2.3. Preparation of Lipid Nanoparticles

Tobramycin-loaded nanoparticles were prepared as described. Briefly, two loaded formulations were elaborated, namely solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) [21]. An emulsion solvent evaporation technique was chosen for the preparation of SLN. Briefly, 10 mg of antibiotic (Sigma-Aldrich, St. Louis, MO, USA) were mixed with a 5% (w/v) Precirol^® ATO 5 (Gattefossé, Madrid, Spain) dichloromethane solution. Then, the organic phase and an aqueous surfactant containing solution (Poloxamer 188 at 1% w/v and Polysorbate 80 at 1% w/v) were mixed and emulsified by sonication at 20 W for 30 s (Branson Sonifier 250, Danbury, CT, USA). The solvent was allowed to evaporate by magnetic stirring for 2 h at room temperature. Subsequently, the resulting SLNs were washed by centrifugation in Amicon^® centrifugal filtration units (100,000 MWCO, Merck Millipore, Billerica, MA, USA) at 2500 rpm for 15 min three times. For the NLC elaboration, a hot melt homogenization technique was selected. In brief, Precirol^® ATO 5 and Miglyol^® 812 (Sasol, Johannesburg, South Africa) were selected as the lipid core. Those lipids were mixed with the API and heated above the melting temperature of the solid lipid. The surfactant solution consisted of 1.3% (w/v) of Polysorbate 80 and 0.6% (w/v) of Poloxamer 188. The lipid and aqueous solutions were heated to the same temperature and then emulsified by sonication for 15 s at 20 W. Nanoparticles were stored at 4 °C overnight to allow lipid re-crystallization and particle formation. Then, a washing step was undergone by centrifugation at 2500 rpm in Amicon^® centrifugal filtration units (100,000 MWCO) three times. All the nanoparticles prepared were freeze-dried with two different cryoprotectants, either d-mannitol or trehalose (15%). In SLN formulations, emulsifiers constituted the aqueous phase of the emulsions, stabilizing the lipid dispersion of the nanoparticles and preventing their agglomeration [22]. Thus, the influence of the emulsifier on the bioactivity of the lipid nanoparticles was examined in two different types of SLN. SLN-tobramycin nanoparticles were prepared using the emulsifiers poloxamer 188 and polysorbate 80, each at 1% w/v. SLN-SDS-tobramycin nanoparticles were prepared using 2% sodium dodecyl sulfate (SDS) as the co-emulsifier. NLCs loaded with tobramycin (NLC-tobramycin) were prepared using a hot melt homogenization technique, following the method described by Pastor et al. [21].

All three types of nanoparticles used in this work (SLN-tobramycin, SLN-SDS-tobramycin, and NLC-tobramycin) were stabilized by trehalose, since in previous research we determined that it was a better cryoprotectant than mannitol [20]. Solid Lipid Nanoparticles and Nanostructured lipid carriers were characterized for size, polidispersity index (PDI) and Z-potential by means of Zetaseiser Nano ZS (Malvern Instruments, Worcestershire, UK). Measurements were based on Dynamic Light Scattering (DLS). Atomic force microscopy images were obtained by using a XE-70 atomic force microscope (Park Systems, Suwon, Korea).