*3.4. Confocal Microscopy Biodistribution of Fluorescent Probes*

After the permeation experiment, skin was observed under a confocal microscope, to evaluate the skin distribution of both fluorochromes. The literature has described a high number of fluorochromes that are included in nanoparticles for localization in the skin. Usually, the selection of the tracking compound is based on the aim of the experiment. In this case, C6 was selected as the drug model and LRD to track the nanocapsules. According to the structure of LRD, the hydrocarbon tail would be inside the lipid core and the rhodamine head would likely be located in the polymer shell or in the interphase lipid–polymer in the nanoparticle structure, as with Span 60. To improve visualization, cell nuclei were stained with Hoechst (blue color). The results obtained from the confocal biodistribution study are shown in Figure 5. The autofluorescence value in the red channel (corresponding to LRD) was 5.9 AU and that of the green channel (corresponding to C6) was 0.3 AU. These values were subtracted from the mean intensity of the samples. Using ImageJ software, linear segments were drawn to analyze the intensity profile as a function of depth (in µm). Yellow color corresponds to the co-localization of both fluorochromes.

**Figure 5.** Confocal Microscopy Fluorescence images and plot intensity profiles of pig skin cross-sections. Green color corresponds to C6 and red to LRB fluorescence. Yellow color corresponds to the overlap of C6 and LRB: (**A**,**B**) C6-loaded LRB-labelled LPNCs. (**C**) Free C6 and LRB control solution. Lines numbered 1-3 in Figure 5A; 1-2 in Figure 5B and 1-2 in Figure 5C correspond to Multichannel intensity plot profiles as function of the depth (µm). Dashed squares numbered 4- 5 in Figure 5A, 3-4 in Figure 5B and 3-4 in Figure 5C correspond to zoomed in regions. The images were captured using 10x magnification. **Figure 5.** Confocal Microscopy Fluorescence images and plot intensity profiles of pig skin cross-sections. Green color corresponds to C6 and red to LRB fluorescence. Yellow color corresponds to the overlap of C6 and LRB: (**A**,**B**) C6-loaded LRB-labelled LPNCs. (**C**) Free C6 and LRB control solution. Lines numbered 1–3 in Figure 5A; 1–2 in Figure 5B and 1–2 in Figure 5C correspond to Multichannel intensity plot profiles as function of the depth (µm). Dashed squares numbered 4–5 in Figure 5A, 3–4 in Figure 5B and 3–4 in Figure 5C correspond to zoomed in regions. The images were captured using 10× magnification.

> Figure 5A,B show the biodistribution patterns of the C6 and LRB fluorophores vehiculated in LPNCs (C6-LRB-LPNCs). Figure 5A,B show the biodistribution patterns of the C6 and LRB fluorophores vehiculated in LPNCs (C6-LRB-LPNCs).

In Figure 5A, three lines are drawn from the stratum corneum to deeper layers of the skin. Line 1 corresponds to the central section of the hair follicle, line 2 corresponds to the follicle edge (outer root sheath area), and line 3 corresponds to a non-follicle section. In the central section (line 1), the intensity of C6 is between 100 and 200 AU in the infundibulum (depth < 100 µm). At depths of 200 µm, the intensity of C6 drops to 50 AU and, as the depth increases, the intensity of the green channel continues to decrease, to about 20 AU at 400 µm (corresponding to the hair follicle isthmus). Beyond 500 µm, in depth (i.e., in the suprabulbar region), the intensity of C6 was low (<10AU), which seems to indicate that C6 does not accumulate in the deeper parts of the follicle. Regarding the intensity in the red color, corresponding to the presence of LRB, a different behavior can be observed. The profile of the center of the follicle (Figure 5A, Line 1) shows an intensity value around 50 AU from the skin surface to 700–800 µm deep. The profile of the border of the follicle (Figure 5A, Line 2) shows a similar profile, with the intensity of the LRB up to 700 µm being around 65 AU. Due to the narrowing of the follicle, the line is outside the follicle, around 1000 µm; however, near the follicle sheath (1200 µm), it can be seen that the intensity of the LRB was around 50 AU. In the non-follicle area (Figure 5A, Line 3), the C6 intensity profile was high to 200 µm in depth, but then decreased quickly, suggesting the accumulation of C6 in the epidermis. It can also be seen that the intensity of LRB outside the follicle at depths greater than 200 µm was clearly lower than that of the follicle lines. In the epidermis, a yellow color can be observed, corresponding to the co-localization of both fluorochromes in this region. In other words, LRB had accumulated throughout the follicle at depths greater than C6.

In Figure 5B, corresponding to another skin section of the permeation with C6-LRB-LPNCs, a section of the hair follicle can be seen. In this case, the section is not longitudinal but a cross-section. The intensity profiles of the different skin annexes and hair follicles followed the same biodistribution for both fluorophores as previously discussed. Accumulation of C6 and LRB was seen up to about 400–500 µm deep. At deeper follicle depths, only LRB accumulation was observed.

Different magnifications were made on sections of the hair follicle, in order to highlight the fluorochrome distribution in the infundibulum (Figure 5A, zoom 4) and in the isthmus area (Figure 5A, zoom 5). In the zooms of Figure 5B, zoom 3 and zoom 4 highlight the transversal hair follicle section with the different fluorochrome distribution previously described.

In Figure 5C, the zoom after permeation of the FREE-C6-LRB control solution can be observed. In this case, the observed biodistribution was different. As they were not delivered in nanoparticles, the fluorophores did not accumulate in the hair follicles. As can be seen in the profiles of lines 1 and 2 of Figure 5C, the intensity of C6 was around 20 AU in the epidermis and decayed at depths greater than 100 µm. The LRB intensity was around 20 AU throughout the entire zoom where you can see the dermis, as well as the hair follicle cross-section.

In addition, when analyzing zooms 3 and 4 of Figure 5C, it can be observed that the intensity of the yellow color in the follicles is almost non-existent, due to the nonaccumulation of C6 in the hair follicle. In zoom 5 of Figure 5C, the accumulation of C6 and LRB in the epidermis of a superficial invagination (<100 µm) can be seen.

These results explain the biodistribution of these two hydrophobic fluorophores carried in the LPNCs. The intensity of the green color, corresponding to the accumulation of C6, was greater in the epidermis and in the upper part of the hair follicle, and decreased with increasing depth in the skin. The images obtained by confocal microscopy seem to indicate that the in vitro skin penetration by C6 in the LPNCs accumulated on the epidermis and the hair follicle infundibulum, but did not penetrate into the deepest parts of hair follicles. This fact suggests that C6 is released when it comes into contact with the stratum corneum lipids, due to its hydrophobic nature, leading to greater accumulation in less deep areas.

The intensity in red color, corresponding to LRB, was less than that of C6 in the epidermis but greater than that of C6 when analyzing the profile deeper into the hair follicle. The fact that an accumulation of LRB was observed at these depths suggests that this fluorophore is found in the lipid nucleus of the nanoparticles and travels with them towards the bulb of the follicle. As described in the literature, ethyl cellulose polymer nanoparticles accumulate in skin annexes, due to their geometry and size, and can deliver small molecules to hair follicles through the transfollicular route, limiting distribution to the rest of the skin and to systemic circulation [33].
