Effects of rAmb a 1-Loaded PLGA-PEG Nanoparticles in a Murine Model of Allergic Conjunctivitis

Abstract

Ambrosia artemisiifolia (Amb a) contains many allergens. Allergic conjunctivitis caused by Ambrosia artemisiifolia and its related allergen-specific immunotherapy (AIT) are seldom studied at present. poly(DL-lactide-co-glycolide)-polyethylene glycol (PLGA-PEG) is a very good nano-carrier, which has been applied in the medical field. In this context, we studied the immunotherapy effect and potential mechanism of recombinant Amb a 1 (rAmb a 1)-loaded PLGA-PEG nanoparticles. A mouse allergic conjunctivitis model was established with Ambrosia artemisiifolia crude extract, and the nanoparticles were used for AIT through direct observation of conjunctival tissue, degranulation of mast cells in conjunctival tissue, serum-specific antibodies, cytokines and other assessment models. The treatment of nanoparticles enhanced the secretion of T-helper 1 (Th1) cytokine Interferon-gama (IFN-γ) and the production of immunoglobulin G (IgG)2a (IgG2a), inhibited the secretion of T-helper 2 (Th2) cytokine Interleukin (IL)-13 and IL-4 and the level of IgE. Especially, degranulation of mast cells and expression of mast cell protease-1 (MCP-1) in conjunctival tissue was reduced significantly. In this study, we proved that the nanoparticles prepared by rAmb a 1 and PLGA-PEG have an immunotherapy effect on allergic conjunctivitis in mice.

Keywords: Amb a 1; allergen; allergen-specific immunotherapy; allergic conjunctivitis; poly(DL-lactide-co-glycolide)-polyethylene glycol (PLGA-PEG).

Figure 1

(a) Field emission scanning electron microscopic image; (b) dynamic light-scattering spectra; (c) in vitro cumulative protein release of rAmb a 1-loaded PLGA-PEG nanoparticles. Abbreviations: PLGA-PEG, poly(DL-lactide-co-glycolide)-polyethylene glycol; rAmb a 1, recombinant Amb a1.

Figure 2

(a) SDS-PAGE analysis of rAmb a 1-loaded PLGA-PEG and rAmb a 1, M: protein marker, 1: rAmb a 1-PLGA-PEG, 2: rAmb a 1; (b) Western blot of rAmb a 1-loaded PLGA-PEG and rAmb a 1 with serum from an Ambrosia artemisiifolia-allergic patient, M: protein marker, 1: rAmb a 1-PLGA-PEG, 2: rAmb a 1. Abbreviations: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PLGA-PEG, poly(DL-lactide-co-glycolide)-polyethylene glycol; rAmb a 1, recombinant Amb a 1.

Figure 3

(a) Ocular signs of allergic conjunctivitis in mice in each group; (b) eye feature scores of mice in each group. The data are shown as mean ± SD from five individual mice (**** p < 0.0001, *** p < 0.001, * p < 0.05, ns: no significant difference). Abbreviations: NG, naive group; AC, allergic conjunctivitis group; AC + rAmb a 1-PP, allergic conjunctivitis + rAmb a 1-PLGA-PEG treatment group; AC + PP, allergic conjunctivitis + PLGA-PEG treatment group; AC + rAmb a 1, allergic conjunctivitis + rAmb a 1 treatment group.

Figure 4

(a) Formalin-fixed conjunctival tissue section with mast cells stained with toluidine blue; (b) degranulation rate of mast cells in mice in each group. The data are shown as mean ± SD from four individual mice (*** p < 0.001, ** p < 0.01, ns: no significant difference). Abbreviations: NG, naive group; AC, allergic conjunctivitis group; AC + rAmb a 1-PP, allergic conjunctivitis + rAmb a 1-PLGA-PEG treatment group; AC + PP, allergic conjunctivitis + PLGA-PEG treatment group; AC + rAmb a 1, allergic conjunctivitis + rAmb a 1 treatment group.

Figure 5

Serum-specific IgE (a) and IgG2a (b) in mice in each group. The data are shown as mean ± SD from five individual mice (*** p < 0.001, ** p < 0.01, ns: no significant difference). Abbreviations: NG, naive group; AC, allergic conjunctivitis group; AC + rAmb a 1-PP, allergic conjunctivitis + rAmb a 1-PLGA-PEG treatment group; AC + PP, allergic conjunctivitis + PLGA-PEG treatment group; AC + rAmb a 1, allergic conjunctivitis + rAmb a 1 treatment group.

Figure 6

IL-4 (a), IFN-γ (b) and IL-13 (c) in each group. Cytokine production in spleen cell cultures. The data are shown as mean ± SD from five individual mice (*** p < 0.001, ** p < 0.01, * p < 0.05. ns: no significant difference). Abbreviations: NG, naive group; AC, allergic conjunctivitis group; AC + rAmb a 1-PP, allergic conjunctivitis + rAmb a 1-PLGA-PEG treatment group; AC + PP, allergic conjunctivitis + PLGA-PEG treatment group; AC + rAmb a 1, allergic conjunctivitis + rAmb a 1 treatment group.

Figure 7

(a) Immunohistochemical method to stain mMCP-1 in paraffin section of mice conjunctival tissue; (b) IOD value in each group. The data are shown as mean ± SD from four individual mice (*** p < 0.001, * p < 0.05, ns: no significant difference). Abbreviations: NG, naive group; AC, allergic conjunctivitis group; AC + rAmb a 1-PP, allergic conjunctivitis + rAmb a 1-PLGA-PEG treatment group; AC + PP, allergic conjunctivitis + PLGA-PEG treatment group; AC + rAmb a 1, allergic conjunctivitis + rAmb a 1 treatment group; IOD, integrated optical density; mMCP-1, mouse mast cell proteinase-1.

Figure 8

Experimental design.