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40 Cell-Penetrating Peptides: Processes and Applications<br />

with the absence of BBB crossing reported by Bolton et al. 76 However, the conditions<br />

within the two experiments are very different. In particular, Rousselle et al. used<br />

radioactive doxorubicine coupled to a penetratin peptide composed of D-amino acids<br />

(2.5 mg/kg) and quantified brain accumulation 30 min after injection. In contrast,<br />

Bolton et al. used a fluorescent compound (2.0 mg/kg) linked to normal penetratin<br />

and quantified its presence in the brain 24 h after injection. The two approaches thus<br />

differ in terms of protocols and also of sensitivity, not to mention the modification<br />

of the vector (D vs. L amino acids).<br />

Drug delivery to the brain is often restricted by the BBB that regulates the<br />

exchange of substances between brain and blood. The protein Pgp, which participates<br />

in the multidrug-resistance phenomenon (see above), has been detected at the luminal<br />

site of the endothelial cells of the BBB. 79 As a consequence, the brain availability<br />

of several drugs and, in particular, of anticancer drugs is extremely low, a likely<br />

explanation for the failure of brain tumor chemotherapy. The crossing of doxorubicin<br />

was analyzed by the in situ brain perfusion technique or by intravenous injection. 78<br />

The amount of penetratin-coupled doxorubicin transported into the brain was 20-fold<br />

higher than that of free doxorubicin, without BBB disruption. These results demonstrate<br />

that penetratin peptides might be used as very efficient and safe means to<br />

deliver drugs across the BBB.<br />

2.5 EXPERIMENTAL PROCEDURES<br />

2.5.1 ANTPHD AND PENETRATIN<br />

2.5.1.1 AntpHD and Penetratin Labeling<br />

In order to be visualized, the AntpHD and penetratin peptides must be coupled to<br />

a fluorochrome (FITC, NBD TRITC, or CY3), to a biotin residue, or radioactively<br />

labeled. AntpHD can be labeled during synthesis in E.Coli with [ 35 S] methionine,<br />

or postsynthesis with FITC (Sigma), CY3 monofunctional dye (Amersham), or sulfo-<br />

NHS-biotin (Pierce). Penetratin can be coupled to fluorochromes, biotine, 15 or radioactive<br />

molecule 78 during synthesis, or postsynthesis as for AntpHD.<br />

2.5.1.1.1 FITC Labeling Postsynthesis<br />

Solutions<br />

1. 1 M sodium bicarbonate buffer, pH 9.3: dissolve 0.84 g NaHCO3 in 9 ml<br />

deionized water. Adjust to pH 9.3 with NaOH and complete to 10 ml with<br />

deionized water. This solution should be stored at 4°C and used within<br />

one week.<br />

2. Phosphate buffer saline, pH 7.2 (Life Technologies).<br />

3. 1 M glycine: dissolve 7.5 g in 80 ml distilled water. Adjust to 100 ml with<br />

distilled water.<br />

4. FITC dye (Molecular Probes).<br />

Steps<br />

1. Dilute 1 mg peptide in 200 µl of 50 mM sodium bicarbonate buffer, pH<br />

9.3 (final concentration).

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