Flt23k nanoparticles offer additive benefit in graft survival and anti-angiogenic effects when combined with triamcinolone

Abstract

Purpose: To determine if nanoparticles delivering plasmids expressing Flt23k (an anti-VEGF intraceptor) can enhance murine cornea transplant survival and whether their effect is synergistic with steroid therapy.

Methods: Biodegradable PLGA Flt23k loaded or blank nanoparticles were prepared using the emulsion solvent evaporation

Method: Graft survival, corneal neovascularization, and corneal lymphangiogenesis were compared among the Flt23k nanoparticles, blank nanoparticles, triamcinolone acetonide, and PBS groups following subconjunctival injection in mice that underwent penetrating keratoplasty. Graft survival, corneal neovascularization, and corneal lymphangiogenesis in a group treated with both nanoparticles and steroid therapy were also analyzed.

Results: The Flt23k nanoparticle group showed less neovascularization, lymphangiogenesis, and graft failure compared with the PBS control group (P < 0.01). The 2-month graft survival rate was 20% in the Flt23k nanoparticle group with no grafts surviving in the PBS group. When the Flt23k nanoparticle was combined with steroid therapy, a significant increase in graft survival was seen compared with both steroid treatment alone (P < 0.05) and steroid combined with blank nanoparticle treatment (P < 0.05). The 2-month graft survival rate was 91.6% in the combination group compared with 47.6% in the triamcinolone-only group and 42.4% in the triamcinolone plus blank nanoparticle group.

Conclusions: Anti-VEGF nanoparticles (Flt23k) have a significant effect on decreasing neovascularization and lymphangiogenesis, resulting in increased graft survival in penetrating keratoplasty. This beneficial effect is synergistically enhanced with steroid treatment.

Conflict of interest statement

Disclosure: Y.K. Cho, None; H. Uehara, None; J.R. Young, None; P. Tyagi, None; U.B. Kompella, None; X. Zhang, None; L. Luo, None; N. Singh, None; B. Archer, None; B.K. Ambati, None

Figures

Figure 1.

The expression of nanoparticles in the cornea as observed by fluorescence microscopy (upper row—objective lens ×2.5) and confocal microscope (lower row—objective lens ×100).

Figure 2.

The presence of nanoparticles in corneal tissue as observed by confocal microscope with high magnification (objective lens ×1000).

Figure 3.

(A) Comparison of graft survival through 8 weeks postoperatively. Flt23k nanoparticle group showed better survival than the PBS group (P = 0.009) until 8 weeks postoperatively. Flt23k nanoparticle group showed better survival when compared with blank nanoparticle group until 3 weeks postoperatively (P = 0.029). (+Sensored data, *P < 0.05, **P < 0.01). (B) Comparison of graft opacity grade for each week. Through 2 to 5 weeks postoperatively, the Flt23k nanoparticle group showed decreased opacification compared with the PBS group (P < 0.05).

Figure 4.

(A) The Flt23k nanoparticle group showed less total neovascularization compared with the PBS group (P = 0.000) and the blank nanoparticle group (P = 0.019). The Flt23k nanoparticle group showed less graft neovascularization than PBS group (P = 0.009), blank nanoparticle group (P = 0.001), and triamcinolone group (P = 0.000). Error bar is SEM. *P < 0.05, **P < 0.01. (B) The Flt23k nanoparticle group showed less total lymphangiogenesis compared with the PBS group (P = 0.002). The Flt23k nanoparticle group also showed less graft lymphangiogenesis compared with PBS group (P = 0.011) and blank nanoparticle group (P = 0.002). (C) Representative pictures of corneal neovascularization (upper row) and lymphangiogenesis (lower row) in each group.

Figure 5.

(A) The Flt23k nanoparticle plus triamcinolone group increased graft survival compared with the triamcinolone group and triamcinolone plus blank nanoparticle group (P = 0.048, P = 0.020, respectively). +Sensored data, *P < 0.05, **P < 0.01. (B) The Flt23k nanoparticle plus triamcinolone group showed less total neovascularization compared with triamcinolone group (P = 0.000) and triamcinolone plus blank nanoparticle group (P = 0.028). The Flt23k nanoparticle plus triamcinolone group showed less graft neovascularization compared with triamcinolone group (P = 0.008). (C) The Flt23k nanoparticle plus triamcinolone group showed significantly less total lymphangiogenesis compared with triamcinolone group (P = 0.043) and triamcinolone plus blank nanoparticle group (P = 0.014). The Flt23k nanoparticle plus triamcinolone group showed less graft lymphangiogenesis compared with the triamcinolone plus blank nanoparticle group (P = 0.028). (D) Representative pictures of neovascularization (upper row) and lymphangiogenesis (lower row) in each group.