Direct translocation of cell-penetrating peptides in liposomes: A combined mass spectrometry quantification and fluorescence detection study
Section snippets
Materials
The glycerophospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were obtained as a powder from Genzyme (Liestal, Switzerland). Egg PC was purchased from Avanti Polar Lipids (Alabaster, AL, USA) and asolectin from Sigma. 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rh-DOPE) was obtained as a 1 mg/ml solution in chloroform from Avanti Polar Lipids.
Biot-(Gly)4-RL9, Biot-([2,2-D2]-Gly)4
Liposome organization and integrity upon peptide addition
Liposome aggregation, fusion, or leakage could strongly affect or invalidate the liposome internalization studies performed. Therefore these liposome properties were investigated using a P/L of 1/100, which was slightly above that used for the internalization studies (P/L used was ∼1/150). As control experiments to follow the state of liposomes in terms of their size and distribution upon peptide addition, dynamic light scattering was used. The results presented in Table 1 show that the
RW9 is able to translocate through synthetic membranes, in contrast to RL9
Translocation of CPPs across pure lipid bilayers has been an important point of discussion in the literature for the past decade. Various studies showed that penetratin [9], [10], [11], [14], polyarginines [11], [14], and other known CPPs [14] were able to translocate into various kinds of lipid vesicles. Recently, several new peptide sequences were screened according to their translocating properties [34]. This allowed the identification of a certain number of new cell-penetrating peptides
Acknowledgments
We thank Rodrigue Marquant for the synthesis of Biot(O2)-(Gly)4-Cys-RL9. We also thank Emmanuelle Sachon, Nicolas Rodriguez, and Dimitra Milioni for fruitful discussion. This work was supported by the Association Nationale pour la Recherche (ANR-Prob DOM). A.W. and L.M. were both recipients of a Ph.D. fellowship from the French Ministère de l’Enseignement Supérieur et de la Recherche.
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