pH-Responsive Alginate-Based Microparticles for Colon-Targeted Delivery of Pure Cyclosporine A Crystals to Treat Ulcerative Colitis

Abstract

Cyclosporine A (CsA) is a potent immunosuppressant for treating ulcerative colitis (UC). However, owing to severe systemic side effects, CsA application in UC therapy remains limited. Herein, a colon-targeted drug delivery system consisting of CsA crystals (CsAc)-loaded, Eudragit S 100 (ES)-coated alginate microparticles (CsAc-EAMPs) was established to minimize systemic side effects and enhance the therapeutic efficacy of CsA. Homogeneously-sized CsAs (3.1 ± 0.9 μm) were prepared by anti-solvent precipitation, followed by the fabrication of 47.1 ± 6.5 μm-sized CsAc-EAMPs via ionic gelation and ES coating. CsAc-EAMPs exhibited a high drug loading capacity (48 ± 5%) and a CsA encapsulation efficacy of 77 ± 9%. The in vitro drug release study revealed that CsA release from CsAc-EAMPs was suppressed under conditions simulating the stomach and small intestine, resulting in minimized systemic absorption and side effects. Following exposure to the simulated colon conditions, along with ES dissolution and disintegration of alginate microparticles, CsA was released from CsAc-EAMPs, exhibiting a sustained-release profile for up to 24 h after administration. Given the effective colonic delivery of CsA molecules, CsAc-EAMPs conferred enhanced anti-inflammatory activity in mouse model of dextran sulfate sodium (DSS)-induced colitis. These findings suggest that CsAc-EAMPs is a promising drug delivery system for treating UC.

Keywords: colon-targeted delivery; cyclosporine A; ionic gelation; microparticles; ulcerative colitis.

Figure 1

(A) Schematic illustration of CsAc, CsAc-AMPs, and CsAc-EAMPs fabrication using three sequential processes: fabrication of CsAc, incorporation of CsAc into the alginate microparticles, and ES coating of the microparticles. (B) SEM and microscopic images of CsA powder, CsAc, and CsAc-EAMPs. Green arrows represent CsAc loaded in CsAc-EAMPs. Yellow and red scale bars represent 10 μm and 50 μm, respectively. CsAc, cyclosporine A crystals; CsAc-AMPs, CsA crystal-loaded alginate microparticles; CsAc-EAMPs, CsA crystal-loaded, Eudragit S 100 coated alginate microparticles; SEM, scanning electron microscopy.

Figure 2

Drug release profiles of CsAc, CsAc-AMPs, and CsAc-EPMPs in media under different pH conditions and the proposed pH-dependent drug release mechanism of CsAc-EAMPs. Data are expressed as means ± standard deviation (n = 3). CsAc, cyclosporine A crystals; CsAc-AMPs, CsA crystal-loaded alginate microparticles; CsAc-EAMPs, CsA crystal-loaded, Eudragit S 100 coated alginate microparticles.

Figure 3

Macroscopic evaluation of colitis. (A) Changing profiles of disease activity index (DAI). (B) Representative colon image on day 14 (last day of the experiment). (C) Colon length. * and *** denote p < 0.05 and p < 0.001 compared to the colitis group. The results are expressed as means ± standard deviation (n = 8). CsAc, cyclosporine A crystals; CsAc-AMPs, CsA crystal-loaded alginate microparticles; CsAc-EAMPs, CsA crystal-loaded, Eudragit S 100 coated alginate microparticles.

Figure 4

Endoscopic examination of the colon on day 14. (A) Representative endoscopic images (B) Murine endoscopic index of colitis severity (MEICS) scores. *** denotes p < 0.001 compared with the untreated colitis group. The results are expressed as means ± standard deviation (n = 8). CsAc, cyclosporine A crystals; CsAc-AMPs, CsA crystal-loaded alginate microparticles; CsAc-EAMPs, CsA crystal-loaded, Eudragit S 100 coated alginate microparticles.

Figure 5

Representative histological images and histological scores of the colon, treated with or without CsA formulations. Orange and red arrows indicate damaged epithelium and infiltrated immune cells, respectively. Scale bar represents 200 µm. *** denotes p < 0.001 compared with the untreated colitis group. Data are expressed as means ± standard deviation (n = 3). CsAc, cyclosporine A crystals; CsAc-AMPs, CsA crystal-loaded alginate microparticles; CsAc-EAMPs, CsA crystal-loaded, Eudragit S 100 coated alginate microparticles.

Figure 6

Representative CLSM images of tissue sections subjected to E-cadherin immunostaining. Scale bar represents 100 µm. CsAc, cyclosporine A crystals; CsAc-AMPs, CsA crystal-loaded alginate microparticles; CsAc-EAMPs, CsA crystal-loaded, Eudragit S 100 coated alginate microparticles; CLSM, confocal laser scanning microscopy.

Figure 7

MPO activity in colon samples treated with or without CsAc formulations. *** denotes p < 0.001 compared with the untreated colitis group. Data are presented as means ± standard deviation (n = 8). CsAc, cyclosporine A crystals; CsAc-AMPs, CsA crystal-loaded alginate microparticles; CsAc-EAMPs, CsA crystal-loaded, Eudragit S 100 coated alginate microparticles; MPO, myeloperoxidase.