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Quantification of Cell-Penetrating Peptides and Their Cargoes 271 With this method, it is possible to monitor in real time the intracellular degradation of the disulfidebond, and hence the cellular uptake of the constructs by increase in apparent fluorescence. Moreover, it eliminates the need to separate membrane-bound and internalized peptide. However, in order to isolate the uptake kinetic constant, it is necessary to find the intracellular reduction rate constant. In this case, it was done by first measuring intracellular glutathione concentration and then measuring reduction of the constructs in the presence of lipid vesicles. Additionally, it is necessary to confirm that the cargo peptide cannot be internalized after extracellular reduction of the construct and to confirm that the increase in fluorescence is not caused by glutathione outflow due to peptide-induced membrane leakage. We used this technique to compare uptake and cargo delivery efficiency of four different CPPs: penetratin, Tat (48–60), transportan, and model amphipathic peptide (MAP). Our data show that MAP has the fastest uptake, followed by transportan, Tat (48–60) and penetratin. Similarly, MAP has the highest cargo delivery efficiency, followed by transportan, Tat (48–60) and, last, penetratin. 2 EXAMPLE 12.3 QUANTIFICATION OF MODEL PEPTIDE S-S CPP CONSTRUCT 2 One equivalent of CPP-(3-nitro-Tyr)-Cys(NPys) and 2 eq. of free thiol containing Abz-Cys-cargo–peptide were dissolved in a deoxygenated mixture of 3:2:1 dimethyl sulfoxide/N-methyl pyrrolidon/0.1 M sodium acetate, pH 4.5. The mixture was stirred for 4 h under nitrogen flow, and subsequently purified on reverse phase HPLC and characterized on a Voyager MALDI-TOF (Applied Biosystems). The yield of the desired heterodimers was approximately 30%. Cells were washed and harvested by scraping into HEPES-buffered Krebbs–Ringer solution (HKR) containing 0.5 g/l glucose and subsequently counted in a Neubauer chamber. In 96-well plates, approximately 250,000 cells in 50 µl were added to wells containing 150 µl of peptide construct dissolved in HKR to reach concentrations of 0.1, 0.5, 1, 5, or 10 µM. The cells were kept suspended by shaking between readings and the temperature was held constant at 37°C throughout the experiment. After 70 min, 5 µl 100 mM dithiothreitol (DTT) was added to reduce all remaining constructs. The fluorescence intensity value obtained after DTT reduction was used to define 100% of reduction. The fluorescence intensity was found to be dependent linearly on construct concentration. Fluorescence was measured (λ ex: 320 nm, λ em: 420 nm) in a Spectramax Gemini XS (Molecular Devices, USA) 96-well fluoroscence spectrophotometer. After the experiment, the cells were harvested by centrifugation and counted again in order to ascertain that no substantial lysis of the cells had occurred. Intracellular glutathione concentration was determined using a glutathione assay kit (Calbiochem, USA) according to the manufacturer’s instruction. Determination of reduction rate constants in the presence of lipid vesicles: Ten mg of phosphatidyl choline and 5 mg of phosphatidyl serine (Sigma, Sweden) were dispersed into 1 ml of HKR by vigorous vortex mixing. The mixture was sonicated with a probe tip sonicator in 25°C for 30 min. The resulting vesicles were purified
272 Cell-Penetrating Peptides: Processes and Applications by gel filtration on sephadex G50; they were diluted to a final concentration of 2 mg/ml in HKR. Construct was added to yield a lipid-to-peptide ratio of 1000:1 and the mixture incubated for 10 min, after which glutathione (100 and 500 µM; 1 and 2 mM) was added. Fluorescence increase was measured in a Hitachi F-2000 spectroflourimeter and the data analyzed fitting a numerical procedure in accordance to the following reaction scheme: k [ Glutathione]+ [ CPP-S-S-Cargo] ⎯→ ⎯ [ Free cargo-SH]+ [ HS-CPP] using Dynafit software 20 (Biokin Ltd., USA). To confirm that the cargo peptide could not be internalized after extracellular reduction of the construct, independent uptake experiments were performed with the unconjugated cargo peptide. After 60 min of incubation, no significant uptake could be measured. To confirm that the increase in fluorescence is not caused by glutathione outflow due to peptide induced membrane leakage, cells were incubated in 5 µM of the inactive [Pro 19 ]–transportan construct together with 10 µM of biotinyl–MAP, the peptide showing most membrane disturbance. Only modest (
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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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Page 242 and 243: 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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