Energy‐dependent endocytosis is responsible for skin penetration of formulations based on a combination of indomethacin nanoparticles and l‐menthol in rat and göttingen minipig

We previously designed a Carbopol gel formulation (N‐IND/MEN) based on a combination of indomethacin solid nanoparticles (IND‐NPs) and l‐menthol, and we reported that the N‐IND/MEN showed high transdermal penetration. However, the detailed mechanism for transdermal penetration of IND‐NPs was not clearly defined. In this study, we investigated whether endocytosis in the skin tissue of rat and Göttingen minipig is related to the transdermal penetration of IND‐NPs using pharmacological inhibitors of endocytosis. The pharmacological inhibitors used in this study are as follows: 54 μM nystatin, a caveolae‐mediated endocytosis (CavME) inhibitor; 40 μM dynasore, a clathrin‐mediated endocytosis (CME) inhibitor; and 2 μM rottlerin, a micropinocytosis (MP) inhibitor. The N‐IND/MEN was prepared by a bead mill method, and the particle size of solid indomethacin was 79–216 nm. In both rat and Göttingen minipig skin, skin penetration of approximately 80% IND‐NPs was limited by the stratum corneum (SC), although the penetration of SC was improved by the combination of l‐menthol. On the other hand, the treatment of nystatin and dynasore decreased the transdermal penetration of indomethacin in rats and Göttingen minipigs treated with N‐IND/MEN. Moreover, in addition to nystatin and dynasore, rottlerin attenuated the transdermal penetration of IND‐NPs in the Göttingen minipigs’ skin. In conclusion, we found that l‐menthol enhanced the SC penetration of IND‐NPs. In addition, this study suggests that the SC‐passed IND‐NPs are absorbed into the skin tissue by energy‐dependent endocytosis (CavME, CME, and/or MP pathways) on the epidermis under the SC, resulting in an enhancement in transdermal penetration of IND‐NPs. These findings provide significant information for the design of nanomedicines in transdermal formulations.