Biomedical Engineering Reference
In-Depth Information
(Joanne et al. 2009 ; Epand and Epand 2009 ). Several studies have demonstrated
the importance of an electrostatic recognition using different approaches. Therefore,
to measure the affinity between the CPPs and lipid model systems and provide a
thermodynamic characterization of such an interaction, ITC and plasmon resonance
methods have been used (Goncalves et al. 2005 ; Binder and Lindblom 2003 ;
Salamon et al. 2003 ; Alves et al. 2009 ; Henriques et al. 2010 ). ITC studies point to
binding affinities in the low micromolar range and positive heat capacities indicat-
ing that electrostatics plays a major role in the interaction. Titration experiments
with increasing amounts of anionic lipids show that at certain anionic lipid concen-
tration and P/L ratios, penetratin is able to bind to both the outer and the inner
leaflet presuming transbilayer distribution of penetratin. Since penetratin has not
been associated with perturbations in membrane integrity, this indicates that, pen-
etratin translocates through the vesicle membrane by an electroporation mechanism
(this will be further discussed below) (Binder and Lindblom 2003 ). Plasmon reso-
nance studies performed with penetratin indicate that binding to planar lipid bilay-
ers is a fast and multistep process, primarily governed by electrostatic interactions
followed by peptide insertion into the hydrophobic membrane core. The peptide
also affected the amount of bound water, lipid-packing density, and bilayer thick-
ness (the latter only at high peptide concentrations) accompanied by a decrease in
membrane capacitance. A considerable enhancement of the binding was observed
in the presence of anionic lipids (Salamon et al. 2003 ; Alves et al. 2009 ; Henriques
et al. 2010 ). Improved binding affinities of penetratin to anionic lipids as compared
with zwitterionic lipids (10-100 fold increase) have also been observed by follow-
ing the intrinsic tryptophan fluorescence intensity of penetratin as a function of the
lipid concentration (Christiaens et al. 2002 ).
4
Kinetics of Internalization in Cells
The kinetics of cell-penetrating peptide internalization has been studied by several
groups. The reported data differ from one peptide to another depending on the cell
type and the method used for peptide tracking.
One of the first studies has shown that 125 I-Biotinyl-transportan internalization
was quick in Bowes melanoma cells and reached the steady-state after 20 min.
Interestingly, the time course was found similar whatever the concentration
(5-500 nM) of the peptide (Pooga et al. 1998). The same group reported the kinetics
of internalization of penetratin (RQIKIWFQNRRMKWKK), transportan, Tat(48-60)
and MAP (KLALKLALKALKAALKLA) (Hällbrink et al. 2001 ). The CPPs were
labelled with a fluorescence quencher (3-nitrotyrosine) and were coupled to a
pentapeptide cargo labeled with a fluorophore (2-amino benzoic acid) via a disulfide
bond. The kinetics was recorded by following the increase in fluorescence intensity
as the disulfide bridge is reduced into the intracellular milieu (Hällbrink et al. 2001 ).
In those experimental conditions, the more hydrophobic the peptides (transportan
and MAP) the faster they internalized in cells, the fluorescence plateau being
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