Sp8x sted

Intracellular trafficking of SP2K polyplexes was observed by using a confocal laser scanning microscope (CLSM). HeLa cells were seeded in confocal imaging dishes at a density of 3 × 10⁵ cells/dish. The media were exchanged with fresh serum-free DMEM after 24 h of incubation. Then, the cells were exposed to the polyplex solutions (3 μg pDNA labeled by YOYO-1 iodide; PEI25K weight ratio = 1, SP2K7 weight ratio = 10). The cells were washed with DPBS twice after 4 h of incubation and stained by LysoTracker Red DND-99 (500 nM). After fixation of cells with 4% paraformaldehyde, cell nuclei were stained by 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) solution (14.3 μM). After washing with DPBS twice, cell images were observed by CLSM (SP8X STED, Leica, Wetzlar, Germany) and processed by the LAS X program.

Intracellular trafficking of the polyplexes was observed by using a confocal laser scanning microscope (CLSM). HeLa cells were seeded on a confocal dish at a density of 3 × 10⁵ cells/dish in 3 mL of medium and incubated for 24 h until the cells achieved 70–80% confluency. After exchange with fresh serum-free medium, the cells were treated with HECP2k/pDNA polyplex solutions (1.5 μg of pDNA/dish, pDNA labeled with YOYO-1 iodide) with optimal weight ratio (50). PEI25k/pDNA polyplex (weight ratio = 1) was used as a control. After 4 h of incubation, the medium was exchanged with fresh medium containing 10% FBS and further incubated for 2 h or 4 h. Then, acidic organelles were stained using Lysotracker Red DND-99 solution (100 nM) and the cells were fixed with 4% paraformaldehyde. Nuclei were labeled with DAPI solution (0.125 μg/mL). After sufficient rinsing of cells, visualization proceeded with a confocal laser scanning microscope (SP8 X STED, Leica, Wetzlar, Germany) and the images were processed with LAS X software.

An equal amounts of HPQ-penta peptide TG beads and a biotin-TG beads mixture (10 mg) was incubated with 10 μL of streptavidin-coated fluorescent NPs (SA-F-NPs, 1.0% w/v, SPHERO™ streptavidin-coated blue particles, ~400 nm in diameter) for 30 min. Then, the resulting TG beads were washed with PBS solution (×3), DI water (×3), and vacuum filtration.
Fluorescence images of TG beads were obtained by a confocal laser-scanning microscope (SP8 X STED, Leica; Germany) with an ultraviolet emission line (405 nm) and detection in the 523 ± 75 nm channel. The fluorescence intensities of the TG beads were analyzed with the Leica Application Suite Advanced Fluorescence software (Leica; Germany)