Soy phosphatidylcholine (PC) was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Meth oxypolyethyleneglycol-di-stearoyl-phosphatidylethanolamine (DSPE-PEG2000, with mPEG MW2000 Da) was obtained from Genzyme (Oxford, UK). Cholesterol (Chol), PBS, dialysis tubing, propidium iodide (PI), RNase and BL were all purchased from Sigma-Aldrich (UK). Methanol, dichloromethane, CyQUANT^® Cell Proliferation Assay kit and Annexin V-FITC/PI Apoptosis Detection kit were both from Thermo Fisher Scientific (Loughborough, UK). RPMI-1640, L-glutamine, penicillin-streptomycin and fetal bovine serum (FBS) were all from Invitrogen Life Technologies (UK). The CellTiter 96^® AQ_ueous One Solution Cell Proliferation Assay (MTS) kit was purchased from Promega (Southampton, UK).

Three types of liposomes with different diameters were prepared. Liposomes were composed of soy PC, cholesterol, and methoxypolyethyleneglycol-di-stearoyl-phosphatidylethanolamine (DSPE-PEG2000; Genzyme). Liposomes were prepared as described elsewhere (35). Briefly, the lipids were dissolved in methanol:dichloromethane 1:2 (v/v) at a PC:Cholesterol:DSPE-PEG2000 molar ratio of 78.9:19.7:1.4 at room temperature. BL was dissolved in the solvent with lipid mixture when formulating the liposomes. Different lipid/BL mass ratios were tested before settling on a fixed ratio of 10:1. The lipid mixtures were deposited on the side wall of the rotary glass vial by removing the solvent with nitrogen. The dried lipid films were hydrated in 10 mM sodium phosphate buffer pH 7.4. This process led to the spontaneous formation of pegylated liposomes. The liposomes were then down-sized by passing through 0.1, 0.2 or 0.4 µm polycarbonate membrane syringe filters (Whatman^®; Whatman, Inc., Clifton, NJ, USA) to produce lipo1, 2 and 3 suspensions, respectively. Free BL was removed by dialysis (14,000 Da cutoff membrane) against 10 mM sodium phosphate buffer pH 7.4 overnight. The size and ζ-potential of liposomes were measured by dynamic light scattering on a Zetasizer-Nano ZS (Malvern Instruments Ltd., Malvern, UK).

Human leukemia K562 cells were purchased from ATCC ^(UK). Cells were cultured in RPMI-1640 media containing 10% fetal calf serum, 100 U/ml of penicillin, 100 mg/ml streptomycin in 75 cm² flasks. The cells were grown in a humidified incubator containing 5% CO₂ and 95% air at 37°C. Cells growing in the log phase and free from mycoplasma was used in this study.

K562 cells were cultured at a density of 6×10⁴ cells/well in 96-well plates overnight and treated with different concentrations of BL and control liposomes for 48 h. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) solution (50 µl) from CellTiter 96^® AQ_ueous One Solution Cell Proliferation Assay kit was added to detect live cells in each well as per manufacturer's instructions. Cells were incubated for 30 min at 37°C with 95% air and 5% CO₂. The absorbance of the solution was measured at 490 nm by FLUOstar Omega (BMG Labtech, Aylesbury, UK). Each treatment was conducted in triplicates. The cell viability was expressed as a percentage of cell viability of liposome treated cells relative to untreated controls.

Cell proliferation assays were conducted with the CyQUANT^® Direct Cell Proliferation Assay Kit (Thermo Fisher Scientific) to avoid interference from the color of BL. K562 cells were cultured at a density of 5,000 cells/well in 96-well plates overnight and treated with different concentrations of free BL or liposomal BL (0.185–100 µM) for 48 h. CyQUANT^® Detection reagent (100 µl) was added to detect live cells in each well as per manufacturer's instructions. Cells were incubated for 60 min at 37°C with 95% air and 5% CO₂. The fluorescence of the solution was recorded at 485 (ex)/520 (em) nm by FLUOstar Omega (BMG Labtech). Cell viability was expressed as a percentage of cell survival of treated cells relative to untreated controls.

Approximately 1×10⁵ K562 cells/well in 96-well plates were incubated with 20 µM free BL or liposomal BL for 48 h. The cells were centrifuged, resuspended in binding buffer and treated with Annexin V-FITC and PI for 5 min before analysis for cell apoptosis. For cell cycle analysis, the cells were harvested, washed and fixed gently (drop by drop) in 70% ethanol and kept at 4°C for 30 min. The cells were then resuspended in PBS containing 40 µg/ml PI and 0.1 mg/ml RNase for cell cycle analysis. After 30 min incubation at 37°C in the dark, 10,000 cells/sample were analyzed were analysed for DNA content by flow cytometry on BD FACSCalibur (BD Biosciences, Oxford, UK) and the cell cycle distribution was determined using a CellQuest software (Becton-Dickinson, Franklin Lakes, NJ, USA). The percentage of cell population that had undergone apoptosis and those in different phases of the cell cycle were assessed accordingly.

The measurement of intracellular ROS was determined by the Cellular Reactive Oxygen Species Detection Assay kit (Abcam, Cambridge, UK), which uses the cell permeant reagent 2′,7′-dichlorofluorescin diacetate (DCFDA), a fluorogenic dye that measures ROS activity within the cell. Briefly, K562 cells were cultured in complete phenol-red free RPMI medium at a density of 1.5×10⁵ cells/well in 96-well plates and were treated with 20 µM BL or liposomal BL for 48 h. After the incubation time, the cell treatments were overlaid with 20 µM DCFDA (100 µl) and incubated at 37°C for further 30 min in the dark. The fluorescence of the treatments was detected at the excitation and emission spectra of 485 and 520 nm, respectively, by FLUOstar Omega (BMG Labtech).

Results were presented as mean ± standard deviation (SD). Statistical significance was tested by paired t-test or one-way ANOVA analysis. Differences between experimental groups were considered significant when the P-value <0.05.

Three groups of liposome suspensions: lipo1 (100 nm), lipo2 (200 nm) and lipo3 (400 nm), were prepared to investigate whether the size of the liposomes contributed to the efficiency of BL encapsulation. BL/PC 1:10 (w/w) was dissolved in the solvent with lipid mixture when formulating the liposomes. Unincorporated BL was removed by dialysis and the concentration of BL in the liposome was determined by dissolving the liposomes in methanol. The absorbance of BL was recorded at 278 nm by FLUOstar Omega (BMG Labtech). Phospholipid (PC) was detected in the liposomes by the Stewart assay (36). Drug encapsulation efficiency was determined by encapsulated BL divided by original BL corrected by PC concentration. The final liposome samples were characterized and the data was shown in Table I, with the drug encapsulation efficiency from high to low: Lipo2 > lipo1 > lipo3.

The size and zeta potential of liposomes were determined by dynamic light scattering (Fig. 1A). Three groups of control liposomes displayed average diameters of 125, 161 and 230 nm, respectively, with polydispersity of 0.1 for lipo1 and lipo2, 0.2 for lipo3. The average diameters for lipo2 and lipo3 were below 200 and 400 nm, which may be due to the quality control of the filters (37). The negative ζ-potential of the liposomes was attributed to one of the liposome components DSPE-PEG2000 which is negatively charged. The stability of liposomes was investigated by comparing the size and zeta potential changes over a one month storage period at 4°C in 10 mM phosphate buffer pH 7.4 (Fig. 1B). The results showed that lipo1 formulation was the most stable, with no statistically significant changes in diameter or ζ-potential over at least a one month period.

Potential cytotoxicity of the liposomes was measured from cell viability relative to control K562 cells treated with medium only. MTS assay demonstrated that cell viability was between 80 and 120% in relation to the control samples (Fig. 2). Statistical analysis showed no evidence of liposome toxicity at phosphatidylcholine concentrations up to 1.6 mg/ml.

The CyQUANT^® Direct Cell Proliferation Assay kit was used to evaluate the cytotoxicity of liposomal BL. The cell proliferation assay was assessed after cells were treated for 48 h with the encapsulated drug. Exposure to free and liposomal BL inhibited leukemia cell line K562 growth in a concentration-dependent manner (Fig. 3). The results showed that there was no significance difference between the antiproliferative effects of liposomal BL and those of free BL at equimolar concentrations. No inhibition effects were observed from three groups of control liposomes, which was consistent with the MTS assay showing no cytotoxicity from the control liposomes.

K562 cells were exposed to three different sizes of control liposomes and their relevant liposomal encapsulated BL for 48 h. Staining procedures using PI or Annexin V-FITC/PI were conducted to evaluate the effect of BL preparations on cell cycle and cell death, respectively, using a flow cytometer. Cells in each sample from different phases (sub-G1, G1, S and G2/M) were presented and their percentage in each phase was calculated and shown in Table II; viable and non-viable (necrotic, early apoptotic and late apoptotic) cells were calculated and shown in Table III. All the BL preparations showed significant increase (P<0.005) in their populations in the sub-G1 phase and significant decrease (P<0.01) in cell viability in comparison of their relative control sample.

The cell permeant reagent DCFDA, a fluorogenic dye that measures ROS activity within cells, was used to investigate whether intracellular ROS production was involved in the mechanism of apoptosis caused by BL. Analysis indicated that all BL preparations induced significant ROS generation (as determined by the fluorescence changes normalized as percentage increase over the untreated cells control level). About 20–30% increase (P<0.05) of ROS production was observed when K562 cells were treated with liposome encapsulated BL, and 50% increase (P<0.001) following treatment with non-encapsulated BL or free BL (Fig. 4). Moreover, there was a significant difference in ROS induction between the treatments with liposomal BL and free BL (Fig. 4).