Energy-dependent endocytosis is responsible for drug transcorneal penetration following the instillation of ophthalmic formulations containing indomethacin nanoparticles
Authors Nagai N, Ogata F, Otake H, Nakazawa Y, Kawasaki N
Received 1 December 2018
Accepted for publication 22 January 2019
Published 18 February 2019 Volume 2019:14 Pages 1213—1227
Checked for plagiarism Yes
Review by Single-blind
Peer reviewers approved by Dr Govarthanan Muthusamy
Peer reviewer comments 2
Editor who approved publication: Dr Thomas Webster
Noriaki Nagai,1 Fumihiko Ogata,1 Hiroko Otake,1 Yosuke Nakazawa,2 Naohito Kawasaki1
1Faculty of Pharmacy, Kindai University, Higashi-Osaka 577-8502, Japan; 2Faculty of Pharmacy, Keio University, Minato-ku, Tokyo 105-8512, Japan
Purpose: We previously found that ophthalmic formulations containing nanoparticles prepared by a bead mill method lead to an increase in bioavailability in comparison with traditional formulations (solution type). However, the transcorneal penetration pathway for ophthalmic formulations has not been explained yet. In this study, we investigated the mechanism of transcorneal penetration in the application of ophthalmic formulations containing indomethacin nanoparticles (IMC-NPs).
Materials and methods: IMC-NPs was prepared by the bead mill method. For the analysis of energy-dependent endocytosis, corneal epithelial (HCE-T) cell monolayers and removed rabbit cornea were thermoregulated at 4°C, where energy-dependent endocytosis is inhibited. In addition, for the analysis of different endocytosis pathways using pharmacological inhibitors, inhibitors of caveolae-mediated endocytosis (54 µM nystatin), clathrin-mediated endocytosis (40 µM dynasore), macropinocytosis (2 µM rottlerin) or phagocytosis (10 µM cytochalasin D) were used.
Results: The ophthalmic formulations containing 35–200 nm sized indomethacin nanoparticles were prepared by treatment with a bead mill, and no aggregation or degradation of indomethacin was observed in IMC-NPs. The transcorneal penetration of indomethacin was significantly decreased by the combination of nystatin, dynasore and rottlerin, and the decreased penetration levels were similar to those at 4°C in HCE-T cell monolayers and rabbit cornea. In the in vivo experiments using rabbits, dynasore and rottlerin tended to decrease the transcorneal penetration of indomethacin (area under the drug concentration – time curve in the aqueous humor [AUCAH]), and the AUCAH in the nystatin-treated rabbit was significantly lower than that in non-treatment group. In addition, the AUCAH in rabbit corneas undergoing multi-treatment was obviously lower than that in rabbit corneas treated with each individual endocytosis inhibitor.
Conclusion: We found that three energy-dependent endocytosis pathways (clathrin-dependent endocytosis, caveolae-dependent endocytosis and macropinocytosis) are related to the transcorneal penetration of indomethacin nanoparticles. In particular, the caveolae-dependent endocytosis is strongly involved.
Keywords: drug delivery system, bead mill, caveolae-dependent endocytosis, clathrin-dependent endocytosis, macropinocytosis
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