2.2. Preparation of 150 nm Solid Lipid Nanoparticles Using Emulsion/Solvent Evaporation

Solid lipid nanoparticles (150 nm) were prepared using an emulsion/solvent evaporation method modified from Naguib et al. [11]. The organic phase was prepared by dissolving 40 mg stearic acid and 30 mg soy lecithin in 1 mL dichloromethane containing 100 μg Nile Red. This organic phase was added directly to the aqueous phase (10 mL containing 1% w/v poloxamer 188 and 50 μL butyric acid), and an ultrasonic probe sonicator (Model 100, Fisher Scientific, Pittsburgh, PA, USA) operated at 75% amplitude for 6 min was used to form the SLNs. The dispersion was kept in an ice bath during SLN formation and then moved to a water bath (48 ± 2 °C) for 15 min with stirring at ~300 rpm. Finally, the dispersion was placed in a fume hood at room temperature with stirring at ~200 rpm for one hour to ensure complete dichloromethane evaporation. The dispersion was filtered through Whatman^® filter paper (# 541, Global Life Sciences Solutions, Pittsburgh, PA, USA) under vacuum to remove any large, non-emulsified solids. This was followed by filtration through a 0.45 μm syringe filter (mixed cellulose esters membrane (SLHA033SS), Merck Millipore Ltd., Carrigtwohill, Ireland). The filtered dispersion was combined with 5 mL Nanopure^® water and placed in an Amicon^® Ultra-15 centrifugal filter unit (Merck Millipore Ltd., Ireland) and centrifuged (Eppendorf (Model 5810R), Hauppauge, Suffolk County, NY, USA) at 500× g for 45 min at 4 °C to separate free dye and excess surfactant from the particles. The solid lipid nanoparticle suspension was filtered through a 0.22 μm syringe filter (MCE, Merck Millipore Ltd., Ireland) to remove any aggregated and/or larger particles.