Eudragit®-engineered pH-responsive probiotic microspheres with enhanced gastrointestinal resistance and prolonged storage stability for inflammatory bowel disease therapy

Abstract

The rising global incidence of inflammatory bowel disease (IBD) is strongly linked to gut microbiota dysbiosis driven by modern lifestyle factors, particularly dietary irregularities and antibiotic overuse. Probiotic supplementation represents a promising therapeutic strategy for microbiota modulation; specifically, Lactobacillus reuteri has been shown to improve microbial community structure, suppress colonization by pathogens, and mitigate intestinal disorders. This study introduces a hierarchically structured double-encapsulated probiotic system, termed “core–shell” AMS@Eud, which integrates calcium alginate microspheres with a Eudragit® resin coating. This design enables pH-responsive release and ensures superior protection of probiotic viability. After optimization, AMS@Eud demonstrated outstanding storage stability, with only 5.28% and 9.09% reduction in L. reuteri viability after 35 days at 4°C and 25°C, respectively, thereby eliminating the need for cold-chain logistics. Under simulated gastrointestinal conditions, the system maintained a high viability of 84.60 ± 2.59% for L. reuteri, whereas free probiotics were entirely inactivated. A comprehensive bio-safety evaluation confirmed its excellent biocompatibility and minimal hemolytic activity, with in vivo studies revealing no adverse effects on major organs. Furthermore, this platform significantly enriched beneficial gut microbes while reducing pro-inflammatory taxa, effectively overcoming two major limitations of conventional probiotic therapy. Thus, AMS@Eud provides a transformative strategy for IBD management through precision microbiota engineering.

Introduction

The human gastrointestinal tract hosts a dynamic ecosystem of trillions of commensal microorganisms that maintain critical symbiotic relationships with host physiology through immunomodulation, metabolic regulation, and epithelial barrier reinforcement.1, 2 The disruption of this delicate microbiota equilibrium, which is driven by modern environmental stressors such as processed diets, antibiotic overuse, and chronic inflammation, has been epidemiologically linked to the increasing global incidence of inflammatory bowel diseases (IBD).3, 4 Current pharmacological strategies, while providing symptomatic relief through immunosuppression (e.g., anti-TNF-α biologics)5 or cytokine signaling inhibition (e.g., JAK inhibitors),6 exhibit concerning limitations: 32%–41% of patients develop hepatorenal complications with long-term use,7 while emerging nanotherapeutics often inadvertently suppress beneficial gut species.8 These therapeutic paradoxes highlight an urgent need for microbiota-sparing interventions that reconcile IBD management with ecological homeostasis restoration.9

Probiotic supplementation presents a compelling alternative, leveraging live microorganisms to competitively exclude pathogens and restore mucosal immunity.10-12 However, translation of this therapeutic paradigm faces fundamental biophysical challenges—a minimum threshold of 106–107 colony-forming units must survive sequential biological barriers including gastric acidity (pH 1.5–3.5), proteolytic enzymes, and bile salt emulsification to achieve intestinal colonization.13, 14 Conventional solutions remain unsatisfactory: strain selection through in vitro acid/bile resistance screening yields limited clinical candidates (<0.1% retention rate),15, 16 while genetically modified probiotics face stringent regulatory hurdles.17, 18 This impasse underscores the necessity for innovative bioengineering strategies that transcend biological evolution’s limitations, enabling reliable probiotic protection without genetic alteration. Lactobacillus reuteri is present in the intestines of nearly all mammals. This strain can regulate the homeostasis of intestinal microbiota, inhibit the proliferation of pathogenic bacteria, and promote the production of immunoglobulin A (IgA) to exert an immunomodulatory effect.19, 20 Additionally, it can inhibit pro-inflammatory bacterial strains by secreting specific substances, downregulate pro-inflammatory signaling pathways, and reduce inflammation levels.21, 22 Furthermore, L. reuteri can upregulate the expression of tight junction proteins (Occludin and ZO-1) in intestinal epithelial cells, thereby enhancing intestinal barrier integrity.23 The exopolysaccharides it secretes can form a protective film on the surface of the intestinal mucosa, which further reduces the damage to epithelial cells caused by inflammatory factors.24 Meanwhile, L. reuteri can specifically bind to mucin (LPxTG-motif proteins) in the intestinal mucus layer by expressing adhesion proteins.25 This strong colonization ability enhances its effectiveness in intestinal therapy.

Recent advances in biomaterial engineering have propelled probiotic encapsulation technologies to the forefront of therapeutic delivery innovation, enabling engineered probiotics to possess enhanced customizable functionalities.26 Meanwhile, protective matrices are employed to mitigate environmental stressors from manufacturing to gastrointestinal transit, so as to address inflammatory or infectious conditions in the human body.27-29 While traditional encapsulation matrices like starch (35%–48% encapsulation efficiency),30 gelatin (≤40% gastric survival),31 and whey protein (72-h bile resistance),32 demonstrate partial protection, their clinical translation remains constrained by fundamental limitations: chitosan’s cationic nature induces 20%–35% probiotic membrane damage,33 while hyaluronic acid carriers show premature 65%–80% payload release in simulated gastric fluid (SGF).34 These material-driven deficiencies collectively result in suboptimal probiotic viability (<50% post-processing) that fails to meet therapeutic thresholds.35 In contrast, hydrogel microspheres have emerged as a promising alternative, combining architectural precision (50–300 μm tunable diameter) with sustained release kinetics (72+ h payload retention).36 Sodium alginate-based systems particularly stand out for oral applications, leveraging reversible Ca2+-mediated gelation and GRAS certification.37 Yet, their inherent mesoporous structure (10–50 nm pore diameter) permits gastric acid infiltration, causing 60%–75% probiotic inactivation within 2 h of gastric exposure.38 Furthermore, conventional alginate beads exhibit burst release profiles (>80% payload discharge in duodenum pH),9 fundamentally misaligned with IBD’s pathophysiological requirements for site-specific, inflammation-modulated delivery. Beyond hydrogel microspheres, emulsion encapsulation and nano-coating technologies are also employed for probiotic protection and the achievement of their targeted delivery, yet they still have limitations.39

This technological impasse has driven the development of multi-material hybrid systems integrating stimuli-responsive components.40 While ROS-sensitive carriers such as thioketal nanoparticles achieve 85% colonic payload release in murine colitis models,41 their complex synthesis (8-step production) and limited storage stability (≤15 days at 25°C) hinder scalability.42 In contrast, pH-responsive Eudragit® polymers offer clinically validated advantages Eudragit® L100-coated systems demonstrate near-quantitative (98.2 ± 1.4%) gastric protection through molecular gastight sealing (≤0.4 nm pore size at pH < 5),43 followed by rapid dissolution (T90 = 8.7 min) at intestinal pH to enable localized probiotic release.44 In addition to the aforementioned technologies, gas shear technology,45 microfluidic technology,36 and even their hybrid form “gas shear-microfluidic technology,”46 also fail to be compatible with industrial-scale production. Our proposed hierarchical AMS@Eud architecture synergistically combines these advantages: the alginate core provides mechanical stability and oxygen-regulated microenvironments, while the Eudragit® shell establishes pH-dependent molecular gatekeeping. This dual-phase design addresses three critical challenges simultaneously-environmental isolation during storage, gastric protection, and spatiotemporal-controlled intestinal release-representing a paradigm shift from conventional single-mechanism encapsulation strategies.

To address these material science challenges, we engineered a hierarchical core-shell microsphere system (AMS@Eud) integrating pH-responsive Eudragit® L100 with calcium alginate through precision biomaterial engineering. This platform synergizes two critical functionalities: acid-resistant molecular shielding through Eudragit®’s pH-dependent polymer conformation (pKa = 5.5), and nutrient-permeable microenvironments via alginate’s Ca2+-stabilized hydrogel matrix. The rational design process (Scheme 1) employed a dual-phase encapsulation strategy: First, water-in-oil emulsion techniques generated uniform alginate microspheres encapsulating L. reuteri with 78.69% efficiency. Subsequent emulsion solvent evaporation created a Eudragit® coating, achieving complete gastric protection (pH < 3) while maintaining intestinal dissolution kinetics. The optimized AMS@Eud system demonstrated unprecedented performance benchmarks: Minimal viability loss (5.28% at 4°C) over 35 days eliminated cold-chain requirements, while simulated gastrointestinal testing revealed exceptional protection (84.60 ± 2.59% survival vs. complete inactivation of free probiotics). Crucially, the system exhibited pH-programmable release profiles—gastric shielding (92.81 ± 2.45% survival in SGF) followed by rapid intestinal activation (89.41 ± 2.91% viability in simulated intestinal fluid [SIF]). In vivo and in vitro studies confirmed the biocompatibility of AMS@Eud, with no adverse effects and a modest increase in mouse body weight during the experimental period. Moreover, the incorporation of prebiotics into AMS@Eud not only enhanced L. reuteri survival during storage and transit but also promoted the proliferation of beneficial intestinal microbiota in mice. This study introduces a feasible and efficient pH-responsive delivery system for precise probiotic administration, providing a promising strategy for IBD therapy and advancing targeted microbiota modulation.

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Excipients mentioned in the research: Eudragit L 100

Ming Teng, Xiaomin Luo, Peng Zhang, Chen Yang, Yuniel Ma, Xinhua Liu, Eudragit®-engineered pH-responsive probiotic microspheres with enhanced gastrointestinal resistance and prolonged storage stability for inflammatory bowel disease therapy, First published: 04 February 2026 https://doi.org/10.1002/bmm2.70066