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

Figures & data

Figure 1 Particle size frequencies and solubility of indomethacin in IMC-NPs.

Notes: Particle size frequencies of indomethacin in IMC-MPs (A) and IMC-NPs (B) as determined by a laser diffraction particle size analyzer. (C) Particle size frequencies of indomethacin in IMC-NPs by the dynamic light scattering method. (D) AFM image of indomethacin in IMC-NPs by the SPM-9700. (E) Solubility of indomethacin in IMC-NPs. n=8. *P<0.05 vs IMC-MPs. The ophthalmic formulation containing 35–200 nm sized indomethacin nanoparticles was prepared by treatment with a bead mill. The drug solubility in IMC-MPs and IMC-NPs was higher than that in IMC-MPs at 84.5 µM and 363.2 µM, respectively.

Abbreviations: IMC-MPs, ophthalmic formulation containing indomethacin microparticles; IMC-NPs, ophthalmic formulation containing indomethacin nanoparticles.

Figure 2 Stability of IMC-NPs 14 days after bead mill treatment.

Notes: Particle size frequencies (A) and AFM image (B) of indomethacin in IMC-NPs by the dynamic light scattering method and SPM-9700, respectively. Changes in concentration (C), particle number (D) and dispersibility (E) of indomethacin in IMC-NPs. n=8. No aggregation or degradation of indomethacin was observed in IMC-NPs, and the size of the indomethacin particles in IMC-NPs remained in the nano order for 14 days.

Abbreviations: IMC-NPs, ophthalmic formulation containing indomethacin nanoparticles; con, concentration.

Figure 3 Transepithelial penetration of indomethacin in IMC-NPs at 4°C and 37°C using HCE-T cell monolayers.

Notes: (A) Changes of TER in HCE-T cell monolayers treated with IMC-NPs 4°C and 37°C. Accumulation (B) and penetration (C) of indomethacin in HCE-T cells treated with IMC-solution, IMC-MPs and IMC-NPs, at 4°C and 37°C. Number (D) and size frequencies (E) of indomethacin nanoparticles in the basolateral side at 4°C conditions. n=6. *P<0.05 vs 37°C conditions for each category. ^#P<0.05 vs IMC solution at 37°C conditions. The accumulation of indomethacin tended to be less at 4°C, and transepithelial penetration was significantly prevented at 4°C.

Abbreviations: IMC-MPs, ophthalmic formulation containing indomethacin microparticles; IMC-NPs, ophthalmic formulation containing indomethacin nanoparticles; con, concentration; TER, transepithelial electrical resistance.

Figure 4 Effect of endocytosis pathways on transepithelial penetration of IMC-NPs in HCE-T cell monolayers.

Notes: Effects of endocytosis inhibitors on the TER (A), accumulation (C) and penetration (E) of indomethacin in HCE-T cell monolayers treated with IMC-NPs. Changes in TER (B), accumulation (D) and penetration (F) of indomethacin in HCE-T cell monolayers treated with IMC-NPs by multi-treatment with nystatin, dynasore and rottlerin (Nys+Dyn+Rot). (G and H) Effect of endocytosis inhibitors on the number of indomethacin nanoparticles in the basolateral side. n=5–8. *P<0.05 vs vehicle for each category. The penetration of indomethacin was significantly decreased by the combination of nystatin, dynasore and rottlerin.

Abbreviations: IMC-NPs, ophthalmic formulation containing indomethacin nanoparticles; con, concentration.

Table 1 Pharmacokinetic analysis of the penetration of IMC-NPs in rabbit corneas treated at cold temperature (4°C) or with endocytosis inhibitors

	Jc (nmol/cm^2/h)	Kp 10^−4 (× /h)	Km (×10^−2)	τ (h)	D (×10^−4 cm^2/h)
Normal (37°C treatment)	150.6±16.2	109.8±9.7	50.8±5.3	0.51±0.03	123.2±11.8
4°C treatment	35.3±3.1*,#	28.1±2.2*,#	10.5±1.6*,#	0.92±0.10*,#	98.7±9.5*,#
Vehicle	158.4±14.7	113.3±10.7	52.4±4.8	0.48±0.04	135.2±13.1
Nystatin	88.8±8.2*,#	63.5±6.8*,#	35.0±3.9*,#	0.57±0.06	113.5±12.7
Dynasore	111.3±9.9*,#	85.3±7.3*,#	47.0±4.3	0.52±0.05	125.4±11.9
Rottlerin	111.6±9.9*,#	85.6±7.1*,#	56.8±5.3	0.54±0.07	147.7±9.1
Cytochalasin D	131.6±12.6	94.2±9.0	50.4±2.9	0.54±0.03	143.7±16.3
Nys + Dyn + Rot	40.1±4.1*,#	36.3±3.2*,#	15.9±1.5*,#	0.89±0.09*,#	99.5±9.9*,#

Notes: Parameters were calculated according to Equations 1$J_c=\frac{K_m\cdot D\cdot C_{\text{IMC}}}{\delta }=K_{\mathrm{p}}\cdot C_{\text{IMC}}$(1) –3$Q_{\mathrm{t}}=J_{\mathrm{c}}\cdot A\cdot \left(t-\tau \right)$(3) (see Materials and methods). The experiments were performed at normal (37°C) and cold (4°C) temperatures. In the study using endocytosis inhibitors, the corneal samples were co-treated with IMC-NPs and inhibitors (0.5% DMSO [vehicle], 54 µM nystatin, 40 µM dynasore, 2 µM rottlerin or 10 µM cytochalasin D). Nys + Dyn + Rot indicates the multi-treated groups, which were treated with 54 µM nystatin, 40 µM dynasore and 2 µM rottlerin. n=5–8.

* P<0.05, vs Normal for each category.

# P<0.05, vs Vehicle for each category.

Abbreviations: D, diffusion constant within the cornea; IMC-NPs, ophthalmic formulation containing indomethacin nanoparticles; J_c, indomethacin penetration rate; K_m, cornea/preparation partition coefficient; K_p, penetration coefficient through the cornea; τ, lag time.

Figure 5 Effect of endocytosis pathways on in vitro transcorneal penetration of IMC-NPs in rabbit corneas.

Notes: (A) Penetration profile of indomethacin in IMC-NPs at 4°C and 37°C. (B) AUC_penetration of indomethacin in IMC-NPs at 4°C and 37°C. Penetration profile (C) and AUC_penetration (D) of indomethacin in IMC-NPs by treatment with individual endocytosis inhibitors. Penetration profile (E) and AUC_penetration (F) of indomethacin in IMC-NPs by multi-treatment with nystatin, dynasore and rottlerin (Nys+Dyn+Rot). n=5–8. *P<0.05 vs 37°C. ^#P<0.05 vs vehicle for each category. AUC_penetration was significantly decreased by the combination of nystatin, dynasore and rottlerin, and the decreased AUC_penetration levels were similar to those at 4°C in rabbit cornea.

Abbreviations: AUC_penetration, area under the drug concentration–time curve in the reservoir chamber; con, concentration; IMC-NPs, ophthalmic formulation containing indomethacin nanoparticles.

Figure 6 Effect of endocytosis pathways on in vivo transcorneal penetration of IMC-NPs in rabbit corneas.

Notes: Intraocular behavior (A) and AUC_AH (B) of indomethacin in IMC-NPs by treatment with individual endocytosis inhibitors. Intraocular behavior (C) and AUC_AH (D) of indomethacin in IMC-NPs by multi-treatment with nystatin, dynasore and rottlerin (Nys+Dyn+Rot). n=4–6. *P<0.05 vs control for each category. Dynasore and rottlerin tended to decrease the AUC_AH. On the other hand, nystatin significantly prevented transcorneal penetration. In addition, the AUC_AH in rabbit cornea with multi-treatment was obviously lower than that in rabbit cornea treated with each individual endocytosis inhibitor.

Abbreviations: AUC_AH, area under the drug concentration–time curve in the aqueous humor; con, concentration; IMC-NPs, ophthalmic formulation containing indomethacin nanoparticles.

Figure 7 Mechanism for corneal penetration after the instillation of IMC-NPs.

Abbreviations: CavME, caveolae-dependent endocytosis; CME, clathrin-dependent endocytosis; IMC-NPs, ophthalmic formulation containing indomethacin nanoparticles; MP, macropinocytosis.