Development and effects of tacrolimus-loaded nanoparticles on the inhibition of corneal allograft rejection

Abstract

Tacrolimus has been widely applied to prevent organ rejection after transplantation. However, the conventional pharmaceutical formulation of tacrolimus limits its applications in ocular therapy due to its hydrophobicity and low corneal penetrability. We optimized tacrolimus-loaded methoxy poly (ethylene glycol-block-poly (d, l)-lactic-co-glycolic acid) nanoparticles (TAC-NPs) by simple and effective nanotechnology as a drug delivery system for corneal graft rejection to overcome these drawbacks. The prepared TAC-NPs were 82.9 ± 1.3 nm in size, and the drug loading and encapsulation efficiency were 8.01 ± 0.23% and 80.10 ± 2.33%. Furthermore, New Zealand rabbits were used to analyze the single-dose pharmacokinetics of the TAC-NPs using high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). In rats with allogenic penetrating keratoplasty, the administration of TAC-NPs dispersion drops improved the TAC concentrations in the aqueous humor and cornea, consistent with a significantly higher effective inhibition of IL-2, IL-17, and VEGF expression compared with conventional 0.1% tacrolimus drops. Meanwhile, we also compared two different topical administration methods (including eye drop and subconjunctival injection) of TAC-NPs to maximize the sustained release characteristic of NPs. In summary, the small-sized TAC-NPs enhanced transcorneal permeation and absorption of TAC and more effectively inhibited corneal allograft rejection, which indicated that biodegradable polymeric nanomaterials-based drug delivery system had great potential for improving the clinical therapy efficacy of hydrophobic drugs.

Keywords: Tacrolimus; immune rejection; keratoplasty; mPEG-b-PLGA; nanoparticles.

Figure 1.

(a) TEM image of TAC-NPs (100,000×, scale bar, 100 nm). (b) In vitro release profiles of TAC-NPs and 0.1% TAC suspension. Pharmacokinetic profile of TAC-NP after a single dose (25 µl, containing 5 µg TAC) topical administrations, including drops (c) and subconjunctival injection (d) in rabbits (data were expressed in mean ± SD, n = 3).

Figure 2.

Clinical manifestations and tacrolimus concentration in rats (a) Clinical manifestations (magnification, 250×) of the anterior segment on postoperative day 14 and 28. (b) Survival curves of rat corneal grafts for all groups. The concentrations of tacrolimus in the corneas (ng/mg) and aqueous humor (ng/ml) on postoperative day 14 (c) and 28 (d). Error bars represent the means ± SD. *A statistically significant difference compared to the control groups at the level of p < .05 using Permutation test. N = 3.

Figure 3.

HE and immunohistochemical staining of corneal grafts (magnification, 200×). (a) HE staining, (b) immunohistochemical staining of CD4⁺ T cells, and (c) immunohistochemical staining of CD8⁺ T cells. Greater numbers of CD4⁺ and CD8⁺ T cells (stained in brown) were observed in the blank control group than in the TAC-NP-treated and 0.1% tacrolimus-treated groups.

Figure 4.

Concentrations of IL-2, IL-17, and VEGF in aqueous humor and cornea on postoperative days 14 (a, b) and 28 (c, d). TAC-NPs inhibited immune reactions more effectively than conventional 0.1% tacrolimus (p < .05). Error bars represent the means ± SD. *A statistically significant difference compared to the control groups at the level of p < .05 using Permutation test. N = 3.