We have synthesized a couple of new MRI contrast agent for recognition of cellular vicinal thiols motif protein thioredoxin.
Preparation of Gd3+ Complex. Ligand (80 µm) was in ultrapure water (10 mL) and the solution was adjusted to ~pH 7 with sodium bicarbonate. Gadolinium chloride hexahydrate (78 µm) was dissolved in 3.0 mL of ultrapure water and added to the solution of ligand in three separate aliquots. After the addition of each aliquot, the pH was adjusted back to a pH between 6.5–7.0 using 0.1 M potassium carbonate solutions. The solution was allowed to stir for 30 min to allow for Gd3+ chelation to occur, dialyzed against ultrapure water for overnight, and lyophilized to yield respective complex. Gd3+ complex .ESI- m/z (M +1) for CA1: calcd. 789.96, found 789.98; ESI- m/z (M +K+) for CA2: calcd. 943.96, found 943.06.
MRI phantom image. MRI phantom images were acquired in 4.7 T MRI instrument (Biospec 47/40, Bruker, Germany). T1 weighted MR image obtained using following scanning parameters : MSME (Multi-slice multi-echo) pulse sequence, TE/TR=8.8/400 ms, matrix size = 192 × 192, FOV=6 × 3 cm, slice thickness = 1 mm. We compared T1 contrast differences between CA1 and CA2 in MCF-7 cell. In addition, we compared brightness of MRI according to trx level in cells.
The contrast agents showed
relaxivity typically single coordinated water molecule to Gd3+ ion center such
as 4.54 ± 0.13 mM-1s-1 and 4.58 ± 0.13 mM-1s-1 for CA1 and CA2 respectively at
60 MHz field. Contrast agents CA1 showed ~140% relaxivity enhancement in the
presence of thioredoxin. 15N-NMR experimental data indicated that CA1 has a
higher binding affinity toward thioredoxin compared to flexible contrast agent
CA2. The contrast agent CA1 has provided T1-weighted phantom images of cancer
cells (MCF-7, A549-Red-Fluc) based on the expression of thioredoxin. Further,
we confirmed thioredoxin- dependent changes of T1-weighted contrast images by
knocked-down the thioredoxin expression in Trx siRNA transfected MCF-7 cells.
The nontoxic nature of the CA1 suggested that it is the first kind of contrast
agent can provide the extent of expression of vicinal thiols motif protein in
live cells noninvasively.
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