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Toxicity and Side Effects of Cell-Penetrating Peptides 249 f) R123, Mitotracker e) MTT MT d) Fluorescein DA (FDA) MTT formazan N (b) FDA Fluorescein g) Lysotracker FIGURE 11.1b Methods used for estimating effects on cell viability. (d) Nonfluorescent, nonpolar fluorescein diacetate (FDA) diffuses into cells across the plasma membrane. The cellular esterases convert FDA into the fluorescent and polar fluorescein retained inside living cells. (e) Soluble MTT is converted into water-insoluble formazan crystals by mitochondrial enzymes of living cells. (f) R123 and Mitotracker ® concentrate into mitochondria and (g) Lysotracker ® into lysosomes of viable cells only. (N = nucleus; MT = mitochondria; LYS = lysosomes.) intercalating dyes like ethidium bromide and, especially, propidium iodide (Figure 11.1.b), which is better suited to fluorescence microscopy, flow cytometry, and cell-sorting techniques, have gained more popularity. 18,19 New derivatives of propidium iodide (PI) are less cell-permeable and practically nonfluorescent when unbound to DNA, thus enabling a more sensitive detection system. Dye exclusion assays are cheap, easy to perform, and well established over decades. However, these methods tend to overestimate viability, since cells unable to attach and proliferate (i.e., nonviable) may exclude the dye. Exclusion of trypan blue 20 (see Table 11.1) and propidium iodide 21 have been used for evaluation of CPPinduced toxicity. 11.2.1.1.2 Cytoplasmic Leakage Another strategy for detection of cell plasma membrane damages is to estimate the outflow of cytosolic enzymes or cell-introduced probes. 22 The release of lactate dehydrogenase (LDH) into cell culture media 23 is one of the most traditional and frequently used leakage assays. LDH activity is measured by the conversion of NAD to NADH in the presence of excess lactate substrate. Release of LDH has been used in different toxicity assays and enables assessment of accumulated leakage over a remarkable time period. The major drawback with this method is the latency of LDH, since leakage of a high molecular weight enzyme is relatively slow as compared to dansyl lysine or PI nuclear staining. 19 Another straightforward assay for determining the amount of viable cells is to measure radioactivity leakage from metabolically prelabeled cells after doping the LYS
250 Cell-Penetrating Peptides: Processes and Applications TABLE 11.1 Estimated Minimal Toxic Concentrations of CPPs In Vitro Cell penetrating peptide Minimal toxic concentration, µM TPE a DGP b Others Cell lines KLALKLALKALKAALKLA-NH2 (MAP) 4 8 1 14 4 (FDAc ) 8 AEC, BMC KLALKALKAALKLA-NH2 Not toxic at 50 20 N/A N/A AEC KLGLKLGLKGLKGGLKLG-NH2 Not toxic at 50 20 N/A N/A AEC GRKKRRQRRRPPQ-NH2 (Tat (48–60)) N/A Not toxic at 20 14 N/A BMC RQIKIWFQNRRMKWKK (penetratin) N/A 20 14 50 (MCd ) 36 U2OS, BMC GWTLNSAGYLLGKINLKALAALAKK IL-NH2 (transportan) N/A 5 14 N/A BMC a Trypane blue exclusion. b 2-deoxyglucose-6-phosphate leakage. c Fluorescein diacetate assay. d Morphological changes. cells with 14 C-thymidine, 35 S-methionine, 32 P, 3 H-uridine, 14 C-nicotineamide, etc. 24 However, these leakage assays do not allow proper detection of pores or temporal disturbances in the plasma membrane, since marker molecules of remarkable size like proteins and DNA cannot leak out through the small or short-living openings. Detection of pores and temporal changes in the plasma membrane can be achieved by a more sensitive leakage assay in which the radioactive chromium ( 51 Cr) isotope is used. Living cells easily take up the reduced form of the isotope 51 Cr 3+ in the form of Na 251 CrO 4 salt. 25 Viable cells oxidize 51 Cr 3+ to the membrane-impermeable 51 Cr 2+ and its liberation enables quantitative estimation of membrane damage or cell death. The retention of 51 Cr 2+ also shows that cells are viable since no radioactivity has been released from the cells. A similar assay to estimate membrane intactness and cell viability by using tritiated 2-deoxyglucose was developed by Walum and Peterson. 22 The glucose analogue 2-deoxyglucose is taken up into cells via glucose carriers and then phosphorylated by hexokinase (Figure 11.1.c). The product, 2-deoxyglucose-6-phosphate (DGP), has been shown not to leave the cell via the plasma membrane when it is intact. 26 The efflux of radioactivity from cells preloaded with tritiated 2-deoxyglucose can be measured according to the method described previously, 22 which is quick, objective, easy to quantify, and does not use as detrimental ion 51 Cr 3+ . However, uptake of glucose (and 51 Cr 3+ as well) varies between different cell lines and could in some cases be too low for detecting subtle changes in plasma membrane permeability. On the other hand, fast changes in membrane permeability induced by CPPs can be detected by the DGP assay. 14
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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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Page 220 and 221: Structure Prediction of CPPs and It
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Cell-Penetrating Peptide Conjugatio
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FIGURE 15.9 (Color Figure 15.9 foll
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Cell-Penetrating Peptides as Vector
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TABLE 16.1 Examples of Transport of
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CPP 2 Cargo or mRNA CAP Antisense A
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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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