2.8. Fluorescence Anisotropy Measurements

To investigate the impact of interaction between dendrons with phospholipid bilayer, the measurement of fluorescence anisotropy using the fluorescence probe 1,6-diphenyl-1,3,5-hexatriene (DPH, Sigma-Aldrich, St. Louis, MO, USA) was performed. Measurement of the steady-state anisotropy of DPH probe is the most common technique for evaluation of ordering of the membrane lipids. The DPH stock solution was dissolved in acetone and stored in the fridge at 4 °C. Before each measurement, the staining solution was prepared, dissolved in PB (pH 7.4), and shaken for 20 min to evaporate acetone. The final concentration of DPH in suspension was 1.5 × 10⁻⁷ M. A luminescence spectrometer LS 45 (PerkinElmer, Waltham, MA, USA) was used for L-format polarization measurements of the steady-state fluorescence anisotropy of DPH. The excitation and emission wavelengths were set to 360 and 430 nm. The measurements were carried out at 37 ± 0.1 °C. Liposomes were incubated with dendrons for 15 min before each measurement. Grating correction factor (G) for the optical system was measured, which is the ratio of the sensitivities of the detection system for vertically and horizontally polarized light [38], where:

I_HV and I_HH represent the fluorescence intensity when the excitation polarizer is in the horizontal position and when the emission polarizer is in the vertical or horizontal position. Every measurement lasted for 2400 s. The steady-state fluorescence anisotropy (r_s) was used for the assessment of membrane fluidity and calculated according to the following equation [38]:

Lower values of fluorescent anisotropy represent higher membrane fluidity.