Hyaluronic acid modified oral drug delivery system with mucoadhesiveness and macrophage-targeting for colitis treatment

Based on the biocompatibility and macrophage targeting of natural polysaccharides, combined with the physiological and pathological characteristics of the gastrointestinal tract and colonic mucosa of ulcerative colitis (UC), we prepare dexamethasone (Dex)-loaded oral colon-targeted nano-in-micro drug delivery systems coated with multilayers of chitosan (CS), hyaluronic acid (HA), and finally Eudragit S100 (ECHCD MPs) using a layer-by-layer coating technique for UC treatment through regulating the M1/M2 polarization of intestinal macrophages. HA/CS/Dex nanoparticles (HCD NPs) are ingested by macrophages via CD44 receptor-mediated endocytosis to regulate M1-to-M2 macrophage polarization and exert anti-inflammatory effects. Moreover, ECHCD MPs show better colon-targeting properties than Dex-loaded chitosan nanoparticles (CD NPs) and HCD NPs which is demonstrated by stronger mucoadhesion to inflamed colon tissues. After oral administration, ECHCD MPs exert significant anti-UC effects. Therefore, ECHCD MPs are proven to be as promising oral colon-targeting drug delivery systems for Dex and have potential application in UC treatment.

Introduction

Ulcerative colitis (UC) is a chronic non-specific inflammatory disease that is marked by chronic inflammation of the colon (Conrad, Roggenbuck, & Laass, 2014). The course of UC is protracted and recurrent, which seriously affects the health and life quality of patients and increases the risk of colitis-related cancer (Khor, Gardet, & Xavier, 2011). The global incidence of UC is rising year by year, nevertheless, its pathogenesis is not yet fully clarified.

While the UC pathogenesis has not been elucidated, the mechanism involves immune responses that attack the colon (Gren & Grip, 2016). Macrophages play a crucial role in the pathophysiological process of UC. Macrophages exist in two different phenotypes: a pro-inflammatory (M1) phenotype and an anti-inflammatory (M2) phenotype. The imbalance between M1 and M2 macrophages is correlated with UC progression (Zhu et al., 2014). M1 macrophages are associated with the upregulation of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β, which exacerbate colon inflammation. Conversely, M2 macrophages secrete anti-inflammatory cytokines and are associated with the resolution of colon inflammation (Parisi et al., 2018; Wei & Besner, 2015). Experimental studies have also found that the effects of UC treatments using drugs might be related to regulation of M1-to-M2 macrophage polarization (Gan et al., 2018; Sun et al., 2020). Dexamethasone (Dex) is an anti-inflammatory corticosteroid that has been used to treat moderate to severe UC (Sood, Midha, Sood, & Awasthi, 2002). Studies have shown that the mechanism of the anti-UC effect of Dex is related to the regulation of M1-to-M2 macrophage polarization (Jiang et al., 2017; Schuliga, 2015). For the nuclear factor-kappaB (NF-κB) signaling pathway involved in M1 macrophage polarization, Dex can inhibit NF-κB induced gene transcription, thereby inhibiting M1 macrophage polarization and alleviating the inflammatory response of UC (Jiang et al., 2017; Schuliga, 2015). However, long-term use of glucocorticoids is often accompanied by serious systemic side effects, such as hypertension and hyperglycemia, and immune suppression (Maltese et al., 2012). Therefore, targeted delivery of Dex to the inflammation site of the colon can improve the local anti-inflammatory effect of Dex and reduce its systemic side effects.

Nano drug delivery system (NDDS) is an effective means to solve this problem. Accordingly, numerous studies reported the use of nanoparticles (NPs) -based drug delivery systems, which specifically accumulate at sites of colon inflammation in experimental murine colitis models (Li et al., 2019; Xue et al., 2018; Zhang et al., 2016). This size-dependent accumulation of NPs at sites of colon inflammation may occur owing to the epithelial enhanced permeability and retention (eEPR) effect (Xiao et al., 2017). From a practical viewpoint, NPs-based drug delivery systems for UC are expected to improve drug efficacy and reduce systemic side effects. However, NPs absorbed in the upper gastrointestinal tract (UGT) and premature drugs released from NPs can compromise the colon-targeting of NPs (Naeem et al., 2020). To overcome the drawbacks of oral colon-targeting drug delivery systems, they can be further encapsulated in colon-targeted microparticles (MPs), namely hybrid nanoparticles-in-microparticles (NPs-in-MPs). MPs with the optimal size range of 10–300 μm have been reported to detain specifically on the inflamed region of the colon (Zhang et al., 2019). Therefore, oral NPs-in-MPs are beneficial to protect the encapsulated NPs during the gastric transit process, avoid absorption of NPs in the UGT and premature drug release from NPs, and promote drug delivery to the inflammatory site of the colon for UC treatment (Naeem et al., 2020).

Many patients require long-term use of drugs due to procrastination and recurrence of UC. Hence, NDDS using natural polysaccharides as carrier material has good biocompatibility (Chen et al., 2020; Han et al., 2019; Lee et al., 2020; Li et al., 2018). Chitosan (CS) is a natural polycationic polysaccharide with biocompatibility, biodegradation, and pH sensitivity. It has been widely applied in the fields of biomaterials because it interacts with anionic biomacromolecules to self-assemble into NPs and form drug carriers (Li et al., 2021; Tronci, Ajiro, Russell, Wood, & Akashi, 2014). It is reported that CS and its degradation product chitosan oligosaccharide can effectively scavenge hydroxyl free radicals and superoxide anions, inhibit the NF-κB signaling pathway and reduce the activity of inducible nitric oxide synthase, thus decreasing the production of NO, reducing oxidative stress and exerting anti-inflammatory effects (Guo et al., 2022; Shi et al., 2019). Additionally, studies have shown that CS and chitosan oligosaccharide have good therapeutic effects on experimental colitis (Niu et al., 2021; Wang, Zhang, Guo, & Li, 2019). Hyaluronic acid (HA), a natural anionic polysaccharide, has been shown to interact with CD44 surface receptors on the inflammatory macrophages to increase cellular uptake (Fakhari & Berkland, 2013). CD44, a transmembrane glycoprotein, is a specific receptor for HA and is highly expressed on the surface of colonic epithelial cells and macrophages in inflammatory tissues (Rosenberg et al., 1995). Therefore, modification with HA on the surface of NPs can increase macrophage uptake. In addition, the physiological characteristics of the colon in UC patients are altered due to intestinal inflammation. A large number of positive charges are accumulated on the injured epithelial surface of the colon due to the depletion of colonic mucosal mucus of inflammatory tissues and the accumulation of positively charged proteins, such as antimicrobial peptides, bactericidal/permeability-increasing proteins, and transferrin (Vidal-Lletjós et al., 2019). Therefore, HA with negative surface charges may be an ideal material for targeted drug delivery to the inflammatory mucosa and macrophages, which can adhere to inflamed mucosa by electrostatic adsorption and avoid the loss of drugs caused by diarrhea in UC patients. In this study, it is hypothesized that HA-modified NDDS with mucoadhesiveness and macrophage-targeting may be a smart oral NDDS for UC treatment.

Here, we develop an NPs-in-MPs system to deliver Dex using ionic crosslinking and electrostatic adsorption methods. Firstly, Dex-loaded chitosan nanoparticles (CD NPs) are prepared by the ionic crosslinking method using CS as carrier material. Then, CD-NPs are wrapped with HA through electrostatic adsorption to obtain HA/CS-Dex-NPs (HCD NPs), to increase the mucosal adhesion and macrophage uptake of HCD NPs, thereby improving the therapeutic effect of Dex. For Eudragit S100 (ES100) coating on HCD NPs, HCD NPs are coated with CS through electrostatic adsorption to obtain CS/HA/CS-Dex-NPs (CHCD NPs). Finally, the above-mentioned NPs are coated with ES100 to obtain ES/CS/HA/CD MPs (ECHCD MPs). Following oral administration, ECHCD MPs can pass through the UGT and reach the colon. As the increase of intestinal pH, the outer layer of the nanoparticles, ES100, begins to dissolve, thus exposing the CHCD NPs. When arriving at the site of the inflammatory colon, CS on the surface of CHCD NPs begins to dissolve in the inflammatory acidic microenvironment, thus exposing the HCD NPs. Then, HCD NPs with negative surface charges can adhere to inflamed tissues and be ingested by macrophages via the CD44 receptor-mediated endocytosis to deliver Dex to macrophages and regulate the M1-to-M2 macrophage polarization, thus exerting an anti-UC effect. Based on the physiological and pathological characteristics of the gastrointestinal tract and colonic mucosa of UC, ECHCD MPs are expected to not only have superior therapeutic efficacy but also considerably reduce systemic drug exposure.

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Materials

Chitosan (CS, deacetylation degree >95 %, viscosity 100–200 mpa.s, MW = 150,000–300,000 Da), sodium tripolyphosphate (TPP), hyaluronic acid (HA, MW = 800,000–1500,000 Da), dexamethasone (Dex), fluorescein5(6)-isothiocyanate (FITC), Eudragit S 100 (ES100), and 2,4,6-trinitrobenzenesulfonic acid (TNBS, 5 % w/v) are purchased from Aladdin Bio-Chem Technology Co. Ltd. (Shanghai, China). All buffer solutions are prepared according to Chinese Pharmacopoeia (2020 edition).

Ying Zhang, Ruirui Ma, Cuiyu You, Xue Leng, Danyang Wang, Shujing Deng, Binyang He, Ziyang Guo, Zelin Guan, Hengyu Lei, Jie Yu, Qinyuan Zhou, Jianfeng Xing, Yalin Dong, Hyaluronic acid modified oral drug delivery system with mucoadhesiveness and macrophage-targeting for colitis treatment, Carbohydrate Polymers, Volume 313, 2023, 120884, ISSN 0144-8617, https://doi.org/10.1016/j.carbpol.2023.120884.

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