Energy-dependent endocytosis is responsible for drug transcorneal penetration following the instillation of ophthalmic formulations containing indomethacin nanoparticles

Noriaki Nagai, Fumihiko Ogata, Hiroko Otake, Yosuke Nakazawa, Naohito Kawasaki

Research output: Contribution to journalArticle

Abstract

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 [AUC AH ]), and the AUC AH in the nystatin-treated rabbit was significantly lower than that in non-treatment group. In addition, the AUC AH 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.

Original languageEnglish
Pages (from-to)1213-1227
Number of pages15
JournalInternational journal of nanomedicine
Volume14
DOIs
Publication statusPublished - 2019 Jan 1

Fingerprint

Endocytosis
Indomethacin
Nanoparticles
Nystatin
Pharmaceutical Preparations
T-cells
Rabbits
Caveolae
Cornea
Clathrin
Monolayers
Area Under Curve
Cytochalasin D
Agglomeration
T-Lymphocytes
Degradation
Aqueous Humor
Phagocytosis
Biological Availability
Experiments

Keywords

  • Bead mill
  • Caveolae-dependent endocytosis
  • Clathrin-dependent endocytosis
  • Drug delivery system
  • Macropinocytosis

ASJC Scopus subject areas

  • Biophysics
  • Bioengineering
  • Biomaterials
  • Pharmaceutical Science
  • Drug Discovery
  • Organic Chemistry

Cite this

Energy-dependent endocytosis is responsible for drug transcorneal penetration following the instillation of ophthalmic formulations containing indomethacin nanoparticles. / Nagai, Noriaki; Ogata, Fumihiko; Otake, Hiroko; Nakazawa, Yosuke; Kawasaki, Naohito.

In: International journal of nanomedicine, Vol. 14, 01.01.2019, p. 1213-1227.

Research output: Contribution to journalArticle

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abstract = "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 [AUC AH ]), and the AUC AH in the nystatin-treated rabbit was significantly lower than that in non-treatment group. In addition, the AUC AH 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.",
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AU - Otake, Hiroko

AU - Nakazawa, Yosuke

AU - Kawasaki, Naohito

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N2 - 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 [AUC AH ]), and the AUC AH in the nystatin-treated rabbit was significantly lower than that in non-treatment group. In addition, the AUC AH 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.

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