Oral Delivery of Astaxanthin via Carboxymethyl Chitosan-Modified Nanoparticles for Ulcerative Colitis Treatment

Abstract

The oral delivery strategy of natural anti-oxidant and anti-inflammatory agents has attracted great attention to improve the effectiveness of ulcerative colitis (UC) treatment. Herein, we developed a novel orally deliverable nanoparticle, carboxymethyl chitosan (CMC)-modified astaxanthin (AXT)-loaded nanoparticles (CMC-AXT-NPs), for UC treatment. The CMC-AXT-NPs were evaluated by appearance, morphology, particle size, ζ-potential, and encapsulation efficiency (EE). The results showed that CMC-AXT-NPs were nearly spherical in shape with a particle size of 34.5 nm and ζ-potential of -30.8 mV, and the EE of CMC-AXT-NPs was as high as 95.03%. The CMC-AXT-NPs exhibited preferable storage stability over time and well-controlled drug-release properties in simulated intestinal fluid. Additionally, in vitro studies revealed that CMC-AXT-NPs remarkably inhibited cytotoxicity induced by LPS and demonstrated superior antioxidant and anti-inflammatory abilities in Raw264.7 cells. Furthermore, CMC-AXT-NPs effectively alleviated clinical symptoms of colitis induced by dextran sulfate sodium salt (DSS), including maintaining body weight, inhibiting colon shortening, and reducing fecal bleeding. Importantly, CMC-AXT-NPs suppressed the expression of pro-inflammatory cytokines like TNF-α, IL-6, and IL-1β and ameliorated DSS-induced oxidative damage. Our results demonstrated the potential of CMC-modified nanoparticles as an oral delivery system and suggested these novel AXT nanoparticles could be a promising strategy for UC treatment.

Keywords: anti-inflammatory; astaxanthin; carboxymethyl chitosan; nanoparticle; ulcerative colitis.

Figure 1

(A) Synthesis scheme of the CMC-AXT-NPs. (B) The appearance photograph of carrier and CMC-AXT-NPs. (C) Particle size distribution, (D) TEM images, (E) EE% and LC% of CMC-AXT-NPs. (F) FT-IR spectra of CMC, AXT-LIPs, and CMC-AXT-NPs. (G) Particle size of carrier and CMC-AXT-NPs after storage at 4 °C for 7 days. (H) In vitro release profile of AXT. Scale bar: 20 nm. Data are presented as mean ± SEM. AXT, astaxanthin; CMC, carboxymethyl chitosan; AXT-LIP, AXT-loaded liposomes; Carrier, CMC-modified AXT-unloaded nanoparticles; CMC-AXT-NPs, CMC-modified AXT-loaded nanoparticles; TEM, transmission electron microscopy; EE, encapsulation efficiency; LC, loading capacity; FT-IR, Fourier-transform infrared spectroscopy; SGF, stimulated gastric fluid; SIF, simulated intestinal fluid.

Figure 2

Cell cytotoxicity assessment. (A) Cell viability of Raw264.7 cells treated with different concentrations of CMC-AXT-NPs for 24 h. (B) Cell viability and (C) the level of LDH in Raw264.7 cells stimulated with LPS. Data are presented as mean ± standard error of the mean (SEM); * p < 0.05, ** p < 0.01, *** p < 0.001. NPs, CMC-AXT-NPs; LPS, lipopolysaccharide; LDH, lactate dehydrogenase.

Figure 3

The inflammation factor levels of (A) TNF-α, (B) IL-6, and (C) IL-1β in Raw264.7 cells treated with LPS. The levels of (D) SOD, (E) GSH, and (F) MDA in Raw264.7 cells stimulated with H₂O₂. Data are presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001. SOD, superoxide dismutase; GSH, glutathione; MDA, malondialdehyde.

Figure 4

(A) Body weight, (B) average weight change, and (C) DAI in different treated groups. (D) Optical photographs of colons and (E) colon length in different treated groups. Data are presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001. DSS, dextran sulfate sodium; DAI, disease activity index.

Figure 5

(A) H&E staining and PAS staining of colon tissues in different treated groups. (B) Histological score of colon tissues in different treated groups. (C) Crypt depth of colon tissues in different treated groups. Scale bar: 100 μm. Data are presented as mean ± SEM; * p < 0.05, *** p < 0.001. H&E, hematoxylin and eosin; PAS, periodic acid–Schiff.

Figure 6

(A) Immunohistochemistry staining of F4/80 and Ly6G of colon tissues in different treated groups. The levels of (B) TNF-α, (C) IL-6, and (D) IL-1β in the serum of different treated groups. Scale bar: 100 μm. Data are presented as mean ± SEM; *** p < 0.001.

Figure 7

The (A) SOD activity, (B) GSH level, and (C) MPO activity in colon tissues of different treated groups. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. MPO, myeloperoxidase.

Figure 8

Schematic representation of UC experiment in vivo.