Combination Nanomedicine Strategy for Preventing High-Risk Corneal Transplantation Rejection

Abstract

High-risk (HR) corneal transplantation presents a formidable challenge, with over 50% of grafts experiencing rejection despite intensive postoperative care involving frequent topical eyedrop administration up to every 2 h, gradually tapering over 6-12 months, and ongoing maintenance dosing. While clinical evidence underscores the potential benefits of inhibiting postoperative angiogenesis, effective antiangiogenesis therapy remains elusive in this context. Here, we engineered controlled-release nanomedicine formulations comprising immunosuppressants (nanoparticles) and antiangiogenesis drugs (nanowafer) and demonstrated that these formulations can prevent HR corneal transplantation rejection for at least 6 months in a clinically relevant rat model. Unlike untreated corneal grafts, which universally faced rejection within 2 weeks postsurgery, a single subconjunctival injection of the long-acting immunosuppressant nanoparticle alone effectively averted graft rejection for 6 months, achieving a graft survival rate of ∼70%. Notably, the combination of an immunosuppressant nanoparticle and an anti-VEGF nanowafer yielded significantly better efficacy with a graft survival rate of >85%. The significantly enhanced efficacy demonstrated that a combination nanomedicine strategy incorporating immunosuppressants and antiangiogenesis drugs can greatly enhance the ocular drug delivery and benefit the outcome of HR corneal transplantation with increased survival rate, ensuring patient compliance and mitigating dosing frequency and toxicity concerns.

Keywords: antiangiogenesis; immunosuppressant; nanoparticle; nanowafer; ocular drug delivery.

Figure 1

Combination treatment of long-acting PLA DSP-NP and topical Axi NW provided 6 month efficacy in preventing HR corneal transplantation rejection on rats.

Figure 2

Characterization of PLGA/PLA DSP-NP. (A) XRD analysis of DSP, DSP–Zn complex, and DSP-NPs. (B,C) Particle size (B) and zeta potential (C) of PLA DSP-NP. (D) TEM image of PLA DSP-NP. (E) Elemental mapping image of PLA DSP-NP. (F) DSP and Zn loading in the DSP-NP formulated with PLGA/PLA polymers with different carboxyl contents. DSP-NP target drug loading is 20 wt % DSP. N = 3 for each formulation. (G,H) ITC titration of the DSP–Zn complex into PLGA-COOH_7 kDa (G) and PLA-2COOH_8.2 kDa (H) dissolved in anhydrous DMSO at 25 °C. (I) Proposed schematic image of DSP-NP.

Figure 3

Development and characterization of Axi NW. (A) Schematic of NW preparation. (B) SEM image of Axi NW. (C) In vitro drug release profile of Axi NW. Mean ± S, n = 3 samples for in vitro drug release analysis.

Figure 4

In vivo PK profiles of Axi after topical administration of Axi NW in rats. Axi concentrations in (A) tears, (B) cornea, (C) conjunctiva, (D) aqueous humor, (E) vitreo–retina, and (F) plasma at 1, 2, 3, 4, 6, 8, 12, and 24 h after topical administration of Axi NW. Mean ± SEM, n = 6 eyes or n = 3 plasma samples.

Figure 5

Construction and characterization of HR corneal recipients. (A) Schedule of HR-recipient construction. (B) Clinical progress of corneal NV at 1, 2, and 5 weeks after suture placement. qPCR analysis of (C) CD31, (D) VEGF A, (E) VEGF R1, (F) LYVE-1, (G) VEGF R3, (H) VEGF R2, and (I) VEGF C in the corneal samples collected at 2 and 5 weeks after suture placement. Statistical analysis for (C–I): one-way ANOVA with a Tukey post hoc test for multiple comparison. (* indicates analysis versus healthy control; *p ≤ 0.05 and ***p ≤ 0.001; # indicates cornea at 2w after suture placement versus cornea at 5w after suture placement; #p ≤ 0.05; ##p ≤ 0.01; and ###p ≤ 0.001). N = 3–6 for each group. All data are plotted from mean ± SEM.

Figure 6

Therapeutic effect of PLA DSP-NP and Axi NW in preventing HR corneal allograft rejection. (A) Schedule for the efficacy study of PLA DSP-NP and Axi NW in preventing HR corneal allograft rejection (HR PKP surgery). (B) Representative images of grafts at 1w, 2w, 1m, 3m, and 6m after HR PKP surgery. (C) Edema, (D) opacity, (E) NV, (F) total grade, and (G) survival curves of grafts received different treatments after HR PKP surgery. (H) Histological images of grafts from different groups; scale bar 100 μm. The black arrow indicates corneal NV, the black asterisk indicates inflammatory cell infiltration, and the red arrow indicates the “bubbly” appearance of the corneal epithelial layer. Statistical analysis for (C–H): two-way ANOVA, followed by Bonferroni’s post hoc test for multiple comparison. (*p ≤ 0.05 versus untreated grafts; **p ≤ 0.01 versus untreated grafts; ***p ≤ 0.001 versus untreated grafts; #p ≤ 0.05 PLA DSP-NP grafts versus combination; and ##p ≤ 0.01 PLA DSP-NP grafts versus combination). All data are plotted from mean ± SEM.

Figure 7

Corneal nerve regeneration after HR PKP surgery. (A) Immunostaining images of corneal nerve marker β-tubulin III, scale bar 200 μm. (B) Corneal nerve regeneration was quantified as the percentage of threshold area positive for β-tubulin III staining in the center part of cornea with the diameter of 3.5 mm. Statistical analysis for B: one-way ANOVA with a Tukey post hoc test for multiple comparison. (*p ≤ 0.05; **p ≤ 0.01; and ***p ≤ 0.001). N = 3 for each group. All data are plotted from mean ± SEM.

Figure 8

Safety evaluation of NW. (A) Schedule of safety study. (B) Corneal microscopy imaging at 1w and 2w after starting the study. (C) Rat BW and (D) IOP during the 2 week follow-up. (E) Rat cornea staining tests and (F) blink rate tests at 1 week and 2 weeks during the study. At 2 weeks after NW administration, scotopic and photopic ERG was tested. Quantification of the amplitude of (G) scotopic a-wave, (H) scotopic b-wave, (I) photopic a-wave, and (J) photopic b-wave. (K) H&E staining of cornea collected at 2 weeks after NW administration. All data are plotted from mean ± SEM (n = 3 for BW monitoring and n ≥ 4 for other tests). Statistical analysis for (B,C,E–H): Two-way ANOVA, followed by Bonferroni post hoc test.