Six-month effective treatment of corneal graft rejection

Abstract

Topical corticosteroid eye drop is the mainstay for preventing and treating corneal graft rejection. While the frequent topical corticosteroid use is associated with risk of intraocular pressure (IOP) elevation and poor patient compliance that leads to graft failure and the requirement for a repeated, high-risk corneal transplantation. Here, we developed dexamethasone sodium phosphate (DSP)-loaded dicarboxyl-terminated poly(lactic acid) nanoparticle (PLA DSP-NP) formulations with relatively high drug loading (8 to 10 weight %) and 6 months of sustained intraocular DSP delivery in rats with a single dosing. PLA DSP-NP successfully reversed early signs of corneal rejection, leading to rat corneal graft survival for at least 6 months. Efficacious PLA DSP-NP doses did not affect IOP and showed no signs of ocular toxicity in rats for up to 6 months. Subconjunctival injection of DSP-NP is a promising approach for safely preventing and treating corneal graft rejection with the potential for improved patient adherence.

Fig. 1.. A single SCT injection of PLGA DSP-NP at POD3 provided short-term reversal of corneal graft rejection.

(A) Treatment scheme of rat corneal allograft rejection. At POD3, a single 10-μl SCT injection of saline, DSP solution (DSP, 10 mg/ml), or PLGA DSP-NP (DSP, 10 mg/ml) was administered (n = 5 per group). Routine clinical evaluation was conducted at POD3, POD10, POD17, and POD28. (B) Representative photos of grafts at POD3, POD10, POD17, and POD28. (C) Corneal opacity, (D) edema, (E) neovascularization, (F) total clinical grade, and (G) graft survival rate. Data are shown as means ± SEM. For (C) to (F), statistical analysis at POD10 was calculated using one-way ANOVA with a Tukey’s post hoc test for multiple comparisons (PLGA DSP-NP compared with Saline: *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001; PLGA DSP-NP compared with DSP solution: #P≤ 0.05, ##P≤ 0.01, ###P≤ 0.001). PKP, penetrating keratoplasty.

Fig. 2.. Reversal of corneal graft rejection with PLGA DSP-NP treatment is associated with down-regulation of pro-inflammatory genes and up-regulation of anti-inflammatory marker at POD10.

Relative expression of (A) IL-1β, (B) IFN-γ, (C) TNF-α, (D) Granzyme B, and (E) IL-4 in cornea tissues at POD10 compared to healthy corneas (n = 6). Gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and analyzed using ΔΔCT method. Results are presented as expression relative to that of healthy cornea tissue. (F) Histology images of grafts at POD10. Scale bars, 100 μm. Black asterisks indicate inflammatory cells. Yellow arrows indicate neovascularization in the cornea samples. Statistical significance was calculated by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison tests (*P < 0.05, **P < 0.01, and ***P < 0.001). Data are shown as means ± SEM.

Fig. 3.. Design and characterization of PLA DSP-NP formulations.

(A) DSP was loaded into PLA-2COOH NPs via zinc ion bridging between the carboxyl groups from PLA-2COOH and phosphate groups from DSP. Transmission electron microscope images of (B) PLA DSP-NP (5.1 kDa) and (C) PLA DSP-NP (8.2 kDa). (D) In vitro drug release profiles of PLA DSP-NP (5.1 kDa) and PLA DSP-NP (8.2 kDa) under sink conditions at 37°C. DL, DSP drug loading.

Fig. 4.. In vivo pharmacokinetic profiles of DSP and DEX after 30-μl SCT injection of PLA DSP-NP (600 μg of DSP) in rats.

DSP and DEX concentrations in (A) conjunctiva, (B) cornea, (C) aqueous humor, (D) vitreo-retina-choroid, and (E) plasma at 6 hours, 1 day, 1 week, 2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, and 6 months after SCT injection of DSP-NP. Data are represented as means ± SEM. n = 5 to 6 eyes or n = 3 plasma samples.

Fig. 5.. A single SCT injection of PLA DSP-NP prevented corneal allograft rejection.

(A) Schedule for the efficacy study of PLA DSP-NP in preventing corneal allograft rejection. (B) Representative images of grafts at PO 1m, PO 3m, and PO 6m. (C) Corneal opacity grade, (D) edema grade, (E) neovascularization grade, (F) total clinical grades, and (G) graft survival rate. (H) Representative histology images of center (top row) and bed (bottom row) area of the cornea at PO 6m. Black asterisks represent the corneal stroma with a lack of corneal keratocytes. Black arrows indicate neovascularization in the corneal samples. Data are represented as means ± SEM. For (C) to (G), statistical analysis at each time points were calculated using one-way ANOVA with a Tukey’s post hoc test with multiple comparisons.

Fig. 6.. A single SCT injection of PLA DSP-NP rescued corneal allografts with signs of early injection and maintained graft survival up to 6 months.

(A) Schedule for the efficacy study of PLA DSP-NP in reversing corneal allograft with signs of early rejection. (B) Representative images of grafts at PO 1 month, 3 months, and 6 months. (C) Corneal opacity, (D) edema, and (E) neovascularization, (F) total clinical score, and (G) graft survival curves. (H) Representative histology images of center (top row) and bed (bottom row) area of the cornea at PO 6m. Black asterisks represent the corneal stroma with a lack of corneal keratocytes. Black arrows indicate neovascularization in the corneal samples. Data are represented as means ± SEM. For (C) to (G), statistical analysis at each time point was calculated using one-way ANOVA with a Tukey’s post hoc test for multiple comparisons. (*P ≤ 0.05 for 100 to 200 μg.)

Fig. 7.. Safety evaluation after a single SCT injection of PLA DSP-NP.

(A) Schedule of safety study. (B) Rat IOP and (C) change in body weight (BW) (%) after single SCT injection of PLA DSP-NP (200, 400, or 800 μg of DSP). Topical 0.1% DSP eye drops (three times/day for 3 weeks) were applied as positive control, and untreated rats were used as negative control. (D) Representative hematoxylin and eosin staining of cornea collected at 6 months after PLA DSP-NP injection. Quantification of the amplitude of (E) scotopic a-wave, (F) photopic a-wave, (G) scotopic b-wave, and (H) photopic b-wave at 6 months. N = 4 independent animals per group were used for body weight monitoring. N = 6 to 8 independent eyes per group for IOP and ERG studies. Data are represented as means ± SEM. Statistical analysis for (B) and (C) and (E) to (H), two-way ANOVA with multiple comparisons was used. (* for comparison with healthy control: *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.)