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40 Cell-Penetrating Peptides: Processes and Applications
with the absence of BBB crossing reported by Bolton et al. 76 However, the conditions
within the two experiments are very different. In particular, Rousselle et al. used
radioactive doxorubicine coupled to a penetratin peptide composed of D-amino acids
(2.5 mg/kg) and quantified brain accumulation 30 min after injection. In contrast,
Bolton et al. used a fluorescent compound (2.0 mg/kg) linked to normal penetratin
and quantified its presence in the brain 24 h after injection. The two approaches thus
differ in terms of protocols and also of sensitivity, not to mention the modification
of the vector (D vs. L amino acids).
Drug delivery to the brain is often restricted by the BBB that regulates the
exchange of substances between brain and blood. The protein Pgp, which participates
in the multidrug-resistance phenomenon (see above), has been detected at the luminal
site of the endothelial cells of the BBB. 79 As a consequence, the brain availability
of several drugs and, in particular, of anticancer drugs is extremely low, a likely
explanation for the failure of brain tumor chemotherapy. The crossing of doxorubicin
was analyzed by the in situ brain perfusion technique or by intravenous injection. 78
The amount of penetratin-coupled doxorubicin transported into the brain was 20-fold
higher than that of free doxorubicin, without BBB disruption. These results demonstrate
that penetratin peptides might be used as very efficient and safe means to
deliver drugs across the BBB.
2.5 EXPERIMENTAL PROCEDURES
2.5.1 ANTPHD AND PENETRATIN
2.5.1.1 AntpHD and Penetratin Labeling
In order to be visualized, the AntpHD and penetratin peptides must be coupled to
a fluorochrome (FITC, NBD TRITC, or CY3), to a biotin residue, or radioactively
labeled. AntpHD can be labeled during synthesis in E.Coli with [ 35 S] methionine,
or postsynthesis with FITC (Sigma), CY3 monofunctional dye (Amersham), or sulfo-
NHS-biotin (Pierce). Penetratin can be coupled to fluorochromes, biotine, 15 or radioactive
molecule 78 during synthesis, or postsynthesis as for AntpHD.
2.5.1.1.1 FITC Labeling Postsynthesis
Solutions
1. 1 M sodium bicarbonate buffer, pH 9.3: dissolve 0.84 g NaHCO3 in 9 ml
deionized water. Adjust to pH 9.3 with NaOH and complete to 10 ml with
deionized water. This solution should be stored at 4°C and used within
one week.
2. Phosphate buffer saline, pH 7.2 (Life Technologies).
3. 1 M glycine: dissolve 7.5 g in 80 ml distilled water. Adjust to 100 ml with
distilled water.
4. FITC dye (Molecular Probes).
Steps
1. Dilute 1 mg peptide in 200 µl of 50 mM sodium bicarbonate buffer, pH
9.3 (final concentration).