Corticosteroid-loaded biodegradable nanoparticles for prevention of corneal allograft rejection in rats

Abstract

Immunologic graft rejection is one of the main causes of short and long-term graft failure in corneal transplantation. Steroids are the most commonly used immunosuppressive agents for postoperative management and prevention of corneal graft rejection. However, steroids delivered in eye drops are rapidly cleared from the surface of the eye, so the required frequency of dosing for corneal graft rejection management can be as high as once every 2h. Additionally, these eye drops are often prescribed for daily use for 1 year or longer, which can result in poor patient compliance and steroid-related side effects. Here, we report a biodegradable nanoparticle system composed of Generally Regarded as Safe (GRAS) materials that can provide sustained release of corticosteroids to prevent corneal graft rejection following subconjunctival injection provided initially during transplant surgery. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles containing dexamethasone sodium phosphate (DSP) exhibited a size of 200 nm, 8 wt.% drug loading, and sustained drug release over 15 days in vitro under sink conditions. DSP-loaded nanoparticles provided sustained ocular drug levels for at least 7 days after subconjunctival administration in rats, and prevented corneal allograft rejection over the entire 9-week study when administered weekly. In contrast, control treatment groups that received weekly injections of either placebo nanoparticles, saline, or DSP in solution demonstrated corneal graft rejection accompanied by severe corneal edema, neovascularization and opacity that occurred in ≤ 4 weeks. Local controlled release of corticosteroids may reduce the rate of corneal graft rejection, perhaps especially in the days immediately following surgery when risk of rejection is highest and when typical steroid eye drop administration requirements are particularly onerous.

Keywords: Corneal rejection; Corneal transplantation; Dexamethasone; Drug delivery; PLGA.

Fig. 1

(A) TEM image and (B) in vitro DSP release profile from DSP-NP.

Fig. 2

Fluorescence images of (A) a rat eye prior to injection and (B) a rat eye following SC administration of fluorescently-labeled nanoparticles. (C) Retention of PS-PEG particles of various diameters after SC administration to rats.

Fig. 3

Sample histology at PO 2 days, 7 days and 14 days showing representative images of rat conjunctiva (A, B, C, G, H, I) and cornea (D, E, F, J, K, L) after SC injection of saline or placebo NP. Conjunctival and corneal epithelia are marked with an asterisk. Foci of mildly increased inflammation were present in the underlying conjunctival substantia propria 2 days after injection but not at later time points (arrow in panels A and G). Original magnification 200×.

Fig. 4

Pharmacokinetic study of free DSP solution and DSP-NP after SC administration to rats. DSP levels (A) at the injection site, (B) in the aqueous, (C) in the vitreous and (D) in blood. (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Fig. 5

Postoperative examination of transplanted corneas. (A) A transplanted cornea immediately following SC injection of nanoparticles (site of injection marked with an asterisk). All grafts were rejected by PO 2 weeks for groups that received weekly SC injection of (B) saline and (C) placebo NP, and by PO 4 weeks for rats that received weekly SC injection of (D) free DSP. All grafts were clear until the end of study (PO 9 weeks) after weekly SC administration of (E) DSP-NP. Arrows show examples of corneal neovascularization in panels B–D. The plus sign shows site of edema in panel D.

Fig. 6

Clinical evaluation of grafts after treatment with SC injection of saline, placebo NP, free DSP or DSP-NP at the study end points (saline group at PO 2 weeks, placebo NP group at PO 2 weeks, DSP group at PO 4 weeks and DSP-NP group at PO 9 weeks). (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Fig. 7

Kaplan–Meier survival curve for transplanted corneal grafts after treatment with SC injection of saline, placebo NP, free DSP or DSP-NP.

Fig. 8

IOP measured on eyes with corneal graft transplantation and undergoing different treatments. Normal eyes without any treatment were used as the control.

Fig. 9

Histological images of transplanted corneas after weekly SC injection with (A, E) saline at PO 2 weeks, (B, F) placebo NP at PO 2 weeks, (C, G) free DSP at PO 4 weeks and (D, H) DSP-NP at PO 9 weeks. The cornea was massively thickened in the control groups (bars, A and B), focally thickened when treated with free drug (bar, C) and normal appearing when treated with DSP-NP (bar, D). Diffuse chronic inflammation (asterisk) and associated blood vessels (arrows) were present throughout the corneal stroma in control animals. Original magnification, 40× (A, B, C, D) and 100× (E, F, G, H).