Oral administration of nanozyme-armed probiotic Escherichia coli Nissle 1917 with ROS scavenging for inflammatory bowel disease therapy

Abstract

Inflammatory bowel disease (IBD) represents a group of chronic, non-specific gastrointestinal disorders characterized by pathological overproduction of reactive oxygen species (ROS). Current therapeutic interventions, including conventional drugs and oral probiotics, are frequently limited by their vulnerability to the harsh gastrointestinal environment. To address these challenges, we have developed a novel engineered bacterium, designated as CeO₂@EcN-L, through the integration of cerium oxide nanozymes (CeO₂) with the probiotic strain Escherichia coli Nissle 1917 (EcN), followed by encapsulation with a pH-responsive anionic copolymer composed of methacrylic acid and ethyl acrylate (Eudragit L100–55). The negatively charged surface of the Eudragit L100–55 coating enables CeO₂@EcN-L to not only selectively adhere to positively charged inflamed colon tissues through electrostatic interactions but also achieve sustained release of therapeutic agents throughout the gastrointestinal tract. Upon accumulation in the inflamed colon, the CeO₂ component demonstrates dual enzymatic activities, mimicking both catalase (CAT) and superoxide dismutase (SOD), which collectively facilitate efficient ROS scavenging and subsequent inflammation alleviation. In a dextran sulfate sodium (DSS)-induced murine IBD model, CeO₂@EcN-L demonstrate remarkable anti-inflammatory efficacy and significantly restore gut microbiota homeostasis. This study establishes a novel, safe, and effective therapeutic strategy for IBD treatment, offering promising potential for clinical translation.

Highlights

• The CeO₂@EcN-L Hybrid, an innovative fusion of E. coli Nissle 1917 and CeO₂ Nanozymes, employs a pH-responsive coating for accurate colon-specific delivery.
• Dual therapy: ROS scavenging, TNF-α/IL-6 suppression, and IL-10 elevation alleviate colitis in DSS-treated mice.
• Gut microbiota modulation: Enriches Akkermansia and Faecalibaculum with high biosafety, supporting IBD treatment translation.

Introduction

Inflammatory bowel diseases (IBD), encompassing ulcerative colitis (UC) and Crohn’s disease (CD), represent a group of chronic, non-specific gastrointestinal inflammatory disorders with complex pathogenesis [[1], [2], [3]]. Clinically, IBD manifests through a spectrum of symptoms including abdominal pain, chronic diarrhea, hematochezia, mucus discharge, and unintended weight loss [4,5]. Notably, long-term IBD patients face a substantially elevated risk of developing colorectal cancer, highlighting the critical need for effective therapeutic interventions [6]. Current clinical treatment for IBD primarily relies on steroidal medications, nonsteroidal anti-inflammatory drugs, and advanced biologics such as monoclonal antibodies [7,8]. However, these conventional therapies often yield suboptimal outcomes due to their non-specific targeting and associated systemic side effects [[9], [10], [11]]. While several targeted therapeutic strategies have been investigated, the hostile gastrointestinal environment continues to pose significant challenges for effective drug delivery [[12], [13], [14], [15]]. The pathophysiology of IBD, though not fully elucidated, is characterized by two prominent features: excessive reactive oxygen species (ROS) production and accumulation of positively charged proteins in inflamed tissues [[16], [17], [18], [19]]. During active inflammation, the intestinal mucosa becomes infiltrated with immune cells that release substantial quantities of ROS and pro-inflammatory cytokines, including interleukins (IL)-1β, IL-6, IL-10, and tumor necrosis factor α (TNF-α). The overproduction of ROS results in oxidative damage to DNA, proteins, and lipids, potentially contributing to the initiation and progression of IBD [[20], [21], [22]]. Furthermore, the disrupted redox balance adversely affects the gut microbiota composition, creating a vicious cycle of inflammation and microbial dysbiosis [[23], [24], [25]]. Consequently, therapeutic strategies that simultaneously target inflammatory sites and mitigate oxidative stress represent a promising avenue for IBD management.

In recent years, nanozymes have captured considerable interest and have been extensively investigated owing to their remarkable characteristics [[26], [27], [28]]. While natural enzymes demonstrate superior catalytic efficiency, their application is limited by inherent instability and high production costs [29,30]. Nanozymes, in contrast to natural enzymes, confer a multitude of benefits such as superior stability, increased durability, and a simpler preparation process [31,32]. Significantly, nanozymes are capable of effectively scavenging ROS by mimicking the activities of superoxide dismutase (SOD), catalase (CAT), or peroxidase (POD), positioning them as promising therapeutic agents for the management of inflammatory diseases [33]. Among these nanozymes, cerium oxide (CeO₂) nanoparticles, a widely studied nanozyme with both CAT- and SOD-like activities, have been utilized in the treatment of ROS-related diseases, including IBD [34]. However, the therapeutic application of bare CeO₂ nanoparticles in IBD is constrained by their positive surface charge and potential systemic toxicity [35], necessitating innovative delivery strategies to enhance their therapeutic efficacy and safety profile.

Probiotics, particularly well-characterized strains such as Escherichia coli Nissle 1917 (EcN), have demonstrated significant potential in IBD management through their immunomodulatory effects and ability to restore gut microbial homeostasis [[36], [37], [38], [39]]. These beneficial microorganisms exert their therapeutic effects by enhancing mucosal barrier function, modulating immune responses, and competing with pathogenic bacteria. Nevertheless, the clinical application of probiotics is hampered by their vulnerability to the harsh gastrointestinal environment and reduced viability in inflammatory conditions characterized by elevated ROS levels [[40], [41], [42], [43]]. To address these limitations, researchers have explored various protective strategies, including the use of pH-sensitive polymers such as Eudragit L100–55—a methacrylic acid-ethyl acrylate copolymer that dissolves at pH > 5.5. This enteric coating technology has shown promise in protecting probiotics from gastric acid while enabling targeted release in the intestinal environment [44,45].

In this study, we developed an innovative probiotic-nanozyme hybrid platform (CeO₂@EcN-L) for targeted IBD therapy. This system integrated the therapeutic benefits of EcN with the ROS-scavenging capabilities of CeO₂ nanozymes, encapsulated within a protective Eudragit L100-55 coating (Scheme 1). Following oral administration, Eudragit L100-55 protected the probiotic-nanozyme complex from gastric degradation, ensuring targeted delivery to inflamed colonic tissues. The system’s therapeutic efficacy was achieved through two complementary mechanisms: (1) electrostatic-mediated adhesion to positively charged inflamed tissues, enabling sustained release and enhanced local accumulation, and (2) ROS scavenging through the dual enzymatic activities of CeO₂ nanoparticles upon polymer dissolution. In a dextran sulfate sodium (DSS)-induced murine IBD model, CeO₂@EcN-L demonstrated remarkable anti-inflammatory effects and significantly improved gut microbial composition, particularly enhancing beneficial genera such as Akkermansia and Faecalibaculum. Comprehensive biosafety assessments through hematological and histological analyses confirmed the system’s favorable safety profile. This study presents a novel therapeutic paradigm that combines the benefits of probiotic therapy with nanozyme technology, offering a targeted, safe, and effective approach for IBD management.

The synthesis of CeO₂@EcN-L was carried out as depicted in Scheme 1a. Escherichia coli Nissle 1917 (EcN), a well-established probiotic strain, was employed as the biological carrier for CeO₂ nanozymes. The synthesis process involved three key steps: (1) metabolic labeling of EcN with 2-azido-2-deoxy-d-glucose to generate azide-functionalized EcN (EcN-N3), (2) functionalization of CeO₂ nanozymes with dibenzocyclooctyne-azide (DBCO) to produce DBCO-CeO₂, and (3) conjugation of the two components via bio-orthogonal strain-promoted azide-alkyne cycloaddition (SPAAC) to form the CeO₂@EcN-L probiotic-nanozyme platform. Transmission electron microscopy (TEM) analysis revealed that native EcN exhibited a characteristic rod-like morphology with smooth surface topology, measuring approximately 2 μm in length and 0.5 μm in diameter (Fig. 1a). Following functionalization, CeO₂@EcN displayed a distinct roughened surface texture with a slight increase in overall dimensions (Fig. S1). Additionally, TEM imaging also confirmed the formation of a continuous CeO₂ coating on the bacterial surface without significant alteration of the core structure (Fig. 1b). Elemental composition analysis via energy-dispersive X-ray spectroscopy (EDX) detected the presence of cerium alongside carbon, nitrogen, and oxygen, providing definitive evidence of successful CeO₂ deposition (Fig. 1c). Dynamic light scattering (DLS) measurements indicated a hydrodynamic diameter for CeO₂@EcN-L, consistent with TEM observations (Fig. 1d). UV–vis spectroscopy analysis revealed a characteristic absorption peak at 327 nm, corresponding to the CeO₂ component (Fig. 1e). Surface charge analysis demonstrated that CeO₂@EcN-L exhibited a zeta potential of −13.3 ± 0.75 mV, a property attributed to the Eudragit L100–55 coating that enhances targeting to positively charged inflammatory sites in the colon (Fig. 1f).

Next, the ROS-scavenging capacity of CeO₂@EcN-L was evaluated against superoxide (●O₂−) and hydrogen peroxide (H₂O₂). CeO₂@EcN-L demonstrated concentration-dependent SOD-like activity (Fig. 2a). Similarly, its catalase (CAT)-like activity was significantly higher than EcN-L, confirming the catalytic contribution of the CeO₂ component (Fig. 2b). Biocompatibility assessment through growth curve analysis in LB medium (pH 6.8) revealed that while CeO₂ loading and Eudragit coating initially delayed bacterial growth, CeO₂@EcN-L achieved comparable optical density (OD600) to native EcN after 8 h of cultivation (Fig. 2c), indicating preserved metabolic activity and viability.

After evaluating the in vitro ROS scavenging activity of CeO₂@EcN-L, we further examined its anti-inflammatory effects at the cellular level. Two representative cell lines were selected for this investigation: RAW264.7 macrophages, a well-established model for studying inflammatory responses, and HT-29 cells, a human colorectal adenocarcinoma cell line that serves as an excellent model for colonic epithelial tissue. The cellular internalization of CeO₂@EcN and CeO₂@EcN-L was first investigated using flow cytometry. Both HT-29 epithelial cells and RAW264.7 macrophages demonstrated efficient uptake of CeO₂@EcN and CeO₂@EcN-L after 12 h of incubation (Figs. 2d & S2). Notably, the Eudragit L100–55 coating did not significantly impair the cellular internalization of EcN, suggesting maintained biological activity of the engineered bacteria.

To further investigate whether CeO₂@EcN-L could further protect cells from ROS-induced injury, we established an in vitro inflammation model using H₂O₂-stimulated RAW264.7 macrophages. Intracellular ROS levels were quantitatively assessed using the ROS-sensitive fluorescent probe 2′,7′-dichlorofluorescin diacetate (DCFH-DA), with fluorescence intensity analyzed by confocal microscopy. As shown in Fig. 2e–f, CeO₂@EcN-L treatment resulted in significantly lower DCFH-DA fluorescence intensity compared to both CeO₂ and CeO₂@EcN groups, demonstrating superior ROS scavenging capacity. The anti-inflammatory activity of CeO₂@EcN-L was further characterized through its effects on key inflammatory mediators.

Collectively, these in vitro findings suggest that CeO₂@EcN-L effectively protects epithelial cells via the following mechanisms: (1) reduction of intracellular ROS levels, (2) mitigation of oxidative stress-induced damage. Therefore, these results suggested that CeO₂@EcN-L could significantly improve the inflammatory microenvironment, highlighting its potential as a therapeutic agent for IBD.

Encouraged by the promising in vitro results, we further investigated the in vivo biodistribution and mucoadhesive properties of CeO₂@EcN-L. To enable real-time tracking, EcN, EcN-L, and CeO₂@EcN-L were fluorescently labeled with Cy5 and administered orally to mice. The spatiotemporal distribution of these formulations was monitored over a 48 h period using an IVIS imaging system (Fig. 3a). Quantitative analysis of fluorescence intensity at various time points revealed distinct biodistribution patterns among the different formulations (Fig. 3b–c). The fluorescence intensity profile of unmodified EcN showed a rapid decline, indicating relatively quick clearance from the gastrointestinal tract. In contrast, both EcN-L and CeO₂@EcN-L demonstrated significantly enhanced fluorescence intensity throughout the observation period, suggesting improved gastrointestinal retention. This enhanced retention could be attributed to two key factors: (1) the protective effect of the Eudragit L100–55 coating, which shielded the bacteria from gastric degradation, and (2) the mucoadhesive properties imparted by the CeO₂ component, which facilitated prolonged interaction with the intestinal mucosa.

To complement the in vivo imaging data, we performed ex vivo analysis of intestinal biodistribution following euthanasia. Quantitative assessment of intestinal fluorescence intensity revealed that CeO₂@EcN-L exhibited the highest fluorescence intensity in the intestine, consistent with the imaging data at earlier time points (Fig. 3d & e). This enhanced localization suggested that CeO₂@EcN-L not only demonstrated superior mucoadhesion but also accumulated to a greater extent than the other formulations. The enhanced gastrointestinal retention and targeted accumulation of CeO₂@EcN-L could be attributed to several synergistic mechanisms: (1) the pH-responsive properties of Eudragit L100–55 enabled selective release in the intestinal environment, (2) the negatively charged surface of the coating facilitated electrostatic interactions with positively charged inflamed tissues, and (3) the nanozyme component enhanced mucoadhesion through surface interactions with the intestinal mucosa. These comprehensive biodistribution studies demonstrated that CeO₂@EcN-L exhibited superior intestinal retention, stability, and targeted accumulation compared to both native EcN and EcN-L. The enhanced mucoadhesive properties and prolonged gastrointestinal residence time made CeO₂@EcN-L as a promising platform for oral probiotic delivery, particularly for applications requiring sustained local action in the intestinal tract. These findings provided strong rationale for further development of CeO₂@EcN-L as a next-generation therapeutic for IBD.

To comprehensively assess the therapeutic efficacy of CeO₂@EcN-L, we established mice colitis model and randomly allocated the animals into five experimental groups: 0.9% NaCl, CeO₂, EcN-L, CeO₂@EcN-L, and a control group (PBS) (Fig. 4a). Throughout the 10-day treatment regimen, we systematically monitored body weight fluctuations and recorded Disease Activity Index (DAI) scores. Comparative analysis revealed that both CeO₂ and EcN-L treatments demonstrated moderate amelioration of colitis-associated pathological manifestations, including mitigation of weight reduction, attenuation of colon shortening, and alleviation of colonic tissue damage (Fig. 4b–f). Particularly noteworthy was the superior therapeutic performance of CeO₂@EcN-L, which manifested in enhanced prevention of weight loss, improved preservation of colon morphology, and substantial reduction in colonic tissue damage, accompanied by a significantly lower DAI score relative to other treatment groups. These results strongly suggested that CeO₂@EcN-L possessed substantial therapeutic potential for managing DSS-induced colitis.

Given the critical role of tissue cytokine profiles as indicators of localized inflammation and their importance in evaluating anti-inflammatory interventions, we investigated the anti-inflammatory mechanisms of CeO₂@EcN-L through quantitative analysis of pro-inflammatory (TNF-α, IL-6) and anti-inflammatory (IL-10) cytokines in colonic homogenates. The results demonstrated that CeO₂@EcN-L treatment significantly attenuated TNF-α and IL-6 levels compared to the PBS control group (Fig. 4g & h), indicating robust suppression of inflammatory pathway activation. In contrast, monotherapy with either CeO₂ or EcN-L only marginally reduced these pro-inflammatory mediators, suggesting limited regulatory capacity when administered individually. Furthermore, we observed a substantial upregulation of IL-10 expression in the CeO₂@EcN-L group (Fig. 4i), implying that this combinatorial treatment not only mitigated inflammatory processes but also potentially enhanced anti-inflammatory cytokine production to facilitate inflammation resolution. These findings collectively demonstrated that CeO₂@EcN-L exerted potent anti-inflammatory effects through a dual mechanism of pro-inflammatory cytokine suppression and anti-inflammatory cytokine induction, effectively modulating the intestinal inflammatory microenvironment.

Probiotics have been extensively documented for their capacity to modulate the gut microbiota and confer significant health benefits to the host. To investigate the specific impact of CeO₂@EcN-L on the gut microbiota in a murine model of DSS-induced colitis, fecal samples were systematically collected from three distinct groups: healthy mice, DSS-induced colitis mice, and CeO₂@EcN-L-treated mice. The microbial diversity within these samples was meticulously assessed using 16S rDNA sequencing techniques. As depicted in Fig. 5a–b, the α-diversity analysis revealed that the CeO₂@EcN-L group exhibited a notably lower Simpson index, indicative of reduced dominance, and a higher Shannon index, reflecting increased species evenness, compared to the DSS-induced colitis group. This suggests that CeO₂@EcN-L treatment significantly enhanced bacterial richness and diversity relative to the DSS group. Moreover, the β-diversity analysis, which was based on Operational Taxonomic Units (OTUs), demonstrated distinct clustering patterns in Principal Coordinates Analysis (PCoA), further highlighting substantial differences in gut microbiota composition between the CeO₂@EcN-L-treated and DSS-induced colitis groups (Fig. 5c). These findings collectively underscore the profound and multifaceted influence of CeO₂@EcN-L on the gut microbial ecosystem in colitis-afflicted mice.

At the genus level, a more granular analysis revealed that CeO₂@EcN-L treatment led to a significant elevation in the abundance of beneficial bacteria (Fig. 5d–e). Notably, the genera Akkermansia and Faecalibaculum were prominently enriched (Fig. 5f–g). Akkermansia, a genus well-known for its critical role in maintaining intestinal epithelial integrity and enhancing gut barrier function, achieves these effects through the metabolism of glycans present in the intestinal mucus layer. Concurrently, Faecalibaculum, a key player in the production of short-chain fatty acids (SCFAs), supports intestinal epithelial cell energy metabolism, promotes cellular proliferation and differentiation, and activates the intestinal immune system, thereby reinforcing mucosal defense mechanisms. Consequently, CeO₂@EcN-L demonstrated a remarkable capacity to not only enhance the abundance of beneficial bacteria but also to reduce the prevalence of pathogenic bacteria in DSS-induced colitis mice. By effectively restoring the richness and diversity of the gut microbiota, CeO₂@EcN-L modulated the microbial composition in a manner that amplified its therapeutic efficacy against colitis, offering a promising avenue for the treatment of this condition.

To thoroughly assess the biosafety of CeO₂@EcN-L, comprehensive biochemical and histopathological analyses were conducted. First, hematological and biochemical parameters were evaluated in treated and untreated mice to determine any potential systemic impacts of CeO₂@EcN-L. Comparative analysis between the CeO₂@EcN-L and control groups revealed no significant alterations in blood cell counts or biochemical markers, demonstrating that CeO₂@EcN-L treatment did not compromise hematological or biochemical homeostasis (Fig. 6a). Furthermore, histopathological evaluation was performed on major organs, including the heart, liver, spleen, lungs, and kidneys, using hematoxylin and eosin (H&E) staining. Microscopic examination of these tissues revealed no evidence of pathological changes or toxicity, indicating that CeO₂@EcN-L did not induce adverse effects at the organ level (Fig. 6b and S3). Taken together, these results underscored the favorable biosafety profile of CeO₂@EcN-L, with no detectable systemic or organ-specific toxicity observed in treated mice.

Read more here

Tiantian Cui, Bing Li, Zhaoyang Lou, Kai Yang, Kaijin Yan, Danhong Ding, Hong Ge, Oral administration of nanozyme-armed probiotic Escherichia coli Nissle 1917 with ROS scavenging for inflammatory bowel disease therapy, Chemical Engineering Journal, 2025, 164949, ISSN 1385-8947, https://doi.org/10.1016/j.cej.2025.164949.