*3.6. Cytotoxicity and Anti-TNFα Efficacy*

Human-transformed keratinocytes (HEK001 cells) were chosen to assess the DEX-LPNC cytotoxicity (Figure 7A). Non-loaded LPNCs, benzalkonium chloride, and untreated cells were tested as controls. For loaded LPNCs, the inverse dilution factors (5000, 25,000, 50,000, 100,000, and 250,000) corresponded to the respective concentrations of 5, 1, 0.5, 0.25, and 0.1 µM of DEX. For benzalkonium chloride, proper concentrations were 1, 0.2, 0.1, 0.05, and 0.02 µM. The cell viability profiles of the tested compounds exhibited a similar pattern: there was a viability reduction, compared to untreated cells, at higher concentrations of loaded and non-loaded LPNCs. To ascertain the reason for this toxicity, pure benzalkonium chloride was evaluated with the same dilution scheme as the LPNCs, demonstrating that the reason for the viability reduction was caused by the preservative. Although this fact was observed, there exist different commercial products that use the same preservative for the topical route, as described in the FDA inactive ingredients data base [37], demonstrating the suitability of the selected preservative system.

To evaluate the in vitro anti-inflammatory efficacy of the LPNCs, TNFα was selected as a tracker, as it is usually involved in most inflammatory alterations of the skin, particularly in AA [38]. Furthermore, in many cases, it has been identified as a promising target for pharmacological modulation [39]. Figure 7B shows the TNFα expression in HEK001 cells after treatment with DEX-LPNCs and FREE-DEX for 24 h after pre-treatment with 10 µg/mL of LPS to induce inflammation. LPNCs were tested at a DEX level equivalent to 0.1 µM, as the cell viability was not reduced at this concentration level. FREE-DEX at the same concentration level was used as control. A significant reduction in TNFα can be seen in both cases. The lower efficacy of the lipomers, compared with the solution, could be caused by the differential release pattern observed in Figure 4: at 24 h, the amount of DEX released from the lipomers was around 75%, compared with 100% in the solution. Similarly, the difference between the FREE-DEX and lipomers was about 27%, such that the drug release limited the anti-TNFα efficacy. This difference is not expected to appear in real applications, considering that the LPNCs have accumulated in hair follicles and, so, a depot effect would appear (not evaluated in cell culture), such that the slow degradation of the lipomers in the follicle would release the drug into the surrounding and deeper area. After multiple administrations, an increased exposition of DEX would take place, compared with the free drug.

**Figure 7.** In vitro cell culture studies: (**A**) HEK001 cell viability with MTT assay. The indicated numbers represent the inverse dilution factors, referring to the composition of synthetized LPNCs. For DEX-loaded LPNCs, the dilution factors correspond to the concentrations of 5, 1, 0.5, 0.25, and 0.1 µM of DEX. For benzalkonium chloride, proper concentrations were 1, 0.2, 0.1, 0.05, and 0.02 µM. Data are represented as the mean ± SEM (*n* = 3) of the cell viability percentage, referring to untreated controls (horizontal lane). Statistical significance was assessed by Student's paired t-test; \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.005. (**B**) TNFα mRNA expression was measured after treating cells for 24 h with 0.1 µM of either free dexamethasone (grey) or DEX-loaded LPNCs (dark gray), without (left) or with (right) a 1-h pre-treatment with LPS (10 µg/mL). TNFα expression is represented as the mean ± SEM (*n* = 3); TNFα expression at control and LPS treatment conditions is indicated with horizontal pink and red lanes, respectively. Statistical significance was evaluated by one-way ANOVA compared to the control (\*) or LPS (+); \*\* *p* < 0.01, ++ *p* < 0.01, ++++ *p* < 0.001. To evaluate the in vitro anti-inflammatory efficacy of the LPNCs, TNFα was selected as a tracker, as it is usually involved in most inflammatory alterations of the skin, particularly in AA [38]. Furthermore, in many cases, it has been identified as a promising target for pharmacological modulation [39]. Figure 7B shows the TNFα expression in HEK001 **Figure 7.** In vitro cell culture studies: (**A**) HEK001 cell viability with MTT assay. The indicated numbers represent the inverse dilution factors, referring to the composition of synthetized LPNCs. For DEX-loaded LPNCs, the dilution factors correspond to the concentrations of 5, 1, 0.5, 0.25, and 0.1 µM of DEX. For benzalkonium chloride, proper concentrations were 1, 0.2, 0.1, 0.05, and 0.02 µM. Data are represented as the mean ± SEM (*n* = 3) of the cell viability percentage, referring to untreated controls (horizontal lane). Statistical significance was assessed by Student's paired t-test; \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.005. (**B**) TNFα mRNA expression was measured after treating cells for 24 h with 0.1 µM of either free dexamethasone (grey) or DEX-loaded LPNCs (dark gray), without (left) or with (right) a 1-h pre-treatment with LPS (10 µg/mL). TNFα expression is represented as the mean ± SEM (*n* = 3); TNFα expression at control and LPS treatment conditions is indicated with horizontal pink and red lanes, respectively. Statistical significance was evaluated by one-way ANOVA compared to the control (\*) or LPS (+); \*\* *p* < 0.01, ++ *p* < 0.01, ++++ *p* < 0.001.

#### cells after treatment with DEX-LPNCs and FREE-DEX for 24 h after pre-treatment with 10 **4. Conclusions**

µg/mL of LPS to induce inflammation. LPNCs were tested at a DEX level equivalent to 0.1 µM, as the cell viability was not reduced at this concentration level. FREE-DEX at the same concentration level was used as control. A significant reduction in TNFα can be seen in both cases. The lower efficacy of the lipomers, compared with the solution, could be caused by the differential release pattern observed in Figure 4: at 24 h, the amount of DEX LPNCs with a high carrying capacity were successfully developed for the active dexamethasone. When studying the biodistribution of the nanoparticles using confocal microscopy, accumulation in hair follicles and cutaneous annexes was observed, thus proving their ability to achieve follicular targeting. These results were confirmed for DEX biodistribution by immunofluorescence, where DEX-LPNCs demonstrated an increase in accumulation in hair follicles, compared to FREE-DEX. The cytotoxicity of the particles was studied, where toxicity (caused by the preservative, benzalkonium chloride) was observed only at high doses. The anti-inflammatory efficacy of DEX-LPNCs, with TNFα as a tracker, was demonstrated. Its high %LC and %EE, good physicochemical properties (size of 115 nm, low polydispersity, and Z-potential of +30 mV), sustained in vitro release profile, localized release in hair follicles, and anti-inflammatory efficacy make this nanoformulation a very interesting candidate to improve the efficacy and reduce the adverse effects of

corticosteroids for diseases in which there is inflammation in the hair follicles, such as alopecia areata. Thanks to the follicular targeting obtained, it could be possible to have a depot effect within the pilosebaceous unit, which could allow us to reduce the frequency of administration compared to classical formulations. The safety and efficacy profiles of the DEX-lipomers should be verified in clinical trials to compare side effects.

**Author Contributions:** Conceptualization, F.F.-C. and E.P.-R.; methodology, F.F.-C., E.P.-R. and S.P.- T.; software, E.P.-R. and A.M.-V.; validation, E.P.-R., F.F.-C.; formal analysis, E.P.-R., A.M.-V. and M.L.-R.; investigation, E.P.-R. and F.F.-C.; resources, F.F.-C. and S.P.-T.; data curation, E.P.-R., F.F.-C.; writing—original draft preparation, E.P.-R.; writing—review and editing, E.P.-R., F.F.-C., A.M.-V., S.P.-T. and M.L.-R.; visualization E.P.-R., F.F.-C., A.M.-V., S.P.-T. and M.L.-R.; supervision, F.F.-C. and S.P.-T.; project administration, E.P.-R. and F.F.-C.; funding acquisition, F.F.-C., E.P.-R. and S.P.-T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received funding from the Generalitat de Catalunya Industrial Doctorate program of the candidate Eloy Pena Rodíguez, expedient number 2019 DI 1989691.

**Institutional Review Board Statement:** Ethical review and approval were waived for this study, due to the pig skin used was obtained from an abattoir. No animals were sacrificed specifically to carry out this research.

**Informed Consent Statement:** Not applicable

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to intellectual property.

**Acknowledgments:** Assistance provided by Manel Bosch from the University of Barcelona Scientific-Technical service, and by Mari Carmen Moreno from Reig Jofre was greatly appreciated. The authors thank Marçal Pastor Anglada and Carlos Nieto Abad for the guidance and advice provided in this project.

**Conflicts of Interest:** F.F.-C., E.P.-R. and M.L.R. are employees of Reig Jofre. The authors are not involved in any commercial or marketing activities of the developed product. Other authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
