2.7.1. Confocal Microscopy Biodistribution of Fluorescent Probes

To study the biodistribution of LPNCs in the skin, confocal fluorescence microscopy was used. For this, C6 was encapsulated in the nanoparticles, to simulate the biodistribution of a hydrophobic small molecule, and LRB was used as fluorophore covalently bound to a lipid (palmitic acid) and included in the lipid core of the LPNCs, in order to track the nanoparticles. The labelled LRB-C6-LPNCs were purified using Visking dialysis tubing with a cut-off of 12,000–14,000 Dalton. The non-encapsulated fraction of both fluorophores was quantified by spectrophotometry (VICTOR Multilabel Plate Reader, Perkinelmer, Massachusetts, USA) and the *%EE* was calculated following Equation (1).

Full thickness pieces of pig skin were warmed and placed in Franz cells, according to Section 2.7. After 18 h of permeation, the diffusion surface was washed with PBS, cut with a scalpel into pieces of about 0.5 cm<sup>2</sup> , and fixed in 4% *w*/*w* paraformaldehyde solution for 5 min. Then, the skin samples were incubated in aqueous solutions of increasing sucrose concentration (5%, 15%, and 25% *w*/*w*) for 15 min in each solution. They were then placed in plastic molds and dipped in OCT to cut on a cryostat Leica CM 3050 S (Leica Biosystems, Barcelona, Spain) to a 50 µm thickness. The slices were collected on poly-lysine-coated

slides and washed with PBS and 0.05% Tween 20 (TPBS) for 5 min, to remove the OCT and permeabilize the samples. On the day of observation, sections were incubated with 15 µL of Hoescht solution (2 µg/mL) for 10 min and washed with TPBS to stain the cell nuclei. The samples were analyzed under a confocal microscope (Leica Microsystems, Wetzlar, Germany). The emission laser wavelengths were 570, 500, and 525 nm and the excitation wavelengths were 561, 488, and 405 nm for LRB, C6, and Hoescht, respectively. About 20 planes were obtained per image, separated by a 3 µm step. Composites of the different planes were created, in terms of the brightest point for each pixel, through the ImageJ tool Z-stack (ImageJ2 v2.35, National Institutes of Health, Bethesda, MD, USA). A skin blank was processed in the same way as the test samples, to quantify skin autofluorescence. The mean intensity was measured with the ImageJ software and was subtracted from the intensity of the red and green channels of the samples.
