Enhanced therapeutic efficacy of budesonide in experimental colitis with enzyme/pH dual-sensitive polymeric nanoparticles

Abstract

Current colon-targeted drug-delivery approaches for colitis therapy often utilize single pH-triggered systems, which are less reliable due to the variation of gut pH in individuals and in disease conditions. Herein, we prepared budesonide-loaded dual-sensitive nanoparticles using enzyme-sensitive azo-polyurethane and pH-sensitive methacrylate copolymer for the treatment of colitis. The therapeutic potential of the enzyme/pH dual-sensitive nanoparticles was evaluated using a rat colitis model and compared to single pH-triggered nanoparticles. Clinical activity scores, colon/body weight ratios, myeloperoxidase activity, and proinflammatory cytokine levels were markedly decreased by dual-sensitive nanoparticles compared to single pH-triggered nanoparticles and budesonide solution. Moreover, dual-sensitive nanoparticles accumulated selectively in inflamed segments of the colon. In addition, dual-sensitive nanoparticle plasma concentrations were lower than single pH-triggered nanoparticles, and no noticeable in vitro or in vivo toxicity was observed. Our results demonstrate that enzyme/pH dual-sensitive nanoparticles are an effective and safe colon-targeted delivery system for colitis therapy.

Keywords: azo-polyurethane; budesonide; colitis; colon-targeted nanoparticles; methacrylate copolymer.

Figure 1

Characterization of ES NPs and Azo-pu ES NPs. Notes: (A) SEM images; (B) size histograms by qNano; (C) DSC thermogram of budesonide, polymers, and NPs; (D) in vitro drug release from ES NPs and Azo-pu ES NPs at different pH values. Abbreviations: NPs, nanoparticles; ES, Eudragit S100; Azo-pu, azo-polyurethane; SEM, scanning electron microscopy; DSC, differential scanning calorimetry.

Figure 2

Clinical activity index during the whole experimental period and macroscopic evaluation of colitis treatment. Notes: (A) Clinical activity-score system for the colitis control group and budesonide-treated groups during the experimental period (error bars not shown for clarity reasons, n=5 animals/group, *P<0.05 compared with colitis control); (B) macroscopic evaluation of the colon; (C) colon length (***P<0.001); (D) open distal colon photographs; (E) colon/body-weight ratio (*P<0.05, ***P<0.001). Healthy group, rats not treated with TNBS; colitis control, rats treated with TNBS; budesonide solution group, colitis rats treated with budesonide in solution form; ES NPs group, colitis rats treated with budesonide-loaded ES NPs; Azo-pu ES NP group, colitis rats treated with budesonide loaded Azo-pu ES NPs administered by oral gavage. Abbreviations: NPs, nanoparticles; ES, Eudragit S100; Azo-pu, azo-polyurethane; TNBS, 2,4,6-trinitrobenzenesulfonic acid.

Figure 3

Histological evaluation of colon tissue from the colitis control and budesonide-treated groups. Notes: Representative microphotographs of healthy control, rats not treated with TNBS; colitis control, rats treated with TNBS; budesonide solution, colitis rats treated with budesonide in solution form, ES NPs, colitis rats treated with budesonide-loaded ES NPs; Azo-pu ES NPs, colitis rats treated with budesonide loaded Azo-pu ES NPs administered by oral gavage. (A) Hematoxylin and eosin staining for microscopic evaluation of the colon sections isolated from healthy control, colitis control, and budesonide-treated groups. Images of tissues are shown with 100× magnification. (B) Histological score of the colitis control and budesonide-treated groups. Data are presented as means ± standard deviation (n=3 animals/group). Azo-pu ES NPs showed statistically significant differences (*P<0.05, ***P<0.001) compared to the colitis control. Abbreviations: NPs, nanoparticles; ES, Eudragit S100; Azo-pu, azo-polyurethane; TNBS, 2,4,6-trinitrobenzenesulfonic acid.

Figure 4

Average MPO activity and cytokine expression in the healthy control group, colitis control group, and treated groups. Notes: (A) MPO assay; (B) IL-6; (C) TNF-α. Statistical comparisons were evaluated between the inflamed control group versus budesonide solution, ES, and Azo-pu ES NPs (*P<0.05, **P<0.01). Abbreviations: NPs, nanoparticles; ES, Eudragit S100; Azo-pu, azo-polyurethane; MPO, myeloperoxidase.

Figure 5

In vivo localization of ES NPs and Azo-pu ES NPs in healthy and inflamed colons. Notes: (A) Confocal images of C-6-loaded Azo-pu ES NPs in healthy control and inflamed control group colon cross sections prepared 24 hours after oral NP administration; (B) quantitative determination of C-6 in healthy and inflamed colon tissue 24 hours after oral administration of ES NPs and Azo-pu ES NPs. Data are presented as means ± standard deviation (n=3 animals/group, **P<0.01). Healthy colon, colon section from healthy rats; inflamed colon, colon section from 2,4,6-trinitrobenzenesulfonic acid-induced colitis rats. Abbreviations: NPs, nanoparticles; ES, Eudragit S100; Azo-pu, azo-polyurethane; C-6, coumarin 6.

Figure 6

Pharmacokinetic and blood glucose-level evaluations of colon-targeted delivery of ES NPs and Azo-pu ES NPs. Notes: (A) Plasma concentration of C-6 after oral administration of C-6 solution, ES NPs, and Azo-pu ES NPs. (B) Blood glucose levels in animals treated with budesonide solution, ES NPs, and Azo-pu ES NPs. Data are presented as means ± standard deviation (n=3 animals/group, *P<0.05, **P<0.01). Abbreviations: NPs, nanoparticles; ES, Eudragit S100; Azo-pu, azo-polyurethane; C-6, coumarin 6; C_max, concentration maximum; T_max, time to C_max; AUC, area under curve.

Figure 7

In vivo and in vitro biocompatibility study of Azo-pu ES NPs. Notes: (A) Representative photomicrographs of the liver and kidney of colitis rats treated with Azo-pu ES NPs. (B) Small intestine and colon hematoxylin and eosin-stained sections (200× magnification) of healthy rats treated with blank Azo-pu ES NPs. Rats without any treatment were regarded as the control group (n=3 animals/group). (C) Changes in body weight of healthy rats treated with blank Azo-pu ES NPs. (D) In vitro cytotoxicity of blank Azo-pu ES NPs in the HT29 and HCT116 cell lines. Abbreviations: NPs, nanoparticles; ES, Eudragit S100; Azo-pu, azo-polyurethane.

Figure 8

Proposed mechanism of budesonide release from the single system (pH-sensitive NPs) and dual system (enzyme pH-sensitive NPs) under gastrointestinal tract-mimicking conditions. Abbreviations: NPs, nanoparticles; Azo-pu, azo-polyurethane.