A Novel 1,2-Dihydroquinoline Anticancer Agent and Its Delivery to Tumor Cells Using Cationic Liposomes

Materials and Methods

Materials. Egg phosphatidylcholine (ePC) was purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). Cholesterol and DOTAP were purchased from Lipoid (Ludwigshafen, Germany). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT,1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 7-nitrobenzofurazan-labeled (NBD-DOPE) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) was obtained from Gibco (Gibco BRL Co. Ltd, Gaithersburg, MD, USA). 4’, 6-Diamidino-2-phenylindole (DAPI) was purchased from Invitrogen Molecular Probes (Carlsbad, CA, USA). HepG2 and SMMC cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA).

Synthesis of compounds. Morita-Baylis-Hillman (MBH) carbonate 2 was prepared according to previously published procedures (20, 21). Reissert compound 1 (R¹=COR) was prepared by the literature procedure (22).

1,4-Diazabicyclo(2.2.2)octane (DABCO) (20 mol%), Reissert compound a (0.2 mmol), MBH carbonate b (1.5 equiv), and CH₃CN or toluene (2.0 ml) were added in a 10 ml round-bottom flask and dried under a nitrogen atmosphere at room temperature. Upon completion, the reaction mixture was concentrated in vacuo. The crude mixture was purified by column chromatography [silica gel, EtOAc/petroleum ether (60-90°C)] to provide product c, d, e or f (Figure 1).

Cell culture. HepG2 and SMMC cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% FBS, 100 IU penicillin and streptomycin at 37°C, and in humidified atmosphere containing 5% CO₂.

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Figure 1.

The scheme of compound synthesis.

MTT screening assay. A total of nine compounds (Figure 2) were evaluated in HepG2 and SMMC cells using the MTT assay (23). Cells were seeded at a density of 1×10⁴ cells/well in a 96-well plate and grown for 24 h. HepG2 and SMMC cells were then incubated with compounds at 250 μg/ml and 1.5 μg/ml per well, respectively. Plates were incubated for 4 h and 100 μl of fresh medium was added to each well. Cells were incubated for another 20 h. Then, 10 μl of MTT solution (5 mg/ml) was added to each well, which was incubated at 37°C for 4 h. Formazan crystals were dissolved by the addition of 100 μl of dimethyl sulfoxide. Cell viability data were obtained by measuring OD₄₉₀ nm on a microplate reader. The results were summarized as the mean±SD of six replicates for each sample. Compound 5 had the highest cytotoxicity among compounds tested, and therefore, was selected for further evaluation.

Preparation of liposomes. Due to its poor water solubility, compound 5 liposomes were prepared by an ethanol injection method. Briefly, neutral liposomes (nLips) were prepared from ePC and cholesterol at the following ratios by mass: ePC:cholesterol (63:35). Cationic liposomes (cLips) were prepared from DOTAP, ePC and cholesterol at the following ratios by mass: DOTAP:ePC:cholesterol (45:18:35). Briefly, 0.3 μmol compound 5 were added to 1.6 μmol lipid mixture. Then the mixture (7 mg/ml) was slowly added to HEPES buffer (20 mM HEPES, pH 7.4) under vortexing to achieve a final ethanol concentration of 14%. The solution was then sonicated with a bath-type sonicator for 20s. The mixture was then passed through a sterile membrane to remove unentrapped compound 5.

For fluorescence labelling of the lipid membrane, NBD-DOPE (1 mole% of total lipids) was added to the lipid mixture to make fluorescence-labelled liposomes, which were stored in the dark at 4°C.

Characterization of liposomes. The particle size and ζ-potential of liposomes were measured with Zeta-sizer Nano ZS (Malvern Instruments Ltd, Malvern, UK) (24). In addition, the size and surface morphology of the cLip–compound 5 were investigated by field-emission scanning electron microscopy (FE-SEM) (JSM-6700F; JEOL, Tokyo, Japan) (25). The sample was fixed on a brass stub using double-sided adhesive tape and was coated with a thin layer of gold and then images were taken at 3.0 kV accelerating voltage.

Cytotoxicity assay (detailed method has already been described above). MTT assay was used to evaluate the viability of HepG2 cells and SMMC cells after treatment with nLip–compound 5, cLip–compound 5 and free compound 5. Cells were seeded at a density of 1×10⁴ cells/well in a 96-well plate and grown for another 24 h. After the medium was replaced with DMEM without FBS, the formulations were then added and the plate was incubated for a further 4 h. Then fresh medium was added and the cells were incubated for another 20 h. Cell viability was determined via the MTT assay. Firstly, 10 μl of MTT (5 mg/ml) reagent was added to each well. After incubation for 4 h, formazan crystals were dissolved by adding 100 μl of DMSO. Cell viability data were obtained by measuring OD490 nm on a microplate reader. The results are summarized as the mean±SD of six replicates for each sample.

Cellular uptake by flow cytometric analysis. The cellular uptake of liposomes by HepG2 and SMMC cells was determined by an EPICS XL flow cytometer (Beckman Coulter Inc., Brea, CA, USA) (26). Cells (1×10⁵) were seeded in a 24-well plate and cultured for 24 h. Then, culture medium was replaced with nLip–compound 5, cLip–compound 5 or free compound 5 diluted in fresh culture medium without FBS and cultured for another 4 h. The cells were then washed three times with phosphate-buffered saline (PBS) and fixed with 350 μl 4% formaldehyde solution. The mean fluorescence intensity of positive cells with NBD-DOPE was analyzed by EPICS XL flow cytometer (Beckman Coulter Corp. Brea, CA, USA) and the data were analyzed with Cell Quest software.

Confocal microscopy and analysis of cellular internalization. Cells (1.5×10⁵) were seeded in a 35 mm glass-bottom culture dishes and cultured for 24 h. Then culture medium was replaced with nLip–compound 5, cLip–compound 5 or free compound 5 diluted in fresh culture medium without FBS and cultured for 4 h. After the incubation, the cells were then washed three times with PBS and fixed with 400 μl 4% formaldehyde for 8 min. The cells were then washed with PBS. Cellular nuclei were stained with 2 μg/ml DAPI for 3 min, and then cells were washed again. Internalization of liposomes was observed on a Zeiss 710 LSMNLO confocal microscope (Carl Zeiss, Jena, Germany) (27).

Statistical analysis. The data were analyzed for statistical significance using Student's t-test and p-values less than 0.05 were regarded as significant. Where indicated, the results are presented as the mean±SD.