3.5. Solid Lipid Nanoparticles Preparation

The lipid phase, containing the solid lipid cetylpalmitate (Gattefossé, Lyon, France) (500 mg) and the stabilizer polysorbate 80 (Tween^® 80 Sigma-Aldrich) (150 mg) was melted at 70 °C, which is above the lipid’s melting point (Table 5). The melted lipid was then dispersed in ultrapure water (4.35 mL) (Milli-Q RG) at the same temperature by high-speed stirring in an Ultra-Turrax T25 (Janke and Kunkel IKA-Labortechnik, Staufen, Germany) followed by sonication (70% amplitude) using a Vibra-Cell™ CV18 (15 min, Sonics and Materials, Newtown, CT, USA). The nanoemulsion was left to cool at room temperature to allow the crystallization of the lipid and consequent formation of the solid lipid nanoparticles [19,44].

Chemical structure of the lipids used in the solid lipid nanoparticles synthesis.

The final parameters chosen were a high shear homogenization of 2 min at 12,000 rpm and a 15 min sonication at an intensity of 70% (the parameters of both techniques were previously optimized to establish the best conditions for the production of a stable formulation with an average size of less than 200 nm) [19,43].

For the nanoparticles conjugated with mAb, 0.5 mg of DSPE-PEG₂₀₀₀-maleimide (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] ammonium salt, Avanti Polar Lipids, Alabaster, AL, USA) and 0.5 mg of LissRhod-PE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) ammonium salt, Avanti Polar Lipids) was added to the lipid phase and then melted at 70 °C.

The particle size and surface charge (zeta potential) of the NPs suspensions were analyzed using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK). The SLNs size measurements were based upon photon correlation spectroscopy. With this technique the hydrodynamic diameter and the polydispersity index (PDI), which is a dimensionless measure for the broadness of the particle size distribution, were obtained. The zeta potential was analyzed by laser Doppler velocimetry using the same instrument.