In vitro cellular uptake assay of CUR

The in vitro cellular uptake of CUR in CUR solution and PLL-DOCA-MPEG-cy5.5/CUR NPs was examined by confocal laser scanning microscopy (CLSM, LSM 510 Meta, Zeiss, Oberkochen, Germany) and flow cytometry (FACS Canto II, BD Biosciences, San Jose, CA). Hep3B cells stained with DAPI were used as a control. Hep3B cells (1 × 10⁵ cells/mL) were seeded on PLL-coated glass coverslips and incubated with the media overnight in an incubator at 37 °C in humid air with 5% CO₂. The cells were treated with CUR solution (3 mL, pH 7.4 PBS) or PLL-DOCA-MPEG-cy5.5/CUR NP solution (3 mL), each in 50 mL of medium (corresponding to 0.09 μM CUR) for 5 and 10 minutes, and then washed with PBS (pH 7.4) three times. The solution-treated cells were fixed in 4% (v/v) formaldehyde solution for 10 minutes and washed with PBS (pH 7.4) three times. The fixed cells were counter-stained with 10 μL ProLong^® Gold antifade reagent with DAPI, as visualized by CLSM at an excitation of 488 nm. For flow cytometry assay, untreated Hep3B cells were used as a control. The analysis was performed using an annexin V-cy3 detection kit (Abcam^®, Cambridge, MA) according to the manufacturer’s instructions. The cells treated with the samples for 5 and 10 minutes (corresponding to 0.09 μM CUR) were washed three times with PBS (pH 7.4) and incubated overnight. The cells were harvested with 0.25% trypsin/EDTA, and then transferred to 5 mL tubes. After addition of 5 μL of annexin V-cy3 and 10 μg/mL of DAPI, the samples were gently mixed and incubated for 30 minutes in the absence of light. Then, 10,000 cells were acquired for flow cytometric analysis.