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FIGURE 15.9 (Color Figure 15.9 follows p. 14.) Left: µNMR image of a mouse brain (isotropic voxel size of 39 µm; TR 150 msec, TE 3.6 msec, FA 34°) with an implanted 9L tumor growing in elliptical shape (arrowheads). Note that i.v.-injected labeled lymphocytes have accumulated in the tumor and significantly reduce the signal intensity (tumor without cells would have similar signal as normal brain with this NMR sequence). Approximately 1500 labeled cells are present within the section shown. Right: HE stains of the most inferior aspect of the tumor extending into the brain (T = tumor; V = ventricle; 7 µm section). Top right: higher magnification HE stain in tumor. Bottom right: high magnification Fe stain. CLIO–Tat-labeled lymphocytes appear dark and are seen in and adjacent to vessels marked with an asterisk. Cell-Penetrating Peptide Conjugations and Magnetic Cell Labels 341
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.
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CELL- PENETRATING PEPTIDES Processe
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Pharmacology and Toxicology: Basic
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Library of Congress Cataloging-in-P
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to the handbook are prominent resea
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REFERENCES 1. Green, M. and Loewens
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Contributors Mats Andersson Microbi
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Erin T. Pelkey Department of Chemis
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Contents Section I Classes of Cell-
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Chapter 16 Cell-Penetrating Peptide
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The Tat-Derived Cell-Penetrating Pe
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24 Cell-Penetrating Peptides: Proce
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3 Transportans Margus Pooga, Mattia
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Transportans 55 Indeed, galparan is
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Transportans 57 especially the endo
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Transportans 59 In the penetration
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Transportans 61 A 21-mer antisense
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Transportans 63 Commonly, the prote
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Transportans 65 antibiotin antibodi
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Transportans 67 For cross-linking o
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Transportans 69 and still retain ef
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Model Amphipathic Peptides 73 its D
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Model Amphipathic Peptides 75 pmol/
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TABLE 4.1 Internalization of Peptid
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TABLE 4.2 Internalization of Peptid
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Model Amphipathic Peptides 81 relat
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Model Amphipathic Peptides 87 A cat
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Model Amphipathic Peptides 89 At th
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Model Amphipathic Peptides 91 16. H
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Signal Sequence-Based Cell-Penetrat
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116 Cell-Penetrating Peptides: Proc
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Interactions of Cell-Penetrating Pe
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Structure Prediction of CPPs and It
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FIGURE 9.7 (Color Figure 9.7 follow
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FIGURE 9.8 The center of the figure
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Biophysical Studies of Cell-Penetra
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Toxicity and Side Effects of Cell-P
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Kinetics of Uptake of Cell-Penetrat
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Page 350 and 351: Cell-Penetrating Peptide Conjugatio
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Microbial Membrane-Permeating Pepti
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Index A Abaecin, 129 Abz radiolabel
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Index 399 Diffraction, 168 Disulphi
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Index 401 structure prediction, 187
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Index 403 pRB proteins, Tat-E1A bin
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Index 405 lipid perturbation (secon
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