U hplgps

The human glioma cell line U87 was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and were cultured using a standard sterile procedure in DMEM (Gibco-BRL, Rockville, IN, USA) containing 10% FBS (Sigma Chemical Co., St Louis, MO, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were maintained with humidity and 5% CO₂ at 37°C.
U87 cells were seeded on a Costar six-well plate (Corning Inc., Corning, NY, USA) at a density of 4 × 10⁵ cells/well in a 2 mL complete DMEM culture medium. After 24 h of incubation, the media was replaced with 800 μL serum-free DMEM per well. DMP/Gli1siRNA (siGli1-1: GCCTGAATCTGTGTATGAA; siGli1-2: GGACAGAA CTTTGATCCTT), DMP–Consi, or DMP in a final volume of 200 μL were subsequently added to designated wells and allowed to incubate for 5 h before replacement with DMEM. Fluorescein isothiocyanate (FITC)-siRNA-transfected cells were observed under a fluorescence microscope (Olympus IX73, U-HPLGPS, Olympus Corporation, Shinjuku, Tokyo, Japan) following an additional 24-h incubation, and transfection efficiency was determined via FACS flow cytometry (BD Biosciences, San Jose, CA, USA).

Since the green fluorescence protein (GFP) is the most frequently used reporter, the plasmids encoding enhanced GFP (pEGFP) were used as the reporting system for analyzing the transfection efficiency in our study. After seeding on the 6-well plate for 24 h, the medium of ID8 cells was replaced with the serum-free DMEM medium with 500 μL per well. The nanoparticles (15 μL) and pEGFP (5 μg) were separately diluted in 250 μL of the serum-free DMEM medium. The medium containing pEGFP was added to the medium containing nanoparticles and incubated for 20 min. Then, the ID8 cells were supplemented with the mixed medium for 4 h incubation. Subsequently, the medium was exchanging with the complete DMEM medium. After 48 h of culture, the transfected ID8 cells were observed under a fluorescent microscope (Olympus IX73, U-HPLGPS, Olympus Corporation, Tokyo, Japan). Moreover, the transfection efficiency was also measured by flow cytometry (ACEA NovoCyte™, ACEA Biosciences Inc., USA).