Inflammatory bowel disease (IBD), encompassing ulcerative colitis and Crohn's disease, causes chronic intestinal inflammation and increases the risk of colorectal cancer. Current treatments, such as aminosalicylates, antibiotics, corticosteroids, and immunosuppressants, often fail to address the root causes—intestinal mucosal damage, impaired barrier function, and gut dysbiosis—and may cause adverse effects. Probiotics, beneficial microorganisms in the gut, have shown promise in treating various diseases, including IBD. Genetically engineered probiotics offer the potential to deliver therapeutic proteins directly to the colon, but challenges remain in ensuring effective delivery and sustained protein production in the harsh gastrointestinal (GI) tract environment. This study addresses these challenges by engineering *E. coli* Nissle 1917 (ECN) to produce ROS-scavenging enzymes and employing a novel coating strategy to enhance their delivery and survival.

The existing literature highlights the role of impaired intestinal mucosal barrier function and intestinal microecology dysbiosis in the etiology and pathogenesis of IBD. While current clinical interventions exist, they often fail to address the root causes of the disease and can lead to significant side effects. Several studies have explored the therapeutic potential of naturally occurring commensal probiotics and genetically engineered probiotics for IBD treatment. However, challenges remain with delivering sufficient therapeutic concentrations to the affected areas and ensuring the survival of the probiotics in the presence of gastric acid and bile salts. The use of biofilm coating to protect probiotics has been investigated, but the search for effective and biocompatible materials continues. This research explores the possibility of using chitosan and sodium alginate, materials approved by the FDA for food additives, as a biofilm coating for delivering genetically modified probiotics to the gut.

This study involved the genetic engineering of *E. coli* Nissle 1917 (ECN) to overexpress catalase (CAT) and superoxide dismutase (SOD), two enzymes that efficiently scavenge reactive oxygen species (ROS). The resultant strain, ECN-PE, was then coated with chitosan and sodium alginate using a layer-by-layer electrostatic self-assembly technique to create ECN-PE(C/A)2. The effectiveness of the coating was evaluated using zeta potential measurements and fluorescence labeling. The viability of ECN-PE(C/A)2 was assessed in simulated gastric fluid and bile salt solutions, and in vivo using bioluminescence imaging with luciferase-expressing ECN and plate counting of bacterial colonies from stomach, intestinal tract and cecum contents. A mouse model of IBD was induced using dextran sodium sulfate (DSS), 2,4,6-trinitrobenzene sulfonic acid (TNBS), and oxazolone. The therapeutic efficacy of ECN-PE(C/A)2 was evaluated by assessing body weight changes, disease activity index (DAI), colon length, colonic tissue histology, myeloperoxidase (MPO) activity, inflammatory cytokine levels (IL-1β, TNF-α, IL-6, IL-10, TGF-β), tight junction protein expression (ZO-1, Occludin), colonic epithelial cell apoptosis (TUNEL assay), and colonic epithelial cell regeneration (EdU assay). The impact on gut microbiota was determined through 16S rRNA gene sequencing. The safety of ECN-PE(C/A)2 was assessed by analyzing blood parameters, serum biochemistry, and organ histology after repeated oral administration. Antibiotic pretreatment was also employed to evaluate the role of the gut microbiome in the therapeutic efficacy.

The chitosan/sodium alginate coating significantly protected ECN-PE from the harsh conditions of the GI tract, resulting in significantly improved survival and colonization compared to uncoated ECN-PE and even compared to ECN coated with Eudragit L100-55, a clinically used enteric coating. ECN-PE(C/A)2 effectively alleviated DSS-, TNBS-, and oxazolone-induced IBD in mice, as evidenced by reduced weight loss, improved DAI scores, increased colon length, decreased colonic tissue damage, and reduced MPO activity. The treatment significantly reduced levels of pro-inflammatory cytokines (IL-1β, TNF-α, IL-6) and increased levels of anti-inflammatory cytokines (IL-10, TGF-β). ECN-PE(C/A)2 also restored the expression of tight junction proteins (ZO-1 and Occludin), reduced colonic epithelial cell apoptosis, and enhanced colonic epithelial cell regeneration. The treatment significantly altered the gut microbiome composition, increasing the abundance of butyrate-producing bacteria *Lachnospiraceae_NK4A136* and *Odoribacter* while decreasing the abundance of *Escherichia-Shigella*. Antibiotic pretreatment reduced the therapeutic efficacy of ECN-PE(C/A)2, indicating the important role of the gut microbiota. Repeated oral administration of ECN-PE(C/A)2 did not induce any adverse effects in mice, as assessed through blood parameters, serum biochemistry, and organ histology.

This study demonstrates the potential of a novel, bioinspired approach to treat IBD using genetically engineered probiotics. The chitosan/sodium alginate coating provides robust protection against the harsh GI tract environment, resulting in markedly improved oral bioavailability of the engineered probiotic ECN-PE. The effectiveness of ECN-PE(C/A)2 in alleviating IBD across multiple models highlights its versatility and broad potential application. The observed modulation of the gut microbiome further underscores the complex interplay between the engineered bacteria, the host immune response, and the gut's microbial ecosystem. The findings suggest that the therapeutic efficacy of ECN-PE(C/A)2 is not solely reliant on ROS scavenging but also on its ability to regulate the composition and activity of the gut microbiota. The safety profile observed with ECN-PE(C/A)2 further supports its potential as a safe and effective therapeutic agent for IBD.

This study successfully demonstrated that a novel, bioengineered probiotic (ECN-PE(C/A)2) effectively treats IBD in a murine model. The double-layered chitosan/sodium alginate coating enhances the bioavailability of the probiotic in the GI tract, improving its therapeutic efficacy. The treatment not only scavenges ROS but also modulates the gut microbiome to reduce inflammation and restore intestinal barrier function. The lack of observed adverse effects highlights the safety of this approach. Future research should focus on clinical trials to evaluate the efficacy and safety of this therapeutic strategy in humans, and explore its potential for treating other intestinal and systemic diseases.

The study utilized a mouse model of IBD, which may not fully replicate the complexities of human IBD. Further research is necessary to translate these promising preclinical findings to human clinical settings. The long-term effects of ECN-PE(C/A)2 on the gut microbiome and host immune system require additional investigation. The mechanism by which ECN-PE(C/A)2 modulates the gut microbiome warrants further exploration.