Biomedical Engineering Reference
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anionic lipid contents (Lamaziere et al.
2007
; Thoren et al.
2005
; Tiriveedhi and
Butko
2007
; Hitz et al.
2006
). They do not induce liposome leakage or other
types of membrane perturbation at low P/L ratios and concentrations around
those required for biological uptake (1-10 mM) (Fuchs et al. 2004; Afonin et al.
2006
). Additionally, no structure change is associated with their membrane
binding and they are only superficially adsorbed on the membrane (Goncalves et al.
2005
; Roux et al.
1988
).
7
Mechanisms of CPP Direct Translocation
There is much evidence that direct translocation through the lipid membrane plays
a significant role in CPP entry into cells. The relative importance of direct translo-
cation and endocytosis seems dependent on conditions such as type of CPP, CPP
concentration, temperature, cargo or cell type but the existence of the direct trans-
location pathways seems now ascertained. This leads to the need to explain by
which mechanism(s) these highly soluble CPPs, all bearing several positive charges
and few or no hydrophobic residues, can cross the hydrophobic core of the mem-
brane bilayer. The order of magnitude of the activation energy for a naked CPP that
would enter this hydrophobic core is given by the Born energy of an ion leaving an
aqueous solution (relative permittivity 80) for the layer formed by the aliphatic lipid
chains of the lipids (relative permittivity 2). This energy for a guadinium ion
2
æ
ö
e
1
1
(radius r
G
= 0.25 nm) is
D=
E
- »
60kT per ion (at T
=
300K).
ç
÷
ion
8
pe
r
è
e
e
ø
0
Gc w
The spontaneous entry of a naked CPP into the bilayer is thus highly unfavorable
from a thermodynamical point of view. Several more refined mechanisms, presented
below, have been proposed and experimentally backed up, and it is worth mention-
ing from the beginning that the question of the translocation mechanisms is currently
still debated and may have no single answer, the direct translocation mechanisms
being CPP or experimental condition dependent.
A first class of proposed mechanism is the neutralization of the positively
charged CPP residues by some hydrophobic counterions that would simultaneously
reduce the Born energy stated above and favor the solubilization of the CPP in the
hydrophobic core of the membrane (Sakai and Matile
2003
, Nishihara et al.
2005
;
Takeuchi et al.
2006
; Wender et al.
2008
) (Fig.
1
). Several potential candidates for
the role of amphipatic counterion lie in membranes such as anionic phospholipids
or sulfated proteoglycans. The proof of concept has been given by Sakai and Matile
(Sakai and Matile
2003
) who showed that polyarginines (~80 residues) initially
dissolved in an aqueous buffer can partition into chloroform when phosphatidylg-
lycerol lipids were added. Rothbard and collaborators conducted a similar experi-
ment with an arginine octamer labeled with fluorescein (Rothbard et al.
2004
).
When they added sodium laurate, the CPP migrated completely from water to an
octanol phase. They also demonstrated the role of the two hydrogen bonds that a
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