Fetal bovine serum (fbs)

Lipofectamine 3000 was bought from Thermo Fisher Scientific (L3000015). The cryopreservation medium (NCRC-10001-50) was purchased from Cyagen Biosciences. CDK4 CRISPR-Cas9 knockout plasmid (sc-400148) was purchased from Santa Cruz. Actin-Tracker Green-488 (C2201S) was bought from Beyotime Biotechnology. Lentivirus expressing luciferase was purchased from HANBIO. A TUNEL assay kit (Red AF647) was obtained from Procell. Picogreen was purchased from Yeasen Biotechnology. A549 and H226 cell lines were obtained from the American Type Culture Collection. A549 and H226 cell lines were cultured in F12K medium and RPMI 1640 medium, which contain 10% fetal bovine serum and penicillin/streptomycin (100 U ml⁻¹; Biosharp) at 37°C in 5% CO₂. Five-week BALB/c nude mice (female/male) were obtained from Zhejiang Vital River Laboratory Animal Technology. Mouse study was conducted in accordance with the protocol approved by Animal Ethics Committee of Zhejiang University (ZJU20230329).

The HepG2 liver cancer cell line (HB-8065) was purchased from American Type Culture Collection, and was cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (Biological Industries; Sartorius AG) at 37°C with 5% CO₂ to maintain a constant cell growth environment. The Huh7 liver cancer cell line (BFN60800691) and HHL-5 normal liver cell line (BFN6072012687) were purchased from BLUEFBIO, and were cultured in DMEM (Biosharp Life Sciences) containing 10% fetal bovine serum at 37°C with 5% CO₂ to maintain a constant cell growth environment. To ensure their identity and purity, the cells were identified using the short tandem repeat method.

To prepare MB-plasmid mixture, 10 µg plasmid and 1.14×10⁹ MB was added to a sterile centrifuge tube, followed by gentle blowing with a pipette tip to aid mixing. The mixture was allowed to stand for 20 min at 4°C. For cell preparation, 2×10⁵ cells were added to a 6-well plate with a sterile 22×22 mm cover glass at the bottom and then placed in a 37°C incubator for 24 h. After 24 h, the original medium [DMEM (Gibco; Thermo Fisher Scientific, Inc.) + 10% fetal bovine serum (Biosharp Life Sciences) + 5% penicillin-streptomycin (Biosharp Life Sciences)] was aspirated and washed with PBS. Tweezers were used to place the coverslip on the cover of the 6-well plate with the cell surface facing the liquid surface. Complete medium (as aforementioned) was added to each well of the 6-well plate until the liquid overflowed. The MB-plasmid mixture was added to the upper layer and placed on the 6-hole plate cover. A small ultrasonic instrument was used to sonicate MBs and cells at the bottom of the 6-well plate. The sonication conditions were 1 MHz at 0.75 W/cm² for 30 sec. The CMB and PTX-CMB group were sonicated under identical conditions. For the group carrying PTX and CRISPR/Cas9 transfection (CRISPR/Cas9-PTX-CMB), the cells were cultured at 37°C for 24 h, and a Puro drug sieve was used for 7-day screening. The expanded culture was used for subsequent experiments.