Cells and medium

Normal human fetal hepatocytes (Hc cells) were obtained from DS Pharma Biomedical Co. (Osaka, Japan) with a certification of informed consent for research and were cultured in CS-C medium (DS Pharma Biomedical Co.). Human hepatocellular carcinoma cells (HuH-7 cells) were purchased from RIKEN CELL BANK (Ibaraki, Japan) and incubated in Dulbecco’s modified Eagle’s medium (DMEM) (FUJIFILM Wako Chemicals, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS; CAPRICORN, Hessen, Germany) and penicillin–streptomycin solution (FUJIFILM Wako Chemicals). In addition, Hc and HuH-7 cells were mixed-inoculated in CS-C medium (DC Pharma Biomedical Co.) such that the percentage of HuH-7 cells to total cells was 0, 25, 50, 75, and 100%. These cells were cultured in a 5% CO2 humidified incubator at 37 °C.

Anti-cancer agent

Doxorubicin hydrochloride (DOX) was purchased from FUJIFILM Wako Chemicals and dissolved in sterile water to a concentration of 10 mM (stock solution) to serve as an anti-cancer reference.

Preparation of HL

HL were prepared by sonicating a mixture containing 90 mol% L-α-dimyristoyl phosphatidylcholine (DMPC; NOF Co., Tokyo, Japan) and 10 mol% polyoxyethylene (n) dodecyl ether (C12(EO)n, n = 25) (Nikko Chemicals, Tokyo, Japan) in a 5% glucose solution. For sonication of HL, a bath-type sonicator (ULTRASONIC-CLEANER-WT-200-M, Tokyo, Japan) was used at 45℃ and 300 W, after which HL were filtered using a 0.20 µm cellulose acetate filter (Advantec, Tokyo, Japan).

The size of HL at 25 °C was determined via the dynamic light scattering method (ELS-8000, Otsuka Electronics, Osaka, Japan). The hydrodynamic diameter of the HL was approximately 34 nm with a narrow size distribution range, and the HL remained stable for more than 3 weeks.

Selection of medium for mixed culture

The medium used for the mixed culture of Hc and HuH-7 cells was evaluated by a cell growth curve using CS-C medium (DS Pharma Biomedical Co.) and DMEM (FUJIFILM Wako Chemicals). The cells were seeded at 1.0 × 104 cells/cm2 and counted using an Auto Cell Counter (ADAM-MC, Digital Bio, Tokyo, Japan) after 1, 2, 3, 5, and 7 d of cell culture. The medium was changed once every 2 days.

Cell membrane fluidity measurement

The membrane fluidity of intact cells was evaluated using the fluorescence depolarization method with the fluorescent probe1,6-diphenyl-1,3,5-hexatriene (DPH; Nacalai Tesque, Kyoto, Japan). The cells were treated with 0.05% trypsin–EDTA and suspended in HBSS (2.0 × 104 cells/ml). DPH was added to the cell suspension at a final concentration of 2 µM and allowed to stand for 15 min at 37℃. The fluorescence polarization (P) of DPH was measured using a fluorescence spectrophotometer (F-7100; Hitachi, Tokyo, Japan).

Accumulation of HL in the cell membrane

HL/NBDPC was prepared by sonicating a mixture containing 86 mol% DMPC, 10 mol% C12(EO)25, and 4 mol% the fluorescent probe 1-palmitoyl-2--sn-glycero-3-phos-phocholine (NBDPC; Avanti Polar Lipids, Inc., AL, USA) in a 5% glucose solution, similar to HL. NBDPC liposomes were prepared by sonication of NBDPC alone in 5% glucose solution.

HuH-7 cells were dye-labeled with 5 µM cell tracking solution (CellTracker™ Red CMFDA, Thermo, Massachusetts, USA) for 30 min. Unlabeled Hc and dye-labeled HuH-7 cells were then collected, mixed, and seeded such that the percentage of HuH-7 cells to the total number of cells was 0, 25, 50, 75, and 100%. Mixed Hc and HuH-7 cells after seeding were cultured at 37° C in a 5% CO2 incubator for 24 h, following which the cells were treated with HL/NBDPC for 1 h at a final concentration of [DMPC] = 300 µM, [C12(EO)25] = 33.3 µM, and [NBDPC] = 13.9 µM in the medium. NBDPC liposomes were treated for 1 h at a final concentration of 13.9 µM in the medium.

The fused accumulation of HL/NBDPC or NBDPC in the membranes of Hc and HuH-7 cells was evaluated by confocal laser microscopy (Olympus, IX83-CSU-X1, Tokyo, Japan) using 488 nm excitation light for NBDPC and 561 nm for CellTracker™ Red CMFDA.

Cell growth inhibition assay

DOX or HL cytostatic assays on Hc and HuH-7 cells were performed using the WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H tetrazolium, monosodium salt) assay (Cell Counting Kit-8, Dojindo, Kumamoto, Japan). Hc or HuH-7 cells were each seeded in a 96-well plate at 5.0 × 103 cells/well and cultured at 37° C in a CO2 incubator for 24 h. DOX (0–100 µM) or HL ([DMPC] = 0–3000 µM]) was then added and cells were cultured for an additional 48 h. The WST-8 solution was then added and incubated for 2 h.

The absorbance was measured at 450 nm using a microplate reader (Thermo Fisher Scientific). The inhibitory activity of DOX or HL on Hc or HuH-7 cell proliferation was assessed by calculating Amean/Acontrol. Here, Amean and Acontrol indicate the absorbance of the water-soluble formazan in the presence and absence of DOX and HL, respectively. Next, the calculated cytostatic rates of DOX and HL were analyzed using GraphPad Prism9, and sigmoid curves and 20%, 50%, and 80% cytostatic concentrations (IC20 values, IC50 values, IC80 values) were calculated.

Confocal laser microscopy

HuH-7 cells were dye-labeled with 5 µM cell tracking solution (CellTracker™ Green CMFDA (5-chloromethylfluorescein diacetate), Thermo) for 30 min. Unlabeled Hc and dye-labeled HuH-7 cells were collected, mixed, and seeded into 35 mm glass bottom dishes (MatTek, P35GC-0-14-C, Massachusetts, USA) such that the percentage of HuH-7 cells to the total number of cells was 0, 25, 50, 75, and 100%. Mixed Hc and HuH-7 cells after seeding were cultured at 37° C in a 5% CO2 incubator for 24 h, treated with DOX (0, 0.1, 1, 10 µM) or HL ([DMPC] = 0, 200, 400, 600 µM), and cultured for 48 h. The cells were then incubated with a nuclear staining solution (TO-PRO™-3 Iodide, Invitrogen) for 15 min and mounted with an antifade agent (SlowFade™ Diamond Antifade Mountant, Invitrogen). They were then observed using a confocal laser scanning microscope (TCS-SP; Leica Microsystems, Wetzlar, Germany) using a 488 nm Ar laser line and a 633 nm He/Ne laser line.

Flow cytometry analysis

HuH-7 cells were dye-labeled with 5 µM cell tracking solution (CellTracker™ Green CMFDA (5-chloromethylfluorescein diacetate), Thermo, C7025) for 30 min. Unlabeled Hc and dye-labeled HuH-7 cells were collected, mixed, and seeded into 60 mm dish (BD FALCON, Arizona, USA) such that the percentage of HuH-7 cells to the total number of cells was 0, 25, 50, 75, and 100%. Mixed Hc and HuH-7 cells after seeding were cultured at 37° C in a 5% CO2 incubator for 24 h, treated with DOX (0, 0.1, 1, 10 µM) or HL ([DMPC] = 0, 200, 400, 600 µM), and cultured for 48 h. Then, the mixed cells were detached with a 0.05% trypsin–EDTA solution, and the cell number was adjusted to 1.0 × 106 cells/sample. Flow cytometry was performed using a CytoFLEX with a 488 nm laser line. Data were analyzed using CytExpert software (Beckman Coulter Inc., California, USA).

Soft agar colony formation assay

Cell tumorigenicity was assessed using a soft agar colony formation assay. Concentrated CS-C medium containing agar prepared according to DS Pharma Biomedical Co., DMEM/Nutrient Mixture F-12 (Thermo Fisher Scientific, Ashland, NC, USA) containing 10% BSA and 50 ng/mL recombinant human FGF-acidic (PeproTech Inc., Rocky Hill, NJ, USA) was used for the assay.

First, 350 µL of 0.5% agar medium (1% agar solution:2 × DMEM/F-12 = 1:1) was added to a 48-well plate (Sumitomo Bakelite Co., Ltd., Tokyo, Japan) and solidified as the lower-layer agar. Next, 0.5% agar medium and cell suspension were mixed in a ratio of 2:1 to form a semisolid to prepare upper agar; 50 µL of upper agar was layered on top of the lower agar. The cells were seeded at 1000 cells/well and cultured for 9 d at 37℃ in 5% CO2 and 95% humidity.

After 9 d of culture, surviving colonies in the top agar were stained with 5 µM Calcein-AM (DojinDo, Kumamoto, Japan). The stained colonies were imaged using a fluorescence microscope (EVOS FL; Thermo Fisher Scientific Inc., Waltham, MA, USA). Colony numbers were analyzed from the photographs using the ImageJ software (National Institutes of Health, Bethesda, Md., USA). A stained colony > 80 µm in diameter, corresponding to > 20 cells, and an area > 5700 µm2 were defined as a tumorigenic colony (Meyskens et al. 1984), which is tumorigenic colonies are formed through cell proliferation.

Statistical processing

The results are presented as mean ± standard deviation (SD). Data were statistically analyzed using Student’s t-test. Differences were considered statistically significant at p < 0.05.