Datensatz

Zusammenfassung

introduction: Molecular imaging of cellular processes requires the interaction of imaging
probes with intracellular targets like proteins, mrna or dna. The plasma membrane of cells poses
an impervious barrier to the intracellular delivery of such probes. Cell penetrating peptides (CPPs)
gained importance as vectors for transport of cargos attached to them into cells primarily by
endocytosis. however, confi nement of biomolecules into endosomes limited their use for intracellular
targeting. We present here the development of a novel cysteine-rich peptide, CyloP-1, that shows
the ability to deliver into the cytosolic compartment of the cell. The transport of gd-doTa, a contrast
agent for magnetic resonance imaging (Mri), after conjugation with CyloP-1 will be discussed as
well.
Methods: The conjugates were synthesized by continuous solid phase synthesis using fmoc
chemistry and n-terminally labeled with fl uorescein isothiocyanate. Cellular uptake of compounds
was confi rmed by fl uorescence microscopy and spectroscopy in nih-3T3 mouse fi broblast cells
plated in 96well plates. internalized fl uorescence was measured in a multiplate reader, and
microscopic images were made to determine the cellular localization of conjugates. Mr analysis of
gd-doTa conjugates was also performed on labeled cells in eppendorf tubes. Mri of cell pellets was
conducted at 3T in a Siemens human Mr scanner using T1- and T2-weighted spin-echo sequences.
relaxation rates were obtained from axial slices as well as images of sagittal slices were made.
results: The novel cysteine rich CPP was derived by Structure activity relationship (Sar)
studies of a sequence from the polypeptide Crotamine [1]. CyloP-1 is markedly distinct showing an
effi cient uptake at low concentrations (2.5 μM) and a cytosolic distribution along with vesicular uptake
(fig. 1a) unlike other common CPPs (e.g. Tat or antennapedia) at these concentrations. The
localization of CyloP-1 into the cytosol was maintained even at 4°C while the vesicular uptake was
much reduced. additional coupling of gd-doTa to this peptide showed profi cient uptake maintaining
the cytosolic localization of the conjugate (fig. 1b). furthermore, fl uorescence spectroscopic data
showed that the internalization effi cacy at 2.5 μM increased signifi cantly by about 50% in comparison
to the conjugate containing the d-form of Tat peptide, known to be an effi cient CPP.
Conclusions: These results demonstrate that our novel peptide might prove useful for
efficient trans-membrane delivery of molecular imaging probes directed to cytosolic targets.