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Figure 3 | Journal of Nanobiotechnology

Figure 3

From: A display of pH-sensitive fusogenic GALA peptide facilitates endosomal escape from a Bio-nanocapsule via an endocytic uptake pathway

Figure 3

Fluorescence images of HER2-expressing SKBR3 and HER2-non-expressing HeLa cells treated with LP, His6-Z HER2 -BNC/LP, and GALA-His6-Z HER2 -BNC/LP encapsulating calcein after incubation for 6 h (A), 24 h (B), and 48 h (C). SKBR3 cells were maintained in RPMI 1640 medium supplemented with 10% (v/v) FBS at 37°C in 5% CO2. HeLa cells were maintained in DMEM medium supplemented with 10% FBS at 37°C in 5% CO2. Approximately 5 × 104 SKBR3 or HeLa cells were seeded in 35 mm glass-bottom dishes. Complex carriers of His-ZHER2-BNC and GALA-His-ZHER2-BNC with LP, in which calcein was incorporated, were prepared. Freeze-dried LP (COATSOME EL-01-A) was dissolved in distilled water (2 ml) containing 100 mM of calcein. After incubation for 1 h at room temperature, gel-filtration chromatography was performed using a PD-10 (GE healthcare). The obtained LP incorporating calcein (100 μl) was added to freeze-dried His-ZHER2-BNC and GALA-His-ZHER2-BNC (100 μg as protein) and incubated at room temperature for 1 h. After washing with serum-free medium, 7.5 μl of the carriers containing calcein were added to 200 μl of the medium and then the cells were cultured for 1 h. After washing with serum-free medium twice, cells were incubated with FBS-containing medium for 5 h, 23 h and 47 h. Cells were observed using a LSM 5 PASCAL laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany) with a 63-fold oil immersion objective lens with excitation using the 488-nm line of an argon laser and emission collection using a 505–530 nm band pass filter for calcein and the 543-nm line of an He-Ne laser and emission collection using a 560-nm long-pass filter for the Lysotracker Scale bar, 20 μm.

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