Preparation of liposomes with phospholipids and study of their photo-interaction with a fluorophore-containing compound

Abstract

Aim. To substantiate the optimal technology for obtaining two types of bio-related liposomes based on the composition of dipalmitoyl-L-α phosphatidylcholine (DPPC) or palmitoyl-sphingomyelin-N-hesadecanoyl-D erythrosphingosylphosphorylcholine (C16-SM) with cholesterol and study their interaction with a fluorophore-containing compound.

Materials and methods. To prepare liposomal vesicles based on phospholipids – DPPC, C16-SM and cholesterol (1:1) the method of reverse-phase evaporation on a rotary vacuum evaporator Bϋchi-210 was used. Ultrasonic homogenization was performed using a Jeken (Codyson) PS-08A sonicator. A fluorescent compound R-203 (5’-isopropyl-3’- ((4-methyl-2-oxo-2H-chromen-7-yl) -carbamoyl) -2-oxospiro-[indoline-3,2’-pyrrolidine] -4’-carbon) with a 7-amino-coumarin (7-AMC) fluorophore was obtained by the synthesis previously described. Liposome particle size determination was measured using laser diffraction on a LA-960 Laser Particle Size Analyzer (Horiba Scientific). The measuring range of the device is 0.01 μm to 5000 μm; guaranteed accuracy is ± 0.6 %. Fluorescence spectra were recorded in the emission range of 365… 650 nm on a FluoroMax®-4 spectrofluorometer (Horiba Scientific) at an excitation wavelength of 350 nm. R-203 fluorescence spectra (λem = 412 nm) were measured in a 500 μl cuvette.

Results and discussion. Target liposomes were obtained based on the composition of DPPC or C16-SM phospholipids with cholesterol in the ratio of phospholipids and cholesterol 1:1. The optimal size of liposomes of about 100 nm was achieved for the composition of C16-SM: cholesterol (1:1). The calculated packaging parameter (γ) allowed us to conclude that in the case of DPPC and C16-SM phospholipids, the parameter γ < 1, i.e. the molecules of these lipids had a cone shape and were packed in phase I type where the molecules could form spherical (vesicles) and / or cylindrical micelles. However, the C16-SM phospholipid has a larger polar area of the molecule (a0), in contrast to the DPPC molecule. Fluorescence spectra of R-203 interaction with DPPC : cholesterol and C16-SM : cholesterol (1:1) liposomes in phosphate-buffered saline pH 7.4 were studied. In the interaction of the fluorescent substance R-203, quenching of the fluorescence intensity was observed according to the Stern–Volmer equation; it was more intense for C16-SM: cholesterol liposomes than in the case of DPPC: cholesterol liposomes.

Conclusions. The optimal technology for obtaining two types of bio-related liposomes based on the composition of DPPC or C16-SM with cholesterol (1: 1) has been proposed using the method of reverse phase evaporation with ultrasound treatment; the control parameters of their technology have been determined. Liposomes based on C16-SM interact more intensively with the 7-AMK fluorophore of the compound R-203 in the buffer.