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46 Cell-Penetrating Peptides: Processes and Applications
Step
1. Peptide–vector internalization is identical to oligonucleotide–vector internalization,
except that 30 µg/ml DNase I can be added to the culture
medium during peptide incubation in order to reduce nonspecific binding
of the vector to DNA released from dead cells.
2.5.3 COMMENTS
AntpHD and penetratin transduction properties can be exploited in all cases when
delivery of exogenous hydrophilic compounds in cells or tissues is required. The
diversity of molecules delivered with these vectors (peptides, proteins, oligonucleotides,
PNAs, and chemical substances) allows several experimental approaches. To
date, a wide range of biological phenomena have been studied, perturbed, or corrected
due to these peptidic vectors (Table 2.3). They are particularly well suited for
dissecting mechanistic aspects of regulations mediated by complex protein–protein
interactions, like signal transduction pathways or cell cycle regulation (see
Section 2.4.3.1). Penetratin vectors are also promising in therapeutics: they have
been employed to correct hyperproliferative disorders often leading to cancerization
(see Sections 2.4.3.1 and 2.4.4.3); they are also good vectors to bypass in vivo tight
barriers and detoxification systems such as, respectively, the BBB and the MDR
system (see Sections 2.4.3.4 and 2.4.4.3). The consequence is an important potentiation
of the cargo molecule in terms of accessibility to the targeted tissue and of
biological effect.
The cell type in which peptides will be delivered is not a critical parameter,
since penetratin-cargo hybrids are efficiently delivered in many cell types: PC12,
rat, and mice primary neurons, 3T3, COS-7, MDCK, K562, SAOS, U2OS, MCF-
7, Hela, ES, myotubes.
In general, whatever cargo is chosen, the solubility of cargo–vector hybrid is
important. Lack of solubility is often due to neutralization of charges between the
two molecules and can be improved by avoiding the use of PBS, increasing the ionic
strength, or adding a small percentage of DMSO.
2.5.3.1 Oligopeptides
Concerning oligopeptides, the efficacy of delivery will depend on structure or conformation
of the fusion hybrid. There is no absolute rule, and oligopeptides up to
156 aa have been successfully delivered in cells so far (see Table 2.3). The concentration
of the coupled product to be used is variable: in general 2 to 20 µM in most
applications; time of treatment is also variable, depending on the biological effect
observed. Shortest times (10 min to 2 h) are preferred when responses are believed
to be rapid, such as for molecules involved in signal transduction, longer times for
prolonged biological effects such as differentiation, proliferation, apoptosis (2 to
48 h). The only data demonstrating in vivo delivery of oligopeptides (mice blood
vessels 75 ) report systemic intraperitoneal injections of 0.3 mg/kg of the fusion peptide
(35 aa) and a monitored short-term biological effect (on inflammation) 45 min
after injection, and a long-term biological effect 24 h after injection (on vascular
leakage).