Balance of Drug Residence and Diffusion in Lacrimal Fluid Determine Ocular Bioavailability in In Situ Gels Incorporating Tranilast Nanoparticles

Abstract

We previously designed ophthalmic formulations (nTRA) containing tranilast nanoparticles (Tra-NPs) with high uptake into ocular tissues. In this study, we used in situ gel (ISG) bases comprising combinations of pluronic F127 (F127) and methylcellulose (MC/F127), pluronic F68 (F68/F127), and Carbopol (Car/F127), and we developed in situ gels incorporating Tra-NPs (Tra-NP-incorporated ISNGs) such as nTRA-F127, nTRA-MC/F127, nTRA-F68/F127, and nTRA-Car/F127. Moreover, we demonstrated the therapeutic effect on conjunctival inflammation using lipopolysaccharide-induced rats. Each Tra-NP-incorporated ISNG was prepared by the bead mill method, the particle size was 40-190 nm, and the tranilast release and diffusion from formulation were nTRA > nTRA-F127 > nTRA-F68/F127 > nTRA-Car/F127 > nTRA-MC/F127. In the Tra-NP-incorporated ISNGs, the tranilast residence time in the lacrimal fluid, cornea, and conjunctiva was prolonged, although the C_max was attenuated in comparison with nTRA. On the other hand, no significant difference in conjunctival inflammation between non- and nTRA-F127-instilled rats was found; however, the nTRA-F68/F127, nTRA-Car/F127, and nTRA-MC/F127 (combination-ISG) attenuated the vessel leakage, nitric oxide, and tumor necrosis factor-α expression. In particular, nTRA-F68/F127 was significant in preventing the conjunctival inflammation. In conclusion, we found that the combination-ISG base prolonged the residence time of Tra-NPs; however, Tra-NP release from the formulation was attenuated, and the T_max was delayed longer than that in nTRA. The balance of drug residence and diffusion in lacrimal fluid may be important in providing high ocular bioavailability in formulations containing solid nanoparticles.

Keywords: in situ gelling system; nanoparticles; ophthalmic delivery; pluronic F-127; tranilast.

Figure 1

Particle size and atomic force microscopy (AFM) images of tranilast (Tra) in ophthalmic formulation. (A) and (B) Particle size frequencies of Tra in ophthalmic dispersions containing Tra-microparticles (A, mTRA) and Tra-nanoparticles (B, nTRA). The data were measured by SALD-7100. (C) Particle size frequencies of Tra-nanoparticles (Tra-NPs) in nTRA measured by NANOSIGHT LM10. (D) AFM images of Tra-NPs in nTRA. Compositions of mTRA and nTRA are shown in Table 1. The Tra particles in mTRA and nTRA were 15–300 µm and 40–190 nm, respectively. The difference in particle size in Figure 1B,C is due to the measuring method (Figure 1B, SALD-7100. Figure 1C, NANOSIGHT LM10).

Figure 2

Particle size frequencies and AFM images of Tra-NPs in nTRA-F127-L (A), nTRA-F127-H (B), nTRA-MC/F127-L (C), nTRA-MC/F127-H (D), nTRA-F68/F127-L (E), nTRA-F68/F127-H (F), nTRA-Car/F127-L (G), and nTRA-Car/F127-H (H). Compositions of each Tra-NP-incorporated ISNG shown in Table 1. The bar in the AFM image indicates 200 nm. The ISG bases were not affected by the particle size, and the particle size frequencies in each Tra-NP-incorporated ISNG were approximately 70–210 nm.

Figure 3

Changes in dispersion stability in each ophthalmic Tra formulation 1 month after preparation. (A) Images of dispersibility in each ophthalmic Tra formulation at 4 and 20 °C. (B–F) Effect of ISG bases on dispersion stability in the nTRA (B), nTRA-F127 (C), nTRA-MC/F127 (D), nTRA-F68/F127 (E), and nTRA-Car/F127 (F). The compositions of the ophthalmic Tra formulations are shown in Table 1. n = 5. * p < 0.05 vs. Tra con. immediately after preparation (0.5%). The low dispersibility of nTRA was improved by the addition of an ISG base at 4 °C, although the Tra concentration in the upper layer of nTRA-MC/F127-H and nTRA-F68/F127-H was significantly poor at 20 °C, and the aggregation and precipitation of Tra-NPs were observed.

Figure 4

Corneal toxicity of each ophthalmic Tra formulation. (A) Changes in the cell viability of HCE-T cells treated with each ophthalmic Tra formulation. Each ophthalmic Tra formulation was applied for 2 min. (B) Effect of repetitive instillation on the rat cornea. The repetitive instillation was performed three times a day for 2 months. The compositions of the ophthalmic Tra formulations are shown in Table 1. n = 8. * p < 0.05 vs. CA-TRA for each category. ^# p < 0.05 vs. nTRA for each category. The corneal toxicities of nTRA, nTRA-F127, nTRA-MC/F127, and nTRA-F68/F127 were lower than that of CA-TRA. On the other hand, the cell was stimulated by the treatment with nTRA-Car/F127, and slight corneal damage was observed in rats repetitively instilled with nTRA-Car/F127-H.

Figure 5

Diffusion of Tra in each ophthalmic Tra formulation through a methacrylate cell. (A) Diffusion behavior of Tra in the ophthalmic Tra-NP-incorporated ISNGs with a low ISG base. (B) Diffusion behavior of Tra in the ophthalmic Tra-NP-incorporated ISNGs with a high ISG base. The compositions of the ophthalmic Tra formulations are shown in Table 1. n = 6–9. *p < 0.05 vs. nTRA for each category. The release of Tra from Tra-NP-incorporated ISNGs decreased with the content of the ISG base, and the Tra release with the combination of pluronic F-127 and another ISG base (MC, F68 and Car) was lower than that of nTRA. The release levels of Tra were nTRA > nTRA-F127 > nTRA-F68/F127 > nTRA-Car/F127 > nTRA-MC/F127.

Figure 6

Changes in Tra contents in the lacrimal fluid and blood of rats instilled with each ophthalmic Tra formulation. (A,B) Tra behavior in the lacrimal fluid of rats instilled with the Tra formulations containing a low (A) and a high (B) ISG base. (C,D) Tra behavior in the blood of rats instilled with the Tra formulations containing a low (C) and a high (D) ISG base. The compositions of the ophthalmic Tra formulations are shown in Table 1. n = 5–10. * p < 0.05 vs. nTRA for each category. The residence time and content of Tra in the lacrimal fluid were enhanced by the addition of an ISG base. In the blood, prolonged residence time and decreased C_max were observed in the combination-ISGs incorporating Tra-NPs.

Figure 7

Changes in Tra contents in the cornea and conjunctiva of rats instilled with each ophthalmic Tra formulation. (A,B) Tra behavior in the cornea of rats instilled with the Tra formulations containing a low (A) and a high (B) ISG base. (C,D) Tra behavior in the conjunctiva of rats instilled with the Tra formulations containing a low (C) and a high (D) ISG base. The compositions of the ophthalmic Tra formulations are shown in Table 1. n = 5–10. * p < 0.05 vs. nTRA for each category. The residence time of Tra content in the cornea and conjunctiva was prolonged by the addition of an ISG base; however, the C_max in the Tra-NP-incorporated ISNGs were attenuated in comparison with nTRA.

Figure 8

Changes in EB exudation (A), NO levels (B), and TNF-α levels (C) in the conjunctiva of conjunctivitis rats 5 h after the instillation of each ophthalmic Tra formulation. The compositions of the ophthalmic Tra formulations are shown in Table 1. n = 5–12. * p < 0.05 vs. Normal for each category. ^# p < 0.05 vs. Control for each category. ^$ p < 0.05 vs. Tra-NP-incorporated ISNGs with a high ISG base for each category. The instillation of combination-ISGs incorporating Tra-NPs attenuated EB exudation, NO levels, and TNF-α levels caused by LPS injection. In particular, nTRA-F68/F127-L significantly prevented the EB exudation, NO levels, and TNF-α levels 5 h after the instillation.

Figure 9

The balance of drug residence time and drug diffusion in lacrimal fluid related to the ocular BA of Tra-NP-incorporated ISNGs.