Synthesis and characterization of beta-sitosterol liposomal nanoparticles: initial approach

Abstract

INTRODUCTION. Beta-sitosterol (BS) phytotherapy is gaining significant interest as a sustainable therapeutic alternative for the management of human cancer. Its development has been limited by its low aqueous solubility. Before establishing its anticancer potential in humans or in murine models of osteosarcoma, we sought to increase its bioavailability in an aqueous medium by using it as a liposomal BS. OBJECTIVE. To develop and characterize liposomal nanoparticles with BS. METHODOLOGY. We prepared liposomal nanoparticles using the thin lipid layer hydration method. The phospholipids dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (PPCP) were mixed in a chloroform-methanol flask with BS in a ratio of 2:8:1 (formulation L1) and with BS and cholesterol in a ratio of 2:8:0.5:0.5 (formulation L2). The solvent was evaporated in a rotary evaporator at 40°C. The resulting thin lipid layers at the bottom of the flask were suspended in water or PBS, sonicated, and finally extruded in 15 passes through 200 nm filters. The morphology and dimensions of the nanoparticles were measured by transmission electron microscopy (TEM); By dynamic light scattering (DLS), their hydrodynamic diameters (Dh, nm), polydispersity indices (PDI), and electrostatic stability or zeta potential (PZ, mV) were obtained; the concentrations of encapsulated BS in liposomal formulations L1 and L2 were obtained by reversed-phase isocratic high-pressure liquid chromatography (HPLC - stationary phase/C18; mobile phase/100% methanol; flow rate: 0.4 mL/min; 40°C; 210 nm) of the nanoparticles lysed in ethanol. RESULTS. There was agreement between the measurements obtained by TEM and their Dh obtained by DDL for the suspensions diluted 1:10 in water or PBS (n=3): for L1/water they fluctuated in a range of 122.8 - 142.8 nm and 123.4 – 174.1 nm, for L1/PBS, while the dimensions of the L2/water nanoparticles fluctuated between 103.5 – 194.4 nm and those of L2/PBS between 103.5 – 184.2 nm. The particle size and IPD values ​​of all liposomal preparations measured in water or PBS (0.198 - 0.474), correspond to the values ​​reported in the literature for this type of particles. However, the colloidal stability (CSS) of most liposomal preparations was well below the expected ±15 mV, ranging from -4.45 to -12.4 mV and -1.40 to -2.87 mV for the L1/water and L1/PBS liposomal formulations, respectively; for the L2/water and L2/PBS formulations, their aggregation tendency ranged from -1.60 to -11.4 mV and from -1.60 to 3.80 mV, respectively. We measured BS incorporation into six liposomal preparations by CLAP: their retention times ranged from 2.510 to 2.828 min; the average drug encapsulation efficiency was only 2.36%. CONCLUSIONS. Our preparations reached the expected particle size for this type of liposomal formulations, although their colloidal stability (PZ) and BS encapsulation value should be optimized.