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Kinetics of Uptake of Cell-Penetrating Peptides 279
CPP out
13.2.1 CELLS
Outside
CPP out M CPP in M CPP in Free
FIGURE 13.1 Schematic presentation of a CPP uptake by cells. CPP out and CPP in represent
the peptide outside and inside the cells, respectively. M denotes the hypothetical membranebound
state of the CPP; free denotes the possible nonbound or cytosolic fraction of the CPP;
bound denotes the state of CPP in interaction with intracellular (cytosolic or nuclear) structures,
e.g., proteins or phospholipid membranes; degradation indicates the possibility for CPP
to be degraded by proteases yielding the shift in all-over equilibrum.
Transport of CPPs across membranes has most frequently been studied with cultured
cells, but, in some cases, also with artificial lipid vesicles, tissues, and even organs
in vivo.
A number of different mammalian cells has been used for internalization studies
of CPPs. Kinetic experiments have been performed with calf aortic, 3 porcine, and
human umbilical vein endothelial cells, human neuroblastoma and hepatoma cells, 4
human leukemia cells, 5,6 human Bowes melanoma cells, 7,8 and human epidermoid
carcinoma cells. 6 No special cell cultivating procedures are needed for internalization
studies. Cells are usually grown to 70 to 80% confluence in flasks or dishes.
It is convenient to detach cells from the surface of flasks or dishes and prepare
a suspension of cells to assure a uniformly accessible cell surface prior to incubation
with CPP. Detachment of cells can be achieved by scraping cells of the surface
(dishes or flasks with the removable upper wall are very convenient), or by short
trypsin treatment. In the first case, some cells will be damaged; in the latter case,
the cell surface proteins might be affected by trypsin. Cells can be collected by mild
centrifugation (usually at 1500 rpm for 10 min) and resuspended in suitable buffer
solution. Prior to incubation of the obtained cell suspension with a CPP, it is
necessary to determine the “concentration” of the cell suspension (number of cells
per volume by means of cell counter or flow cytometer) as well as to estimate the
average cell volume.
13.2.2 INCUBATION OF CELLS WITH CPP
Inside
Plasma membrane
Degradation Degradation
CPP in Bound
Degradation
During the incubation of cells with a CPP, a fraction of the peptide will be taken
up by the cells, while another fraction of CPP molecules will be bound to the cell