Vesicle size-dependent translocation of penetratin analogs across lipid membranes.
The recent discoveries of serious artifacts associated with the use of cell fixation in studies of the cellular uptake of cell-penetrating peptides (CPPs) have prompted a reevaluation of the current understanding of peptide-mediated cellular delivery. Following a report on the differential cellular uptake of a number of penetratin analogs in unfixed cells, we here investigate their membrane translocation abilities in large and giant unilamellar vesicles (LUVs and GUVs, respectively). Surprisingly, in contrast to the behavior in living cells, all peptides readily entered the giant vesicles (>1 microm) as proved by confocal microscopy, while none of them could cross the membranes of LUVs (100 nm). For determination of the location of the peptides in the LUVs, a new concept was introduced, based on sensitive resonance energy transfer (RET) measurements of the enhanced fluorescence of acceptor fluorophores present solely in the inner leaflet. An easily adopted method to prepare such asymmetrically labeled liposomes is described. The membrane insertion depths of the tryptophan moieties of the peptides were determined by use of brominated lipids and found to be very similar for all of the peptides studied. We also demonstrate that infrared spectroscopy on the lipid carbonyl stretch vibration peak is a convenient technique to determine phospholipid concentration.
Department of Chemistry and Bioscience, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden. danielp@phc.chalmers.se
Daniel
D
Persson
Per E G
PE
Thorén
Elin K
EK
Esbjörner
Mattias
M
Goksör
Per
P
Lincoln
Bengt
B
Nordén
D001692:Biological Transport; D002352:Carrier Proteins; D057846:Cell-Penetrating Peptides; D003374:Coumarins; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D008051:Lipid Bilayers / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism*; D008246:Lysophospholipids; D010316:Particle Size; D010455:Peptides; D010539:Permeability; D017550:Spectroscopy, Fourier Transform Infrared; D014364:Tryptophan
D002352:Carrier Proteins; D057846:Cell-Penetrating Peptides; D003374:Coumarins; D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D008081:Liposomes; D008246:Lysophospholipids; D010455:Peptides; C403208:lysoMC; D014364:Tryptophan; C414409:penetratin