Te2000 s

Before settling the cells onto Ad-SiMWS, K562 3.21 cells were first starved in serum-free RPMI medium in 75-cm² cell culture flask for 10 hours to synchronize cells to G₀/G₁ phases of cell cycle (28 (link)) and then centrifuged to remove medium at the rate of 300g for 5 min. Approximately K562 3.21 cells (1 × 10⁵ in 1.0 ml of serum-containing RPMI medium) were introduced into each well of an eight-well plate, where a 2.5 × 3 cm² Ad-SiNWS was placed. Last, the Cas9•sgRNA⊂SMNPs (containing 3.0 μg of Cas9 protein) in 1.0 ml of serum-containing RPMI medium were added to each well. The cells were coincubated with SMNPs for a certain period. Every 48 hours, 1.0 ml of medium was removed via pipette and then a new 1.0 ml of serum-containing RPMI medium was added to each well. After washing with PBS, the cells in the well were immediately fixed with 2% paraformaldehyde (PFA) and then stained with DAPI. Microscopy-based image cytometry was used to detect the cellular uptake performances of different formulations. After each treatment, the GFP signal was quantified with a fluorescent microscope equipped with a charge-coupled device CCD camera (Nikon TE2000S, Japan).

Wound healing assays were used to analyze the migratory ability of MDA-MB-231 cells transfected with Linc-OIP5 siRNA. A total of 3×10⁵ cells per well were seeded into 24-well plates and cultured in DMEM with 1% FBS at 37°C in 5% CO₂ for 48 h to allow the cells to adhere and form a confluent monolayer. Subsequently, the monolayers were scratched using the tip of a 10 µl pipette tip. The scratched wound was rinsed three times with PBS to remove debris. Cells were incubated at 37°C in 5% CO₂ and monitored for 24 h. Wound healing was monitored by taking digital images from three different fields of view and from three independent samples at 0, 12, and 24 h after scratching. Scratch-wound images were captured using an inverted light microscope (TE2000-S; Nikon Corporation; magnification, ×40).