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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 expression. 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