Aplicación del método de expertos para la evaluación del modelo y el procedimiento

2.1.11.1 Cytotoxicity against A2780 Human Ovarian Cancer Cells

The cytotoxicity of iridium complexes against A2780 human ovarian cancer cells

was performed by Dr. Ana M. Pizarro (Department of Chemistry, University of

Warwick). The following sections outline the general cell experiments used in this

2.1.11.1.1 Materials and Maintenance

The A2780 human ovarian cancer cell line was obtained from the European

Collection of Cell Cultures (ECACC, Salisbury, U.K.). The cells were maintained

in RPMI 1640 medium, which was supplemented with 10% fetal calf serum, 1%

glutamine and 1% penicillin/streptomycin. Experiments were performed with cells

within 15 passages from each other. All cells were split two to three times a week

when around 80–95% confluence was reached using 0.25% trypsin/EDTA.

2.1.11.1.2 In Vitro Growth Inhibition Assay

IC50 values (concentration at which 50% of the cell growth is inhibited) against

A2780 human ovarian cancer cells were determined as part of this thesis.

Plates containing 8 rows × 12 columns, in total 96 wells with 300 µL capacity

were used. The A2780 cancer cells were plated at a density of 5000 cells/well. All

cells were grown for 48 h at 310 K in 5% humidified atmosphere, before addition of

IrIII complexes. Cisplatin (CDDP) was bought from Sigma and was included in every assay as control.

Stock solutions of the iridium complexes or CDDP were made up in 5% DMSO

and saline, by initial dissolution in DMSO followed by dilution with saline.

Sonication was sometimes used to facilitate complete dissolution. These stock

solutions were diluted with medium to give final concentrations of 400 µM, 200

µM and 20 µM (0.5% DMSO final concentration). Aliquots of 50 µL of these

Chapter 2: Experimental Methods and Materials

150 µL of media, so that the final concentrations were 100 µM, 50 µM and 5 µM

(0.125% DMSO final concentration), respectively. These three concentrations were

used in an initial screen for activity, and candidates were classified as either:

a) inactive <50% growth inhibition at 100 µM,

b) modest 80% to 50% growth inhibition at 50 µM,

c) potent >50% growth inhibition at 5 µM.

IC50 values were obtained by testing at typically six different concentrations,

ranging from 0.05 µM to 100 µM. Each assay includes cisplatin which acts as a

positive and comparative control as well as 16 wells free of drug solution, which

acts as the 100% cell survival control. Each concentration of cisplatin or complex

was tested in triplicate so that three compounds plus cisplatin could be tested per

plate. Each assay was done in duplicate, so that each concentration was tested a

total of six times.

After 24 h exposure (at 310 K in a 5% CO2 humidified atmosphere), the drug

containing medium was removed, the cells washed with 50 µL phosphate buffered

saline (PBS) and 200 µL of fresh medium was added. The cells were left to grow

for three doubling times (72 h) at 310 K in a 5% humidified atmosphere.

The remaining biomass after this time was determined using the sulforhodamine

B (SRB) assay.15 Cells were fixed to the 96-well plate by adding 50 µL 50% trichloroacetic acid (TCA) to each well (final concentration 10%) and incubating at

assistance of a hairdryer until no standing moisture was visible. The TCA-fixed

cells were stained by adding 50 µL of 0.4% (w/v) sulforhodamine B (SRB)

dissolved in 1% acetic acid, and left to stand for 30 min at ambient temperature.

The excess dye was removed with four quick rinses of 1% acetic acid and again

dried with the assistance of a hairdryer until no standing moisture was visible. The

cell-bound dye was solubilised by addition of 150 µL of 10 mM Tris base

(tris(hydroxomethyl)aminoethane, pH 10.5) and plates were left standing at ambient

temperature for 1 h to allow the solutions to become homogeneous. The optical

density was then measured at 540 nm on a BioHit BP800 plate reader. IC50 values

were obtained from plots of % cell survival against the Log of the drug

concentration and fitted with a sigmoidal equation using ORIGIN 8.0.

2.1.11.2 NCI/DTP Cytotoxicity

Five iridium complexes studied in Chapters 3 and 4 were further evaluated by the

National Cancer Institute Developmental Therapeutics Program (NCI/DTP, U.S.A.)

for in vitro cytotoxic test against ca. 60 human cancer cell lines. The protocol for

the determination of cytotoxicity on the 60 cell line panel can be found at

http://dtp.nci.nih.gov/branches/btb/ivclsp.html. The DTP homepage can be accessed at http://dtp.cancer.gov/.

Chapter 2: Experimental Methods and Materials

2.2

Synthesis and Characterisation of Starting Materials

The ligands tetramethyl(phenyl)cyclopentadiene (CpxphH) and tetramethyl(biphenyl)cyclopentadiene (CpxbiphH), and iridium dimers of the type [(η5-Cpx)IrCl2]2 (Cpx = pentamethylcyclopentadienyl, Cp*; tetramethyl(phenyl)-

cyclopentadienyl, Cpxph; tetramethyl(biphenyl)cyclopentadienyl, Cpxbiph), Figure 2.1, were synthesised and characterised, in which the ligand CpxphH16 and the dimer [(η5-C5Me5)IrCl2]2 (1),17 were prepared according to literature methods.

CpxH Cp*H CpxphH CpxbiphH Ir Cl Cl Ir Cl Cl Cpx Cpx dimer Cpx Cpxph Cpxbiph 2 3 Cp* 1

Figure 2.1. Cyclopentadiene ligands and iridium dimers studied in this work.

The derivative CpxbiphH, in which one of the methyls of Cp* is replaced by a biphenyl group, has not been previously reported. All the synthesised ligands and

dimers were fully characterized by 1H NMR spectroscopy and CHN elemental analysis.