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(Figure 1–B). Both lymphocyte cell lines (Bv173 and MOLT4) and peripheral bloodmononuclear cells (PBMC) showed very high levels of peptide entry. These experimentswere done using both NrTP1-RhB and NrTP5-RhB, and the results are very similar bothfor the profile in multilamellar giant vesicles and cells.AB200200Fluorescence / AU15010050Fluorescence / AU1501005000 5 10 15 20 25distance / μm00 5 10 15 20 25distance / μmFig. 1. Translocation of NrTP1-RhB into giant multilamellar vesicles (POPC + DPPE-NBD 1%) and Bv173 (pre-B cell leukemia) cell line. Graphs represent fluorescenceintensity along a longitudinal line drawn on the vesicle or cell, respectively. Panel A showsthe co-localization of NBD (---) (488 nm laser) with NrTP1-RhB 15 mM (—) (561 nmlaser). Each spike of NBD fluorescence corresponds to a lipid bilayer. Panel B shows theco-localization of the nuclear dye Hoeschst (---) (405 nm laser) with NrTP1-RhB 15 mM(—), (561nm laser).Finally, a conjugate of NrTP (NrTP6-C) bound to β−galactosidase (from E. coli) wasprepared by chemical synthesis. This conjugate maintains enzymatic activity and is stableat 4ºC for several days, retaining its activity after -20ºC storage. Internalization studies forthe delivery of β-galactosidase into HeLa cells were conducted with the above mentionedconjugate. Efficient translocation of the enzyme was detected in a cell free extractfluorescence based assay (Figure 2).Fluorescence / a.u. 440 nm1500100050000 15 30 45 60 75 90time / minThe work done so far with this new family of CPP has revealed strong interaction andtranslocation with lipid model systems. Moreover, and as a proof of concept that these cellpenetratingpeptides are good carriers for the delivery of large molecules into the cellinterior, we have successfully observed that NrTP can translocate β-galactosidase into cells.AcknowledgmentsPartial funding and M.R. PhD grant (SFRH/BD/37432/2007) by Portuguese Ministry of Science (FCT-MCTES) and by Spanish Ministry of Science and Innovation (MICINN, grant BIO2008-04487–CO3)are acknowledged.References1. Nicastro, G. Eur. J. Biochem. 270, 1969-1979 (2003).2. Kerkis, A., et al. FASEB J. 18, 1407-1409 (2004).3. Nascimento, F.D., et al. J. Biol. Chem. 282, 21349-21360 (2007).4. Rádis-Baptista, G., et al. J. Med. Chem. 51, 7041-7044 (2008).5. Santos, N.C., et al. Biochim. Biophys. Acta. 1612, 123-135 (2003).Fig. 2. Progression curves of β-galactosidaseenzymatic activity. Fluorescence intensity ismeasured at 440 nm upon addition of enzymeto 0.5 mM of MUG. The plot represents the invitro activity of the conjugate (NrTP6-C-βgalactosidase)when it is present at 0.5 nM (●),2 nM (■), 5 nM (▲) and 7 nM (│).Theprogression curve of a cell free extractresulting from the incubation of 0.3 mM ofconjugate with HeLa cells (○).409