Hesperidin-loaded Eudragit S100 nanoparticles alleviate ulcerative colitis by repairing intestinal barrier and modulating gut microbiota

Abstract

Ulcerative colitis (UC) is a chronic inflammatory bowel disease with no curative therapy. Hesperidin (HDN) exhibits potent anti-inflammatory and antioxidant properties, but its poor solubility limits its therapeutic application. Here, we developed a colon-targeted nano-formulation by encapsulating HDN in pH-responsive Eudragit S100 nanoparticles (HDN-EUD NPs). The nanoparticles were optimized using Box-Behnken Design, yielding uniform spherical morphology with a mean particle size of 174.4 nm and encapsulation efficiency of 83.98%. HDN-EUD NPs remained stable in simulated gastric and intestinal fluids but rapidly disintegrated in simulated colonic fluid, releasing 74% of HDN within 2 h. At the cellular level, HDN-EUD NPs exhibited excellent biocompatibility and significantly enhanced protection against H₂O₂-induced oxidative stress and apoptosis. In vivo biodistribution confirmed prolonged colonic retention of HDN-EUD NPs. In a DSS-induced UC mouse model, HDN-EUD NPs treatment significantly alleviated disease symptoms, as evidenced by attenuated body weight loss, reduced disease activity index (2.06 vs. 3.61), and restored colon length (6.8 cm vs. 4.5 cm) compared to the DSS group. Mechanistically, HDN-EUD NPs repaired the intestinal barrier by upregulating tight junction proteins ZO-1 and Occludin, downregulated pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and myeloperoxidase activity (from 3.56 to 1.02 U/g) in colon tissue, and restored gut microbiota balance by increasing beneficial bacteria (Prevotellaceae, Bifidobacteriaceae) while decreasing harmful taxa (Sutterellaceae, Bacteroidaceae). Biosafety assessments revealed no obvious toxicity. Overall, this colon-targeted nano-formulation effectively delivered HDN to the colon and enhanced its therapeutic efficacy, representing a promising preclinical candidate for UC therapy.

Introduction

Ulcerative colitis (UC) is a chronic idiopathic inflammatory bowel disease, characterized by continuous mucosal inflammation that typically starts in the rectum and extends proximally (Bera et al., 2025; Le Berre et al., 2023). Since the start of the 21st century, the global incidence of UC has steadily increased, drawing significant attention from the medical and public health communities worldwide (Chen et al., 2018). This trend is particularly prominent in newly industrialized regions such as parts of Asia, Africa, and South America (Loayza et al., 2023).

Due to its high recurrence rate, challenging treatment, prolonged duration, and associated cancer risks, UC is often referred to as “green cancer” (Wangchuk et al., 2024). While the precise etiology of UC remains unclear, emerging evidence suggests that its pathogenesis is driven by complex, multifactorial interactions. These include dysbiosis of the gut microbiota, dysfunction of the intestinal epithelial barrier, genetic predispositions, and dysregulated immune responses, which together contribute to the initiation and progression of the disease (Sun et al., 2022). Clinically, UC is characterized by diarrhea, abdominal pain, mucopurulent bloody stools, and weight loss. Treatment strategies such as aminosalicylic acid, corticosteroids, immunosuppressants, and tumor necrosis factor inhibitors are tailored according to the severity of the disease, aiming to reduce inflammation and promote tissue repair. For mild-to-moderate UC, 5-aminosalicylic acid (5-ASA) is typically the first-line pharmacotherapeutic agent. However, long-term use of 5-ASA is associated with various adverse effects, including nephrotoxicity, hepatotoxicity, and multiorgan dysfunction (Gao et al., 2022). Other therapeutic options are also associated with several limitations, including low bioavailability, poor patient tolerance, and a range of adverse reactions induced by long-term administration. Therefore, there is an urgent need to develop novel, safe, effective, and economic strategies to treat UC.

There is increasing interest in using natural compounds for UC intervention, primarily due to their relatively lower side effect profiles compared to conventional therapeutic agents. Both preclinical studies and clinical trials have demonstrated significant anti-inflammatory effects of flavonoids in the context of UC. Hesperidin (C28H34O15, HDN) is a natural flavanone glycoside, widely found in citrus fruits (Rutaceae family) (Pereira et al., 2025). In the pharmaceutical and food industries, HDN has garnered considerable attention for its anti-inflammatory, antioxidant, lipid-lowering, and insulin-sensitizing properties (Quiles et al., 2022). Specifically, HDN exhibits intestinal protective effects via antioxidative and anti-inflammatory mechanisms, modulating inflammatory responses induced by Clostridium infection, and rectifying CD4+/CD8+ T-cell imbalance (Ruiz-Iglesias et al., 2020). It can also regulate the intestinal flora and promote the intestinal health. Estruel Amades et al. found that adding 200 mg/kg HDN to rat feed increased the total bacterial count in the cecum and the proportion of beneficial bacteria such as Lactobacillus and Bifidobacterium, thus contributing to intestinal homeostasis (Estruel-Amades et al., 2019). Furthermore, HDN could enhance colonic fluid absorption, mitigate oxidative damage, and reduce intestinal inflammation in murine models (Mirzaei et al., 2023). Despite these confirmed therapeutic effects, the clinical translation of HDN is hindered by the poor aqueous solubility, low bioavailability, and physicochemical instability in the gastrointestinal (GI) tract (Hoang et al., 2026). Therefore, innovative formulations of HDN are crucial for enhancing its therapeutic potential.

In recent years, conventional formulation approaches including cyclodextrin inclusion, solid dispersions, microparticles, gels, and microemulsions have been used to improve solubility, stability and absorption of HDN. Furthermore, novel nano-formulations are also developed to enhance the pharmacokinetics and efficacy of HDN, such as nanosuspensions, solid-lipid nanoparticles, liposomes, exosomes, and nanofibers (Hoang et al., 2026). Among them, the oral colon-targeted drug delivery systems are considered as an innovative pharmaceutical technology for UC therapy. In contrast to current treatment strategies, they can prevent premature drug release in the upper gastrointestinal tract (stomach and small intestine) and enable site-specific delivery to the colon, where HDN can exert its local or systemic therapeutic effects (Kumar et al., 2024; Zhou et al., 2025). Polymers, such as polysaccharide (e.g., alginate, chitosan, and pectin) and polyacrylic acid derivatives (e.g., Eudragit and Carbomer) have been applied for colon-targeted drug delivery systems (Kang et al., 2024; Li et al., 2025). Among them, Eudragit S100 (EUD) is the most widely used colon-targeted excipient. EUD is a pH-sensitive copolymer, which remains insoluble in acidic gastric environments but dissolves at colonic pH (> 7.0). Colon-specific Eudragit S100 nanoparticles (EUD NPs) have been widely applied to deliver anti-inflammatory agents, such as cyclosporin A, 5-ASA, Periplaneta americana extract. The nanoparticles can enhance the solubility of anti-inflammatory agents by incorporating them in an amorphous form, improve their stability by shielding them from acidic and enzymatic degradation, and ensure the targeted release in the colon—resulting in a significantly enhanced therapeutic effect against UC (Li et al., 2022a; Nikam et al., 2023). However, studies on EUD NPs loaded with HDN for the treatment of UC are remain limited up to now. Importantly, the current nano-formulations for UC therapy often lack systematic optimization of preparation parameters to achieve the best HDN loading (DL) and encapsulation efficiency (EE).

In this work, we aimed to develop an oral colon-targeted nano-formulation by encapsulating HDN within Eudragit S100 nanoparticles (HDN-EUD NPs) to improve its therapeutic effect against UC. In order to achieve high DL and EE, Box-Behnken design (BBD) was used to optimize the key parameters. BBD is a statistical tool based on mathematical techniques used to investigate individual factor effects, parameter interactions, and process optimization. As a form of response surface methodology (RSM), BBD offers the key advantages of avoiding extreme experimental conditions and minimizing the number of required runs (Khan et al., 2024). As shown in Fig. 1, we hypothesized that HDN-EUD NPs could remain stable in stomach and small intestine, and disintegrate in the colon, thereby achieving the site-specific release of HDN. Subsequently, the released HDN exerting anti-inflammatory and antioxidant effects to ameliorate UC. This formulation could address several key challenges: it improved HDN’s poor solubility, enhanced colloidal stability, achieved high HDN loading (DL) and encapsulation efficiency (EE), and released HDN in the colon. Accordingly, in vitro and in vivo experiments confirmed that HDN-EUD NPs precisely released HDN in the colon, inhibited cellular apoptosis, repaired intestinal barrier damage, and modulated the gut microbiota, thereby alleviating DSS-induced UC.

Download the full article as PDF here Hesperidin-loaded Eudragit S100 nanoparticles alleviate ulcerative colitis by repairing intestinal barrier and modulating gut microbiota

or continue reading here

Materials

HDN was obtained from Macklin (Shanghai, China). Eudragit S100 was acquired from Evonik Nutrition & Care Gmbh (Berlin, Germany). Polyvinyl alcohol (PVA) and dextran sulfate sodium (DSS) were procured from MP Biomedicals (Santa Ana, CA, USA). Cell Counting Kit-8 (CCK-8) and 4% paraformaldehyde fixation solution were obtained from Beyotime Biotech (Shanghai, China). Bovine serum albumin (BSA) was purchased from Seven Biotech Co., Ltd. (Beijing, China). Polysorbate 80 (Tween 80) was obtained from Adamas-Beta Co., Ltd. (Shanghai, China). Citrate antigen retrieval buffer (pH 6.0), EDTA antigen retrieval buffers (pH 8.0 and pH 9.0), phosphate-buffered saline (PBS), tissue autofluorescence quencher, and 4′,6-diamidino-2-phenylindole (DAPI) staining reagent were provided by Servicebio Technology Co., Ltd. (Wuhan, China). Propidium iodide (PI) and Calcein-AM were bought from Solarbio Science & Technology Co., Ltd. (Beijing, China). TNF-α, IL-1β, and IL-6 enzyme-linked immunosorbent assay (ELISA) kits were obtained from Elabscience Biotechnology Co., Ltd. (Wuhan, China). All other chemicals were of analytical grade and obtained from commercial suppliers.

Jiazheng Zhou, Junjie Li, Monong Su, Xingxuan Zhou, Yan Yang, Yuxin Wang, Xiaohan Li, Jingyu Wang, Liang Wang, Jianbin Zhang, Hesperidin-loaded Eudragit S100 nanoparticles alleviate ulcerative colitis by repairing intestinal barrier and modulating gut microbiota, International Journal of Pharmaceutics: X, Volume 11, 2026, 100524, ISSN 2590-1567, https://doi.org/10.1016/j.ijpx.2026.100524.

Are you looking for excipients in commercial quantities?