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Cell-Penetrating Peptide Conjugations and Magnetic Cell Labels 337 5 Fe bound (ng/10 cells) 5 Fe bound (ng/10 cells) 3000 2000 1000 3000 2000 1000 0 0 0 0 b. 10 10 20 5 Fe added (µg/10 cells) (a) 20 5 Fe added (µg/10 cells) (c) 30 30 CLIO-Tat CLIO CLIO-Tat CLIO FIGURE 15.5 Quantitative uptake of CLIO–Tat into different cell types. Different cell types were incubated with increasing concentration of 111 In-CLIO–Tat or 111 In-CLIO for 1 h at 37°C. Mean ± SD of uptake is plotted for: (a) CD34+ cells, (b) mouse neural progenitor cells (C17.2), (c) human CD4+ lymphocytes, and (d) mouse splenocytes. (From Lewin, M. et al., Nat. Biotechnol., 18, 410, 2000. With permission.) Without the assistance of Tat peptide, the CLIO particle cannot pass through cell membrane efficiently. 15.3.1.2 Label Distribution in Dividing Cell Populations 40 40 50 50 The next two questions we addressed were (1) what is the intracellular magnetic half-life of CLIO–Tat in cells and (2) what is the fate of cellular CLIO–Tat in actively dividing cells? To answer the first question we performed time dependence studies using MR imaging of labeled lymphocytes. Results from these studies showed that the transverse relaxation time (T2, T2*) of magnetically labeled lymphocytes returned to normal approximately 7 to 14 days (depending on amount internalized) after labeling, indicating that the magnetic core of CLIO–Tat is biodegraded. 5 Fe bound (ng/10 cells) 5 Fe bound (ng/10 cells) 3000 2000 1000 0 3000 2000 1000 0 0 0 c. 10 10 CLIO-Tat 5 Fe added (µg/10 cells) CLIO 20 (b) 20 30 30 40 CLIO-Tat 40 5 Fe added (µg/10 cells) (d) 50 50
338 Cell-Penetrating Peptides: Processes and Applications Counts 0 40 80 120 160 200 10 0 Before labeling After CLIO-tat labeling 40 hrs later M2 10 1 LX1.002 LX1-CLIO-TAT.003 LX1-CLIO-TAT.003 10 2 FL1-Height 10 3 10 4 Counts 0 40 80 120 160 200 10 0 FIGURE 15.6 CLIO–Tat (containing an FITC label) appears to be distributed in roughly equal amounts to daughter cells after cell division. To answer the second question we labeled actively dividing human LX1 tumor cells (doubling time of 20 h) with CLIO–Tat and performed FACS analysis before labeling, immediately after labeling, and 40 h (2 division cycles) after labeling (Figure 15.6). Data from these studies support the hypothesis that CLIO–Tat is equally distributed to progeny cells, similar to the case for many cellular fluorescent stains. 15.3.1.3 CLIO–Tat Is not Toxic to Cells at Concentrations Used for Magnetic Labeling 10 1 In order to determine the dose at which Tat labeled particles become toxic to cells, a trypan blue exclusion assay was initially performed. For these studies, mouse lymphocytes were incubated with Tat-labeled particles ranging from 7 to 700 nmol peptide per 10 6 cells for 3 h followed by trypan blue staining. Tat-labeled particles were nontoxic to cells at incubation concentrations
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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 308 and 309: Kinetics of Uptake of Cell-Penetrat
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Page 350 and 351: Cell-Penetrating Peptide Conjugatio
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Page 370 and 371: Cell-Penetrating Peptides as Vector
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Page 380 and 381: CPP 2 Cargo or mRNA CAP Antisense A
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Page 400 and 401: Microbial Membrane-Permeating Pepti
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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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