Colon-targeted delivery of cyclosporine A using dual-functional Eudragit® FS30D/PLGA nanoparticles ameliorates murine experimental colitis

Abstract

Background: Colon-targeted oral nanoparticles (NPs) have emerged as an ideal, safe, and effective therapy for ulcerative colitis (UC) owing to their ability to selectively accumulate in inflamed colonic mucosa. Cyclosporine A (CSA), an immunosuppressive agent, has long been used as rescue therapy in severe steroid-refractory UC. In this study, we developed CSA-loaded dual-functional polymeric NPs composed of Eudragit^® FS30D as a pH-sensitive polymer for targeted delivery to the inflamed colon, and poly(lactic-co-glycolic acid) (PLGA) as a sustained-release polymer.

Methods: CSA-loaded Eudragit FS30D nanoparticles (ENPs), PLGA nanoparticles (PNPs), and Eudragit FS30D/PLGA nanoparticles (E/PNPs) were prepared using the oil-in-water emulsion method. Scanning electron microscope images and zeta size data showed successful preparation of CSA-loaded NPs.

Results: PNPs exhibited a burst drug release of >60% at pH 1.2 (stomach pH) in 0.5 h, which can lead to unwanted systemic absorption and side effects. ENPs effectively inhibited the burst drug release at pH 1.2 and 6.8 (proximal small intestine pH); however, nearly 100% of the CSA in ENPs was released rapidly at pH 7.4 (ileum-colon pH) owing to complete NP dissolution. In contrast to single-functional PNPs and ENPs, the dual-functional E/PNPs minimized burst drug release (only 18%) at pH 1.2 and 6.8, and generated a sustained release at pH 7.4 thereafter. Importantly, in distribution studies in the gastrointestinal tracts of mice, E/PNPs significantly improved CSA distribution to the colon compared with PNPs or ENPs. In a mouse model of colitis, E/PNP treatment improved weight loss and colon length, and decreased rectal bleeding, spleen weight, histological scoring, myeloperoxidase activity, macrophage infiltration, and expression of proinflammatory cytokines compared with PNPs or ENPs.

Conclusion: Overall, this work confirms the benefits of CSA-loaded E/PNPs for efficiently delivering CSA to the colon, suggesting their potential for UC therapy.

Keywords: colon-targeted nanoparticles; cyclosporine A; sustained and pH-sensitive release; ulcerative colitis.

Figure 1

Morphology and size analysis of CSA-loaded PNPs, ENPs, and E/PNPs: (A) SEM images; (B) size histograms. Abbreviations: CSA, cyclosporine A; PNPs, poly(lactic-co-glycolic acid) (PLGA) nanoparticles; ENPs, Eudragit FS30D nanoparticles; E/PNPs, Eudragit FS30D/PLGA nanoparticles; SEM, scanning electron microscopy.

Figure 2

pH-dependent in vitro drug-release profiles and size analysis of CSA-loaded PNPs, ENPs, and E/PNPs: (A) in vitro drug release; (B) size analysis at different pH values. Abbreviations: CSA, cyclosporine A; PNPs, poly(lactic-co-glycolic acid) (PLGA) nanoparticles; ENPs, Eudragit FS30D nanoparticles; E/PNPs, Eudragit FS30D/PLGA nanoparticles.

Figure 3

Representative ex vivo images of the GIT after oral administration of blank and DiR-loaded NPs, showing colon-targeted drug delivery and accumulation potential of PNPs, ENPs, and E/PNPs. Abbreviations: GIT, gastrointestinal tract; NPs, nanoparticles; DiR, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide; PNPs, poly(lactic-co-glycolic acid) (PLGA) nanoparticles; ENPs, Eudragit FS30D nanoparticles; E/PNPs, Eudragit FS30D/PLGA nanoparticles.

Figure 4

In vivo quantification of CSA-loaded nanoparticles in the GIT: (A) PNPs; (B) ENPs; (C) E/PNPs. n=3 mice per time point; ***p<0.001. Abbreviations: CSA, cyclosporine A; GIT, gastrointestinal tract; PNPs, poly(lactic-co-glycolic acid) (PLGA) nanoparticles; ENPs, Eudragit FS30D nanoparticles; E/PNPs, Eudragit FS30D/PLGA nanoparticles.

Figure 5

Body weight changes and DAI during the experimental period and macroscopic assessment of colitis after CSA-loaded nanoparticle treatment: (A) body weight changes (%); (B) DAI; (C) colon length; (D) colon length/weight ratio; and (E) spleen weight. Error bars are not shown in parts (A and B) for reasons of clarity; n=5 mice/group; *p<0.05, **p<0.01, ***p<0.001 compared with colitis control. Abbreviations: DAI, disease activity index; CSA, cyclosporine A; DSS, dextran sodium sulfate; PNPs, poly(lactic-co-glycolic acid) (PLGA) nanoparticles; ENPs, Eudragit FS30D nanoparticles; E/PNPs, Eudragit FS30D/PLGA nanoparticles.

Figure 6

Histological evaluation of colon tissue. (A) H&E staining for microscopic evaluation of the colon sections isolated from healthy control, colitis control, and CSA-loaded nanoparticle-treated groups. Images of tissues are shown at 100× magnification. Arrowheads indicate disrupted epithelium, black arrows indicate inflammatory cell infiltration, and yellow arrows indicate re-epithelialization. (B) Histological score. Data are presented as mean±SD (n=3 animals/group). *p<0.05, ***p<0.001 compared with colitis control. Abbreviations: CSA, cyclosporine A; PNPs, poly(lactic-co-glycolic acid) (PLGA) nanoparticles; ENPs, Eudragit FS30D nanoparticles; E/PNPs, Eudragit FS30D/PLGA nanoparticles.

Figure 7

Immunofluorescence staining of macrophages in the colon tissue of a healthy control group, colitis control group, and CSA-loaded nanoparticle-treated groups. Representative confocal microscopy images. Blue, DAPI nuclear staining; red, macrophages (F4/80). White arrows indicate macrophage infiltration. Abbreviations: CSA, cyclosporine A; PNPs, poly(lactic-co-glycolic acid) (PLGA) nanoparticles; ENPs, Eudragit FS30D nanoparticles; E/PNPs, Eudragit FS30D/PLGA nanoparticles.

Figure 8

Proinflammatory cytokine levels and MPO activity in the healthy control group, colitis control group, and CSA-loaded nanoparticle-treated groups. (A) TNF-α; (B) IL-6; and (C) MPO activity assay. Statistical comparisons were evaluated between the colitis control group and CSA-loaded NP groups (*p<0.05, ***p<0.001). Abbreviations: TNF-α, tumor necrosis factor-alpha; MPO, myeloperoxidase; CSA, cyclosporine A; PNPs, poly(lactic-co-glycolic acid) (PLGA) nanoparticles; ENPs, Eudragit FS30D nanoparticles; E/PNPs, Eudragit FS30D/PLGA nanoparticles.