*3.5. Immunohistofluorescence Biodistribution*

Confocal laser microscopy is usually employed to study the biodistribution of nanoparticles in different tissues; unfortunately, this is an indirect measurement, as there is no direct tracking of the API. There exist other techniques to study topical biodistribution, such as skin layer separation [34], cyanoacrylate tape-stripping [35], or dermatopharmacokinetics [36]. Techniques such as cyanoacrylate-stripping allow to study hair follicle targeting but usually have a lower spatial resolution than IHF and could have problems, such as analytical interferences, quantification limit issues, lack of complete recovery, or matrix effects. By means of the IHF, it is possible to visualize if there is accumulation in the different anatomical regions of the hair follicle.

Due to this and to confirm the data obtained in the previous section, an IHF experiment was carried out. Rabbit anti-DEX IgG and Alexa Fluor 488 goat anti-rabbit were employed, such that direct visualization of the drug could be performed.

After the IHQ experiment, a different behavior was observed, with respect to the DEX-FREE (Figure 6C,D) and LPNCs-vehiculated DEX (Figure 6A,B). The visualization filter used in ImageJ was Green Fire Blue, where the blue color corresponds to the lowest intensity and the yellow-green color to the highest intensity. Figure 6B shows a longitudinal section of the hair follicle. In panels A, C, and D of Figure 6, cross-sections of the hair follicles are observed. Surface plots were obtained, in order to compare the intensity in a more visual way.

In the same way as in Section 3.6, different lines were drawn to compare the intensity profiles. Line 1 in Figure 6A corresponds to the intensity across the lateral zone of a hair follicle. In this case, an accumulation of DEX was seen (intensity around 140 AU at 500 µM depth). Line 2 corresponds to permeation outside the follicle, for which it was observed that DEX remained restricted to the epidermis and its invaginations, with an intensity of 60 AU. In Figure 6B, three lines are drawn. Lines 1 and 2 correspond to the central zone and the outer sheath of the follicle, respectively. Although the maximum intensity (approximately 100 AU) was observed at around 250 µm, DEX accumulation went to 700 µm, corresponding to the hair follicle bulb (Figure 6B, Zoom 1 and 2). Line 3 corresponds to an area outside the follicle; in this case, the intensity dropped to 25 AU when entering beyond 100 µm and, again, it was observed that DEX remained restricted to the area of the epidermis and stratum corneum. The intensity along the follicle remained around 40 AU—almost double compared to outside the follicle. Surface plots of both images were also made, to obtain a visual image of the accumulation of DEX. It is clear that accumulation in hair follicles has occurred.

Regarding the permeation of FREE-DEX, the behavior was different. Lines 1 and 2 of Figure 6C,D are very similar and show that DEX did not accumulate in hair follicles when it was formulated in this solution. Beyond 50 µm in depth (epidermis), the intensity was low (about 20 AU). This behavior is shown in the surface plots in Figure 6C Zoom 3 and Figure 6D Zoom 3, where FREE-DEX did not accumulate in follicles, only in the stratum corneum and part of the epidermis.

**Figure 6.** IHF images of a pig skin cross-section. Green color corresponds to high intensity and blue to lower intensity fluorescence of Alexa 488 secondary antibody (Ab)–Primary Ab–DEX complex: (**A**,**B**) DEX-loaded LPNCs; (**C**,**D**) hydroalcoholic DEX control solution. Figures 6A 1-2, Figure 6B 1-3, Figure 6C 1-2 and Figure 6D 1-2 correspond to green intensity plot profiles as function of the depth (µm). Figure 6A 3, Figure 6B 4, Figure 6C 3 and Figure 6D 3 correspond to surface plots. The images were captured using 10× magnifications and the Lookup Table (LUT) used to show the pictures was Green Fire Blue. **Figure 6.** IHF images of a pig skin cross-section. Green color corresponds to high intensity and blue to lower intensity fluorescence of Alexa 488 secondary antibody (Ab)–Primary Ab–DEX complex: (**A**,**B**) DEX-loaded LPNCs; (**C**,**D**) hydroalcoholic DEX control solution. Figure 6A 1–2, Figure 6B 1–3, Figure 6C 1–2 and Figure 6D 1–2 correspond to green intensity plot profiles as function of the depth (µm). Figure 6A 3, Figure 6B 4, Figure 6C 3 and Figure 6D 3 correspond to surface plots. The images were captured using 10× magnifications and the Lookup Table (LUT) used to show the pictures was Green Fire Blue.

When comparing these results with those obtained by confocal microscopy, it can be observed that the biodistribution of DEX was similar to that of LRB, as it accumulated in the deep areas of the follicles (>400 µm). These results corroborate the high encapsulation efficiency results and suggest that it is possible to achieve a controlled and sustained release in the hair follicle by encapsulation in LPNCs. As a result, a depot effect in the pilosebaceous unit is expected. This effect could, on the one hand, reduce the frequency of administration (from the 12 h usually used in topical corticosteroids to 24 h) and then could reduce the associated adverse effects. On the other hand, a depot effect and the release of the drug in the site of action would improve the treatment efficacy and reduce or avoid the systemic or intralesional administration of the drug, reducing the systemic adverse effects (which are usually worse than the adverse effects of topical administration) and patient discomfort caused by injections (in the intralesional therapy). These facts should be evaluated in future clinical trials.
