Formulation and Characterization of NLC Nanocarriers

Nanostructured lipid carrier (NLC) formulation was produced as follows^{38 (link),39 (link),44 (link)}. Trimyristin (Sigma-Aldrich, St. Louis, MO), squalene (SAFC Supply Solutions, St. Louis, MO), sorbitan monostearate (Spectrum Chemical Mfg. Corp., New Brunswick, NJ), and the cationic lipid DOTAP (Lipoid, Ludwigshafen, Germany) were mixed and heated at 60 °C in a bath sonicator to create the oil phase. Polysorbate 80 (MilliporeSigma, Burlington, MA) was separately diluted in 10 mM sodium citrate trihydrate and heated to 60 °C in a bath sonicator to create the aqueous phase. After dispersion of components in each phase, a high-shear mixer (Silverson Machines, East Longmeadow, MA) was used at ~5000 rpm to mix the oil and aqueous phases. Particle size of the mixture was then further decreased by high-pressure homogenization by processing at 30,000 psi for ten discrete passes using an M110P Microfluidizer (Microfluidics, Westwood, MA). The NLC product was then filtered through a 0.22 µm PES filter and stored at 2–8 °C until use.