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

Abstract

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.

Novel Sustained-Release Drug Delivery System for Dry Eye Therapy by Rebamipide Nanoparticles

“…Moreover, we found that drug supplementation from REB-NPs is sustained and that REB-NPs appears to provide a useful therapy for dry eye (Figure 7). There are many methods for preparing solid drug nanoparticles by break down and build up, and we also previously reported a break down-based method using special additives [12][13][14][15][16]. The selection of additives is important for preparing REB-NPs, and MC and HPβCD lead to an enhancement in the crushing force and dispersion stability, respectively [12][13][14][15][16].…”

“…Our previous study showed that bead mill treatment with MC allows a decreased particle size to the nano level, and that the addition of HPβCD prevents aggregation of the nanoparticles [12][13][14][15][16]. In addition, we also reported that carbopol is suitable as a base for dermal formulations [13].…”

“…Ophthalmic dispersions containing rebamipide nanoparticles were prepared following the previous reports [12][13][14][15][16]. Briefly, rebamipide powder (particle size 741 ± 12.7 nm) purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) was mixed with 2-hydroxypropyl-β-cyclodextrin (HPβCD, Nihon Shokuhin Kako Co., Ltd., Tokyo, Japan) and type SM-4 methylcellulose (MC, Shin-Etsu Chemical Co., Ltd., Tokyo, Japan) in distilled water, and the dispersions were milled at 5500 rpm for 1 min × 30 times using 0.1 mm zirconia beads and a Micro Smash MS-100R (TOMY SEIKO Co. Ltd., Tokyo, Japan).…”

“…Solid drug nanoparticles come with the added benefits of possible cellular targeting and improvement in cellular uptake and have been used widely as nanotechnology-based delivery systems. We previously designed solid nanoparticles created by a breakdown method (bead mill), and reported on their low toxicity and high transdermal penetration via endocytosis when used in nano-formulations [12][13][14][15][16]. It is expected that the application of solid nanoparticles to the eyelid may be a possible route to sustained drug supplementation to the ocular surface and provide a novel strategy for ophthalmic DDS.…”

Development of Sustained-Release Ophthalmic Formulation Based on Tranilast Solid Nanoparticles

“…In addition, the size distribution and number of nanoparticles in the TL-NPs-Gel were measured using a NANOSIGHT LM10 (QuantumDesign Japan, Tokyo, Japan) with the viscosity, wavelength and measurement time set to 1.27 mPa·s, 405 nm and 60 s, respectively. Atomic force microscopic (AFM) images were provided by a SPM-9700 (Shimadzu Corp., Kyoto, Japan) according to previous report [33].…”