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Toxicity and Side Effects of Cell-Penetrating Peptides 251 11.2.1.2 Viability Assays Cell viability assays are based on counting living active cells instead of damaged or dead cells, which are counted in methods based on exclusion and leakage. These viability assays estimate the functioning of whole cells, usually assuming the intactness of the plasma membranes. Mainly, the activity of enzymes, pumps, and transporters is used for measuring cellular function, even under unfavorable conditions. 11.2.1.2.1 Enzymatic Assays Many enzymatic viability assays are based on the activity of cytosolic esterases that convert neutral nonpolar esters of dyes that diffuse into the cells to colored or fluorescent substances. The products, which are polar or poorly soluble, cannot leave a cell via intact plasma membrane. The diacetyl derivative of fluorescein 27 was the first and, for a long time, the most commonly used compound for measuring viability enzymatically (Figure 11.1.d). An alternative compound for wider application is now provided by acetoxymethyl ester of calcein. This substance has the advantages of longer cellular retention time and lower pH sensitivity as compared to fluoresceinyl derivatives. The functionality of mitochondria is assessed in the MTT assay described in the early eighties by Mosmann as a rapid colorimetric method for measuring cellular growth and survival. 28 It is based on the cleavage of a yellow, water-soluble tetrazolium dye, MTT, by succinate dehydrogenase (Figure 11.1.e). The tetrazolium ring is cleaved only in active mitochondria, resulting in a purple, water-insoluble MTT formazan product whose absorbency can be measured after dissolving the formed crystals in an organic solvent. MTT cleavage occurs only in active cells; the reaction is quick and no washing steps are required, which makes the method convenient and popular for measuring cytotoxicity, proliferation, and activation. However, a number of factors can significantly influence the cleavage of MTT, 29 such as cellular uptake and metabolism of glucose, cellular concentration of the reduced pyridine nucleotides NADH and NADPH, pH of media, and type of cell line. These parameters must be considered when establishing MTT assay conditions. Both methods — the retention of fluorescein and the MTT assay — have been used to study effects of CPPs on cell viability 8,21,30 (see Table 11.2). 11.2.1.2.2 Uptake Assays Maintenance of organelles and their specific environment is essential for cell survival. Some dyes sensitive to minor pH changes can diffuse into cells and will concentrate in acidic organelles, thereby reporting maintenance of organelle intactness and functionality. Acridine orange and neutral red can be used to reveal and assess the functioning of lysosomes. 31 The dye rhodamine 123 (R123), which is sensitive to changes in membrane potential, concentrates in active mitochondria resulting in an intense fluorescence signal (Figure 11.1.f). This enables detection of mitochondria membrane intactness by the membrane potential and also visualization of these organelles. 32 Loss of R123 fluorescence is a rapid indicator of decreased cell viability and precedes the onset of propidium iodide staining of the nuclei. New derivatives of Mitotracker ® and
252 Cell-Penetrating Peptides: Processes and Applications TABLE 11.2 CPP-Induced Effect on Cell Viability as Determined by the MTT a Method Cell-penetrating peptide Conc., µM Time, h Viability Cell line KLALKLALKALKAALKLA-NH2 (MAP) 10 0.5 50% 9 C(Acm b )GRKKRRQRRRPPQCc (Tat (48–60)) 100 24 80% 31 C(Acm b )FITKALGISYGRKKRRQRRRPPQCc (Tat (37–60)) 100 24 40% 31 GRRRRRRRRRPPQ-C c (R9-Tat (48–60)) 100 24 70% 42 GRQIKIWFQNRRMKWKKG (Gly-penetratin) 30 72 100 35 a 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide. b Cys-acetamidomethyl. c Fluorescein labeled cysteine. d Mouse macrophage cells. e Lymphoblastoid cell line. Lysotracker ® series offer more sensitive and selective ways for measuring the maintenance and functionality of mitochondria and lysosomes (Figure 11.1.f,g) and thereby cell viability. 11.2.1.2.3 Ion Pump Assays Intracellular calcium concentrations reflect the ability of cells to respond to receptormediated events. Chelators such as fura 2, indo-1, indo-2, and their acetoxymethyl derivatives that fluoresce upon binding calcium can be used to monitor changes in intracellular calcium concentration as a cellular response to different compounds. The concentration at which the compound of interest starts to reveal detrimental effects on particular cells can only be reliably estimated by combining different cytotoxicity and viability assays. 11.2.2 CPP TOXICITY IN VITRO The effects of CPPs on cell viability and toxicity have mainly been assessed by using three different short-term in vitro assays: dye uptake and exclusion, 20 MTT assay, 30 and deoxyglucose leakage. 14 11.2.2.1 Model Amphipathic Peptides (MAPs) AEC HeLa HeLa RAW264.7 d IB4 e The 18 amino acids-long model amphipatic peptide (MAP; Table 11.1) contains only amino acids Lys, Ala, and Leu. This peptide was created to study cellular uptake mechanisms of a perfect amphipathic helix. MAP shares many characteristics (e.g., charge and amphipathicity) with antimicrobial peptides and its toxicity is therefore expected to be rather high. The toxicity of MAP and its analogues has been examined on cell cultures by using both trypan blue exclusion and leakage of fluorescein. Indeed, by using trypan blue exclusion, initial indications of membrane perforation of calf aortic endothelial cells (AEC) were detectable from 4 µM peptide concentration (Table 11.1). 8 The decrease in cellular activity to hydrolyze fluorescein diacetate was
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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 222 and 223: 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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