crc press - E-Lib FK UWKS

264 Cell-Penetrating Peptides: Processes and Applications
because it is indeed translocated by the aid of the transport peptide. The most
frequently applied reporter group in in vitro studies so far is biotin, which is detected
with fluorescently labeled streptavidin–avidin visualized in a fluorescence microscope
or quantified by, for example, fluorescence-activated cell-sorting (FACS) analysis
or fluorescence spectrophotometry.
Various cargo molecules, oligonucleotides (ONs), peptide nucleic acid (PNA),
drugs, peptides, and proteins have been successfully transported by CPPs into a
great selection of cell types (for review, see Lindgren et al. 1 ). More recently, CPPs
have also been applied in in vivo studies. Chemical conjugation by disulfide bonds
or in-chain synthesis (for peptides and PNA) are the two main methods for conjugation
of cargo to the CPP. In addition, recombinantly expressed fusion proteins
have been used for the Tat protein and Tat peptides. The importance of detachment
of the cargo from the transport peptide in case of intracellularly cleavable disulfide
bonds is a debated matter. It could be argued that if the CPP is not disturbing the
cargo’s intracellular activity a detachment is not necessary. The CPP could, in fact,
provide a useful protection against degradation.
Despite all of the quantification methods available today, with exception of direct
antibody detection, it is the amount of a cargo–reporter group that is measured and
not the amount of the CPP, as would be desired. In experiments where only a
qualitative indication of intracellular delivery is sufficient, biotin is a useful label,
but when quantification of the uptake is essential, fluorescence labeling of the CPP
or cargo is more convenient.
In this chapter, various methods to quantify the efficiency of CPP uptake are
compared. Emphasis is on methods in which the main interest has been to characterize
the CPP itself. There are also a great number of publications where the focus
has been on activity of the cargo delivered by the CPP; these are only briefly reviewed
in this chapter.
12.2 QUANTIFICATION METHODS
The concentration range of CPP used during uptake experiments is crucial for exact
quantification. A cell-penetrating peptide is defined as a peptide primarily entering
the cell — not via endocytosis or by pore formation. Several CPPs cause pore
formation or have lythic activity, e.g., transportan and model amphipatic peptide
(MAP), but it is negligible 2 at low concentrations (lower than 5 µM). In contrast,
endocytosis contributes to the uptake of Tat fragments. 3 As described in Chapter 11,
dye leakage assays or tritiated-glucose leakage can be used to detect and quantify
membrane disturbing effects of CPPs.
Another important challenge when quantifying CPP uptake is to distinguish
between membrane- or cell-associated peptide and intracellular CPP. For example,
Oehlke et al. (see Chapter 4) have used a method in which the membrane-bound
peptide is modified in order to discriminate it from intracellular MAP. 4 In the studies
of 2-amino benzoic acid (Abz)-labeled transportan (Figure 12.1), a trypsin treatment
has been applied. 5 Furthermore, acid wash and oil centrifugation, 6 and reductionsensitive
7-nitrobenz-2-oxo-1,3-diazol (NBD) fluorophore 7 have been utilized for the
distinction between membrane-associated and internalized CPP. However, inactivation