2.8. Bilayer Characterization by DPH Fluorescence Anisotropy

Bilayer characterization has been carried out on liposomes and RIF-liposomes by measuring the DPH fluorescence anisotropy, which is a parameter interpreted as a membrane microviscosity (viscosity in the bilayer interior) or fluidity [23]. DPH-loaded liposomes were prepared by co-dissolution in the mixture of organic solvents of lipids and probe (2 × 10⁻⁴ M), then the same preparation method described for empty liposomes in 2.2 was followed. DPH-liposome solution was filtered through cellulose filter of 450 nm cutoff, and its fluorescent measurements were performed with excitation λ_exc = 350 nm and detecting the fluorescence intensity at λ_em = 428 nm, using luminescence spectrometer (LS5013, PerkinElmer, Waltham, MA, USA) [24]. The fluorescence anisotropy (r) was determined by using Equation (2):

where I_VV, I_VH, I_HV, and I_HH are fluorescent intensities, and subscript V (vertical) and H (horizontal) represent the orientation of polarized light and G = I_HV/I_HH factor is the ratio of sensitivity of detection system for vertically and horizontally polarized light.