Poster Presentation 2014 International Biophysics Congress

Entry of cell-penetrating peptide transportan 10 into a single vesicle by translocating across lipid membrane and its induced pores (#450)

Md. Zahidul Islam 1 , Hirotaka Ariyama 1 , Jahangir Md. Alam 2 , Masahito Yamazaki 2
  1. Graduate School of Science and Technology, Shizuoka University, Shizuoka, Japan
  2. Nanomaterials Research Division, Research Institute of Electronics, Shizuoka University, Shizuoka, Japan

The cell-penetrating peptide, transportan 10 (TP10), can translocate across the plasma membrane of living cells and thus can be used for the intracellular delivery of biological cargo such as proteins. Here we investigated the entry of TP10 into a single giant unilamellar vesicle (GUV) and the TP10-induced leakage of fluorescent probes using the single GUV method. GUVs of 20% dioleoylphosphatidylglycerol (DOPG)/ 80% dioleoylphosphatidylcholine (DOPC) were prepared, and they contained a water-soluble fluorescent dye, AF647, and smaller vesicles composed of 20% DOPG/80% DOPC. The interaction of carboxyfluorescein (CF)-labeled TP10 (CF-TP10) with these loaded GUVs was investigated using confocal microscopy. The fluorescence intensity of the GUV membrane increased with time to a saturated value, then the fluorescence intensity due to the membranes of the smaller vesicles inside the GUV increased prior to leakage of AF647. This result indicates that CF-TP10 entered the GUV from the outside by translocating across the lipid membrane before CF-TP10-induced pore formation. The rate constant of TP10-induced pore formation in lipid membranes increased with an increase in TP10 concentration. The presence of high concentration of cholesterol suppresses a little the activity of CF-TP10 to enter the GUV. Large molecules such as Texas Red dextran 40000, and vesicles with a diameter of 1−2 μm, permeated through the TP10-induced pores or local rupture in the lipid membrane. These results provide the first direct experimental evidence that TP10 can deliver large cargo through lipid membranes, without the need for special transport mechanisms such as those found in cells.