Shin-3 cells with these
complexes did not induce similar T1/T2 changes inside the cells.
no significant enhancement in
r2
was detectable for
gd-biotin1 and -2 at 3T while
r1 increased by 10-14%. This might be due to the
uptake of the complex into the cells by endocytosis.
as the agent remains trapped in the vesicles, the
contact with water molecules is limited, preventing the relaxivity enhancement as it was observed in
buffer.
Conclusions: The in vitro relaxivity data shows that the two complexes although only slightly
vary in the linker connecting biotin and
gd-do3a but shows very different behaviour toward their
binding affinity to avidin and thus on the observed relaxivity.
amongst the two complexes,
gd-biotin1
showed the most promising r2/r1 behaviour in the presence of avidin.
at 1.5 T, a marked increase in
both r1 and r2 was observed which indicates that the agent could be used as T1 agent as well as T2
agent. The structural design also offers the stability against biotinidase degradation which is
important for the in vivo applications. The results obtained with Shin-3 cells indicate that such
complexes should only be used for extracellular tagging of cell and internalization should be avoided in order to maintain the strong T2 effects of the probe.
