Oral Delivery of Nanoparticles Loaded With Ginger Active Compound, 6-Shogaol, Attenuates Ulcerative Colitis and Promotes Wound Healing in a Murine Model of Ulcerative Colitis

Abstract

Background and aims: Oral drug delivery is the most attractive pathway for ulcerative colitis [UC] therapy, since it has many advantages. However, this strategy has encountered many challenges, including the instability of drugs in the gastrointestinal tract [GT], low targeting of disease tissues, and severe adverse effects. Nanoparticles capable of colitis tissue-targeted delivery and site-specific drug release may offer a unique and therapeutically effective system that addresses these formidable challenges.

Methods: We used a versatile single-step surface-functionalising technique to prepare PLGA/PLA-PEG-FA nanoparticles loaded with the ginger active compound, 6-shogaol [NPs-PEG-FA/6-shogaol]. The therapeutic efficacy of NPs-PEG-FA/6-shogaol was evaluated in the well-established mouse model of dextran sulphate sodium [DSS]-induced colitis.

Results: NPs-PEG-FA exhibited very good biocompatibility both in vitro and in vivo. Subsequent cellular uptake experiments demonstrated that NPs-PEG-FA could undergo efficient receptor-mediated uptake by colon-26 cells and activated Raw 264.7 macrophage cells. In vivo, oral administration of NPs-PEG-FA/6-shogaol encapsulated in a hydrogel system [chitosan/alginate] significantly alleviated colitis symptoms and accelerated colitis wound repair in DSS-treated mice by regulating the expression levels of pro-inflammatory [TNF-α, IL-6, IL-1β, and iNOS] and anti-inflammatory [Nrf-2 and HO-1] factors.

Conclusions: Our study demonstrates a convenient, orally administered 6-shogaol drug delivery system that effectively targets colitis tissue, alleviates colitis symptoms, and accelerates colitis wound repair. This system may represent a promising therapeutic approach for treating inflammatory bowel disease [IBD].

Keywords: Ulcerative colitis; drug delivery system; therapy.

Figure 1.

Preparation and characterisation of 6-shogaol loaded polymeric nanoparticles. A: Schematic illustration of process through which PLGA/PLA-PEG-FA nanoparticles [NPs-PEG-FA] were fabricated using a versatile single-step surface-functionalising technique. B: The morphology of PLGA/PLA-PEG nanoparticles [NPs-PEG] was characterised by transmission electron microscopy [TEM], and their size and zeta potential were measured by dynamic light scattering [DLS] using a Malvern Zetasizer Nano ZS90 Apparatus. C: The morphology, size, and zeta potential of PLGA/PLA-PEG-FA [NPs-PEG-FA] were characterised.

Figure 2.

NPs-PEG-FA is biocompatible. To evaluate the biocompatibility of NPs-PEG-FA in vivo, mice were orally administered wNPs-PEG-FA [15 mg in PBS] or phosphate-buffered solution [PBS] [control] for 7 days. Experiments were performed on five mice per group [n = 5] throughout. A: Body weight change. Body weights were measured in each group and normalised by the corresponding initial body weight. B: Blood was collected by intracardiac puncture with a syringe, and haematological analyses were performed using an automatic haematology analyser [VetScan HM5; Abaxis, CA, USA]. The following haematologic parameters are shown: WBC, total white blood cells; LYM, lymphocytes; MON, monocytes; NEU, neutrophils; RBC, red blood cells; HGB, haemoglobin; PLT, platelets; ALT, alanine aminotransferase; and AST, aspartate aminotransferase. C: Blood was collected and biochemical analyses were performed using a biochemical analyser [VetScan VS2; Abaxis, CA, USA] with a comprehensive diagnostic profile reagent rotor. The following biochemical parameters were assessed: ALT, alanine aminotransferase; ALB, albumin; ALP, alkaline phosphatase; AMY, amylase; CA, calcium; CRE, creatinine; GLOB, globulin; GLU, glucose; PHOS, phosphorus; TBIL, total bilirubin; TP, total protein; and BUN, urea nitrogen. D: haematoxylin and eosin [H＆E] staining of histological sections was used to assess the toxicity of NPs-PEG-FA toward gastro-intestinal tissues [stomach, duodenum, jejunum, ileum, and colon].

Figure 3.

NPs-PEG-FA are taken up by macrophages and epithelial cells through folate receptor-mediated endocytosis. A: Colon-26 cells and activated Raw 264.7 cells were incubated with NPs-PEG/DiL or NP-PEG-FA/DiL for 6 h. Here, DiL was a fluorescent dye and used as a tracker. The colours are as follows: blue channel, DAPI; green channel, FITC; and red channel, DiL. Panels: a, colon-26 cells incubated with NPs-PEG/DiL; b, colon-26 cells incubated with NPs-PEG-FA/DiL; c, activated Raw 264.7 cells incubated with NPs-PEG/DiL; and d, activated Raw 264.7 cells incubated with NPs-PEG-FA/DiL. B and C: Flow cytometry was used to quantify the fluorescence intensity of DiL in colon-26 cells [B] and activated Raw 264.7 cells [C] treated as described above and incubated for 6 h. In all experiments, n = 5; the scale bar in [A] = 20 µm.

Figure 4.

Folate mediates the uptake of NPs-PEG-FA by macrophages and epithelial cells. Colon-26 cells and activated Raw 264.7 cells were incubated with NPs, NPs-PEG, NPs-PEG-FA-1, NPs-PEG-FA-2, or NPs-PEG-FA-3. NPs-PEG-FA-1, 2, and 3 were defined as 10%, 15%, and 20% [w/w] of PLA-PEG-FA in total polymers. After 1, 3, or 5 h of incubation, the cells were rinsed three times with cool phosphate-buffered saline [PBS] and harvested for flow cytometry analysis; uptake efficiency was evaluated according to the fluorescence intensity [n = 5].

Figure 5.

NPs-PEG-FA uptake is increased during colitis in mice. A: DiR-loaded NPs-PEG and NPs-PEG-FA were orally administered to colitic mice, and images were obtained at 6 h and 12 h post-administration using an in vivo imaging system [IVIS]. B: Fluorescence intensities were quantified and compared in the colon at 6 h and 12 h post-dosing [n = 3]. C: Colitis tissue uptake of orally administered NPs-PEG-FA in hydrogel at 12 h post-dosing. Fixed colitis tissues were stained with FITC-phalloidin [green channel] and DAPI [blue channel] for visualisation of actin and nuclei, respectively. Scale bar = 50 µm.

Figure 6.

Protective effect of NPs-PEG-FA/6-shogaol on dexrtran sodium sulphate [DSS]-induced colitis. A: enzyme-linked immunosorbent assay [ELISA] was used to quantify faecal lipocalin-2 [Lcn-2] levels in mice on Days 1, 3, 5, and 7 of DSS treatment with or without co-administration of NPs-PEG-FA/6-shogaol. B: Bioluminescence images of mice treated with an inflammation probe [XenoLight^TM; MA, USA], as captured by IVIS. Mice were intraperitoneally [i.p.] injected with inflammation probe [200 mg/kg; 150 µL/mouse]. At 5 min post-injection, images were obtained using a 5-min exposure time [for improved sensitivity]. C: The bioluminescence intensities of mice were quantified and compared between groups [n = 5]. D: At the end of the experiment, mice were sacrificed and the spleen/body weight of each group was compared [n = 5]. E: Colon histology was examined by haematoxylin and eosin [H＆E] staining. At the end of experiment, colons were collected from the different treatment groups, 6-µm thick tissue sections were prepared, H＆E staining was applied, and histological assessment was performed. Inflammatory cells in the lamina propria are indicated by arrowheads. Representative sections are shown [n = 5/group]. Scale = 20 µm. Panels: a, control mice; b, DSS-treated mice; c, DSS plus NPs-PEG-FA/6-shogaol treated mice; and d: DSS plus free 6-shogaol-treated mice. F: The mRNA expressions of various genes were quantified by real-time polymerase chain reaction [PCR] [n = 5]; ***p < 0.001.

Figure 7.

Therapeutic effect of NPs-PEG-FA/6-shogaol on DSS-induced colitis. A: enzyme-linked immunosorbent assay [ELISA] was used to quantify faecal Lcn-2. During the dextran sodium sulphate [DSS] colitis phase, mice of all groups were given DSS water. During the recovery phase, mice were given plain water and the appropriate groups were orally treated with NPs-PEG-FA/6-shogaol or free 6-shogaol for 7 days [n = 5]. B: Bioluminescence images of mice were obtained using the inflammation probe and IVIS, and intensities were compared [n = 5]. C: Haematoxylin and eosin [H＆E] staining was used for histological assessment. Inflammatory cells in the lamina propria are indicated by arrowheads [n = 5]. Panels: a, control mice; b, DSS-treated mice; c, DSS plus NPs-PEG-FA/6-shogaol-treated mice; and d, DSS plus free 6-shogaol-treated mice. D: The mRNA expressions of various genes were quantified by real-time polymerase chain reaction [PCR] [n = 5]. Symbols: *p < 0.05; ***p < 0.001; and ns, no significant difference.