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Toxicity and Side Effects of Cell-Penetrating Peptides 257 Transportan and penetratin were used for delivery of a 21-mer antisense PNA to down-regulate the rat galanin receptor subtype 1 ex<strong>press</strong>ion. 52 No signs of toxicity were observed when 4.5 µg of the cell-penetrating peptide–PNA constructs were repeatedly (three times in 12-h periods) administered intrathecally into lumbar spinal cords of female Sprague–Dawley rats. 52 Bolton and colleagues have investigated the uptake and spread of fluorescently tagged penetratin in adult rat brain. 53 Intrastriatal injection of 10 µg of penetratin caused neurotoxic cell death and recruitment of inflammatory cells as judged by immunocytochemistry. Lowering the dose to 1 µg or less reduced toxicity and also recruitment of inflammatory cells. 53 A recent study investigated delivery of β-galactosidase (β-gal) by an 11-amino acid-long peptide called the protein transduction domain (PTD) of Tat (amino acids 47–57), into mice tissue. 54 Following 4 to 8 h intraperitoneal administration (i.p.) of 100 to 500 µg of Tat PTD-β-gal, the enzyme activity was detectable in most tissues: liver, kidney, heart, muscle, lung, spleen, blood, etc. Even the brain showed strong β-galactosidase activity 4 h after i.p. injection of Tat-β-gal. Transduction of β-galactosidase into brain did not disrupt the blood–brain barrier of mice since Evans blue-albumin coinjected with Tat-β-gal could not penetrate into brain. Importantly, no signs of gross neurological problems or systemic distress were observed after 14 consecutive days of injection of a mouse with 1 mg of Tat PTD fusion protein per kilogram of body weight. 54 Rothbard and co-workers conjugated the systemic and poorly cell-permeable drug cyclosporin A to a hepta-arginine oligomer (cyclosporin–Arg 7) to increase its cellular uptake. 55 One ear of dermatitis-induced mice was painted with a solution of cyclosporin–R 7 construct or with only the arginine–oligomer. The constructs entered the dermal cells where cyclosporin was released from the transporter peptide and reduced the inflammation dose dependently. The effect was local, cyclosporin was not detected in serum, and no reduction of inflammation of untreated ear was observed. Arginine–oligomer had no effect on the inflammation and no evidence of adverse consequences was seen during the studies. 55 In summary, it is of great importance to evaluate CPPs’ toxicity before applying them as drug transporters. Different strategies have been utilized for evaluating the modulation of cellular functions by CPPs. The initial cellular interaction of these peptides takes place at the cell plasma membrane and effects on the plasma membrane may alter homeostasis of the entire cell. Assays based on dye exclusion or uptake and leakage of cytoplasmic components have mainly been used for evaluating CPP-induced membrane toxicity. In general, amphipathic and positively charged peptides, like MAP, seem to be more toxic than other CPPs according to plasma membrane permeability assays (see Table 11.1). These CPPs show higher effect on the cellular viability that might be the outcome of damaged cell plasma membranes (see Table 11.2). After initial membrane interaction, some CPPs may interact with DNA. Every compound that binds to genetic material must be considered a potential genotoxic agent; however, so far, no data are published concerning CPPs and genotoxicity. In conclusion, more in vivo investigations are needed to clarify the CPP concentrations tolerated without toxicity problems. Unexpected side effects that are not
258 Cell-Penetrating Peptides: Processes and Applications revealed as toxicity or an inflammatory response cannot be excluded, even though no signs of toxicity or inflammation are detected. One of the most serious aspects could be immune response against the transport peptide. 54 The primary structure (e.g., multiple positive charges and bulky aromatic side chains in penetratin and transportan) of several CPPs suggests these to be rather immunogenic. 56,57 On the other hand, short peptides are not usually inducing an immune response. The development and availability of several CPPs with different primary structures will hopefully help to avoid toxicity problems in the future. ACKNOWLEDGMENTS This work was supported by grants from the EC Biotechnology Project BIO4-98-0227, Swedish Institute Visby Program, Swedish Royal Academy of Sciences, Swedish Research Councils TFR and NFR and Estonian Science Foundation (ESF4007), and the James McDonnel Foundation. ABBREVIATIONS AEC Aortic endothelial cells BMC Bowes melanoma cells CPP Cell penetrating peptide DGP Deoxyglucose-6-phosphate FDA Fluorescein diacetate HUVEC Human umbilical vein endothelial cells LDH Lactate dehydrogenase MAP Model amphipathic peptide MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide PI Propidium iodide PNA Peptide nucleic acid PTD Protein transduction domain TPE Trypan blue exclusion VEGF Vascular endothelial growth factor REFERENCES 1. Schwarze, S.R., Hruska, K.A., and Dowdy, S.F., Protein transduction: unrestricted delivery into all cells? Trends Cell Biol., 10, 290, 2000. 2. Schwarze, S.R. and Dowdy, S.F., In vivo protein transduction: intracellular delivery of biologically active proteins, compounds and DNA, Trends Pharmacol. Sci., 21, 45, 2000. 3. Lindgren, M. et al., Cell-penetrating peptides, Trends Pharmacol. Sci., 21, 99, 2000. 4. Ganz, T. and Lehrer, R.I., Antimicrobial peptides of vertebrates, Curr. Opin. Immunol., 10, 41, 1998. 5. Simmaco, M., Mignogna, G., and Barra, D., Antimicrobial peptides from amphibian skin: what do they tell us? Biopolymers, 47, 435, 1998.
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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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Cell-Penetrating Peptide Conjugatio
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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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