3.3. DNA Binding Studies by UV–Vis Spectroscopy

Absorption spectra were performed at room temperature for varying concentrations of CT-DNA (0, 6.25, 12.5, 18.75, 25, 31.25, 37.5, 43.75 to 50 µM) and constant concentrations of G2.5COO(DACHPt)₁₆ metallodendrimer (2 µM), DACHPtCl₂ (9 µM), or oxaliplatin (9 µM). The metallodendrimer, DACHPtCl₂, and oxaliplatin solutions were prepared in ultrapure water and then diluted in a 5 mM Tris-HCl, 50 mM NaCl pH 7.4 buffer. The stock solutions of DNA were directly prepared in this buffer. DNA purity was assessed by UV–vis spectroscopy using the absorbance values ratio at 260 nm and 280 nm (it should be between 1.8–1.9 to make sure it is sufficiently protein-free). The obtained ratio was 1.9, indicating that the DNA was pure. The compounds and the CT-DNA were incubated for 5 min at room temperature. The absorbance was measured in a PerkinElmer UV–vis spectrometer Lambda equipment, using the buffer as blank. Two independent experiments were carried out for the metallodendrimers, DACHPtCl_2, and oxaliplatin. The intrinsic binding constant, K_b, of the compounds with DNA, was determined using the Benesi–Hildebrand equation [36]: A₀/(A−A₀) = ε_G/(ε_H-G − ε_G) + ε_G/(ε_H-G − ε_G) × 1/(K_b[DNA]), where K_b is the binding constant, [DNA] is the DNA concentration, A₀ and A are the absorbance values of the free compound and compound–DNA complex, and ε_G and ε_H-G are their absorption coefficients, respectively. Binding constant values were then obtained from the ratio of the y-intercept to the slope in the plots A₀/A−A₀ vs. 1/[DNA]. Additionally, the Gibbs free energy (ΔG) associated with the process of DNA binding was calculated using the usual equation: ∆G = −RTlnK_b, where T is the temperature in Kelvin and R the gas constant.