The gastrointestinal tract is extensively colonized by a diverse microbial community. Composed of the mucus layer, gut microbiota, epithelial cell layer, and immune cells in the lamina propria, the intestinal mucosal barrier plays a pivotal role in maintaining immune tolerance, preventing pathogen invasion, and regulating nutrient absorption [1,2]. The gut mucus separated microbial community from epithelial cells, which is the first-line barrier to defense against pathogens invasion [3,4]. For example, the colon mucus layer is organized with an inner colon mucus layer that is impenetrable to bacteria and an outer mucus layer that is expanded to allow microbiota colonization. Colonic mucus is a viscoelastic hydrogel composed of O-glycosylated mucins (e.g., MUC2), ions, lipids, antimicrobial peptides (e.g., lysozyme), and immunoglobulins [5]. Alternations in thickness and composition of the gut mucus enhanced the permeability of pathogenic bacteria or toxins cross gut barrier, which are implicated in several diseases such as inflammatory bowel disease, neurological disorder, sepsis and even multiple organ dysfunction syndrome [[6], [7], [8], [9]].

Inflammatory bowel disease (IBD) is a group of chronic, relapsing gastrointestinal disorders, primarily comprising Crohn's disease (CD) and ulcerative colitis (UC). Due to their complex pathophysiology, the management of IBD has become a global challenge [[10], [11], [12]]. Current treatments include usage of aminosalicylates, corticosteroids or both to relieve temporary symptom, failing to address underlying issues such as intestinal barrier disruption and microbial imbalance [13]. Recently, biologic antibodies, e.g., infliximab, Ustekinumab were effective for remission of intractable colitis. However, these therapies often undergone loss of response and become ineffective in some patients [14]. For example, a substantial proportion of patients treated with infliximab do not achieve remission, and approximately 25 % patients experienced severe complications, including acute inflammation, pneumonia, or cancer [15]. Thus, there is an urgent need to develop safer and more effective therapeutic strategies.

Gut microflora dysbiosis driven the formation of gut mucosal wound via depleting mucus and disassembling epithelial tight junction, while gut barrier loss worsened microflora dysbiosis by allowing pathogenic blooms [16]. In context of IBD pathogenesis, gut microflora dysbiosis was characterized by the overwhelming pathogenic bacteria and excessive production of toxins such as endotoxins, which further exacerbated zonulin secretion [17]. Zonulin is a key protein secreted by intestinal epithelial cells, which regulated the permeability of gut epithelial barrier. Under physiological condition, zonulin reversibly opened paracellular pathway to allow the selective passage of nutrients [18]. However, zonulin was excessively produced under the pathogenic stimulus such as bacteria or toxins, resulting in the disassembly of the epithelial tight junction proteins and thus enhancing gut permeability [19,20]. Excessive zonulin production was highly associated with pathogenesis of IBD [21]. It was previously reported that zonulin impaired gut barrier integrity via activating myosin light chain kinase (MLCK) signals [22,23]. MLCK, a serine/threonine protein kinase, phosphorylates myosin light chain (MLC) to induce the epithelial actin-myosin cytoskeleton contraction, leading to the redistribution and degradation of TJs (ZO-1, occludin and claudins) [24,25]. Activating Zonulin-MLCK/p-MLC signals increased intercellular gaps and allowed pathologic microbiota or toxins to penetrate the intestinal epithelial, triggering or exacerbating immune-inflammatory responses [26,27]. Larazotide acetate (LA), a synthetic octapeptide competitively inhibited zonulin receptor to restore tight junctions, thus repairing the intestinal mucosal barrier [28]. It has been demonstrated that LA inhibited the gliadin-induced transepithelial electrical resistance reduction and TJs opening in Caco-2 cells [29]. Besides, LA has been reported to alleviate the spontaneous colitis in IL-10 gene-deficient mice via reducing small intestinal permeability [30]. Moreover, LA has also been demonstrated to reverse the colitis symptoms of DSS-induced Zonulin transgenic Hp2 mice by repairing the intestinal barrier [31]. Due to its exceptional ability to restore the intestinal barrier, LA had entered Phase III clinical trials for celiac disease treatment [16]. Overall, LA might be a promising therapeutic candidate to combat the chronic colitis.

Barrier therapies, resting on a physical non-drug mode of action to manage the clinical syndrome of colitis, are short-term interventions providing transient but meaningful support to mucosal integrity. Recently, barrier therapies products, e.g., Orafate®, Gelclair®, MuGard®, Ziverel®, Gelenterum®, ProctiGard® have been either Community European marked or licensed by Food and Drug Administration (FDA) for colitis. Among them, hydrogel has been widely used as biomimetic mucus to shield the ulcered mucosa, due to its high-water content and three-dimensional network structure. For example, dopamine/thiol-modified hyaluronic acid-based biomimetic hydrogels have been utilized to treat UC [32]. Interestingly, a thiol modified hyaluronic acid solution was cross-linked to form an artificial mucus layer on diseased site in response to reactive oxygen species (ROS) after intragastric administration, reducing the inflammatory immune reaction of colonic mucosa in enteritis mice. Similarly, decellularized extracellular matrix (ECM) hydrogels derived from porcine small intestine submucosa have demonstrated efficacy in repairing epithelial barriers by mimicking the native tissue microenvironment. Additionally, inulin hydrogels with nanozymes and pirfenidone have also been employed to address the fibrotic complications of IBD [33]. However, these hydrogels face critical limitations in colitis treatment. For example, the inadequate mimicry of native mucus physicochemical properties (e.g., poor repairing activities) for most of hydrogels had the suboptimal barrier restoration. Moreover, due to the overwhelming pathogenic bacteria in IBD, these hydrogels without inherent antimicrobial activity have the limited efficacy to combat colitis.

To overcome these limitations, we developed a dual-crosslinked mucus-mimetic hydrogel (HSMP-LA) from thiol/maleimide-modified hyaluronic acid, loaded with ε-polylysine (ε-PL) and larazotide acetate (LA) (Fig. 1). Hyaluronic acid (HA) provided biocompatibility, muco-adhesion, and hydration capacity, mimicking the extracellular matrix to support mucosal healing. Antimicrobial properties of ɛ-PL against Gram-positive, Gram-negative bacteria and fungi have been previously proven in previous studies. Its antimicrobial properties have been directly connected with its free amino (-NH3+) groups, which electrostatically interact with the negatively charged cell membrane of bacteria [34]. Besides, the cationic nature of ε-PL was also expected to reinforce the adhesion of HSMP hydrogel to intestinal mucosa via electrostatic bridging and mucus interpenetration [35]. LA acting as a zonulin receptor antagonist has been reported to actively restore tight junctions and epithelial integrity [36]. HSMP-LA was expected to overcome prior limitations through the following three key innovations. First, dual “click” crosslinkings of thiol-maleimide and disulfide formed a robust yet dynamic network, enabling strong adhesion to inflamed tissues while resisting peristaltic clearance; second, the antimicrobial action of ε-PL for HSMP created a microenvironment conducive to mucosal healing; third, the sustained-release LA directly targeted zonulin-mediated tight junction disruption, a mechanism different from conventional hydrogels. In this study, cysteamine-grafted hyaluronic acid (HS) and 3-maleimidopropionic acid-grafted hyaluronic acid (HM) were firstly synthesized and carefully characterized. The blank HSMP hydrogel without LA was firstly prepared by dual crosslinking of thiol-maleimide and disulfide. The composites of HSMP hydrogel were further screened by rheological test. Subsequently, in vitro shielding effect, adhesion properties and antimicrobial ability of HSMP hydrogel were also characterized. Besides, the activity of LA in HSMP was confirmed on Caco-2 cells by detecting intercellular tight junctions. Moreover, the therapeutic potential of HSMP-LA was also investigated on DSS-induced colitis mice. Finally, the therapeutic mechanism of HSMP-LA was explored by regulating zonulin-MLCK/p-MLC signals.