2.9. Nonspecific adhesivity and cellular internalization analysis

1 × 10⁵ CCRF-CEM (Ramos, A549 or HEK293) cells were incubated with SPN, SPN-DNA or SPNRes-DNA(6 μg mL⁻¹) in a binding buffer at 37 °C for 30 min, followed by washing for 3 times with washing buffer to remove the excess mixture (1000 rpm, 4 min). The efficiency of nonspecific adhesivity and cellular internalization was evaluated by flow cytometry.

For experiments to test the inhibitory effect, cells were pretreated with the inhibitors for 20 min, followed by the addition of a solution of SPN, SPN-DNA₁₀ or SPN-DNA₄₀ (Dynasore: 50 μg mL⁻¹, CPZ: 20 μg mL⁻¹, Nystatin: 20 μg mL⁻¹, LY294002: 20 μg mL⁻¹). The inhibition efficiency of cellular internalization was evaluated by flow cytometry and confocal laser scanning microscopy (CLSM).

For experiments to control cellular internalization of SPN-DNA_40S–S or SPN-DNA_40PCL, the NPs were incubated with different concentrations of TCEP for 1 h or be exposed under UV irradiation for different time at 37 °C. The efficiency of cellular internalization was evaluated by flow cytometry.

The washing buffer was composed of DBPS containing 4.5 g L⁻¹ glucose and 5 mM Mg²⁺. The binding buffer was composed of the washing buffer containing 0.1 mg mL⁻¹ tRNA.