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342 Cell-Penetrating Peptides: Processes and Applications
(TR 150 msec, TE 3.6 msec, FA 34°, voxel size: 39 × 39 × 78 µm) clearly showed
that the labeled cells are detectable in the tumor bed and that very low cell numbers
for the first time can be visualized by MR imaging.
15.3.2 INTERNALIZATION OF PARAMAGNETIC CHELATES
The above described cellular internalization of nanoparticles is a viable option for
labeling cells for MR imaging, particularly when T2 weighted sequences are
obtained. An alternative strategy, however, is to internalize small paramagnetic
molecules into cells that can chelate lanthanides serving as paraqmagnetic or fluorescent
reporters. We hypothesized that it should be feasible to internalize macrocyclic
lanthanide chelators into cells using the same HIV–Tat-derived peptide
sequence used for the CLIO–Tat attachment (Figure 15.10). In order to deliver
various lanthanide elements into cells, we have attached 1,4,7,10-tetraazacyclododecane-N,N’,N’’,N’’’-tetraacetic
acid (DOTA) to the Tat peptide as described in
Example 15.1 (Figure 15.10–top). 33 The Tat–DOTA was then subsequently labeled
with gadolinium (Gd), dysprosium (Dy), or Indium-111 ( 111 In).
Fresh lymphocytes (1 × 10 6 cells) obtained from mouse spleen were incubated
with various amounts of Tat–DOTA– 111 In (25, 50, 100, 150, or 200 nmol) in 1 ml
of RPMI 1620 medium at 37°C for 60 min. After incubation, cells were washed
three times with Hanks buffered saline solution and then resuspended in RPMI 1620
medium. In parallel, DOTA–In 111 without Tat peptide was used as a control. As
shown in Figure 15.10–center, uptake increased with increasing amounts of
Tat–DOTA conjugate without reaching saturation at 200 mol of peptide per million
cells. On the contrary, uptake of DOTA–In 111 was negligible at all concentrations.
To determine whether quantitative differences in cellular uptake between labeled
and unlabeled lymphocytes would translate into differences by MR signal intensity,
we labeled mouse lymphocytes by incubation with 150 µg Gd per 10 6 cells. MR
imaging phantoms were prepared by placing 20 µL cell pellets into agarose and then
sealing them with additional agarose to avoid air-induced susceptibility artifacts.
The phantoms were then subjected to MR imaging at 1.5 T (Signa 5.5; GE Medical
Systems, Milwaukee, WI) using a 5-in. surface coil. The imaging protocol consisted
of a coronal T1 weighted spin echo (SE) sequence with a TR/TE of 300/11, and a
slice thickness of 3 mm. The imaging results (Figure 15.10–bottom) showed that
cells can be labeled paramagnetically and that the labeled cells appear hyperintense.
These T1 markers would potentially be beneficial to improve signal-to-noise ratios
for tracking cells using T1 weighted MR imaging techniques. Conversely, if the
Tat–DOTA was labeled with Dy, the signal of Tat–DOTA–Dy-labeled cells was lower
because of the predominant T2/T2* effect of the latter, which may be desirable for
certain imaging studies.