Oral creatine-modified selenium-based hyaluronic acid nanogel mediated mitochondrial energy recovery to drive the treatment of inflammatory bowel disease

Abstract

The damnification of mitochondrion is often considered to be an important culprit of inflammatory bowel disease (IBD), however, there are fewer reports of mechanisms of mitochondria-mediated IBD treatment. Therefore, we first proposed to reboot mitochondrial energy metabolism to treat IBD by capturing the double-sided factor of ROS and creatine (Cr)-assisted energy adjustment. Herein, an oral Cr-modified selenium-based hyaluronic acid (HA) nanogel (HA_Se-Cr nanogel) was fabricated for treatment of IBD, through ROS elimination and energy metabolism upgradation. More concretely, due to IBD lesion-specific positive charge and the high expression of CD44, HA_Se-Cr nanogel exhibited dual targeted inflammatory bio-functions, and ROS-driven degradation properties in high-yield ROS levels in inflammation areas. As expected, multifunctional HA_Se-Cr nanogel could effectively ameliorate IBD-related symptoms, such as mitochondrial biological function restoration, inhibition of M1-like macrophage polarization, gut mucosal reconstruction, microbial ecological repair, etc., thus excellently treating IBD. Overall, the proposed strategy underlined that the great potentiality of HA_Se-Cr nanogel by restarting mitochondrial metabolic energy in colitis lesions, providing new a pavement of mitochondrion-mediated colitis treatment in clinical applications.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12951-024-03007-0.

Introduction

Inflammatory bowel disease (IBD), as a complicated, multifactorial, immune-mediated gastrointestinal inflammatory disease with high morbidity, and IBD carries lifelong morbidity [1], has became social and economic huge on human health life systems [2].Typically, the two predominant IBD are Crohn’s disease (CD) and ulcerative colitis (UC) depending on the clinical pathophysiology [3, 4]. Regrettably, complex cross-inducing factors such as uncontrolled diet lifestyle, misuse of antibiotics, genetic inheritance, etc., can severely induce the possibility of IBD [5], in contrast, also severely hinder IBD treatment. Currently, biologics, immunomodulators, aminosalicylates and glucocorticoids [6, 7] are the most commonly used therapy, yet, IBD treatment is still not adequately addressed. As we all know, IBD is presently incurable and characterized by alternating periods of exacerbation and remission [3, 5]. Moreover, these problems such as long-term administration of cytotoxicity, off-target toxicity, and increased risk of carcinogenesis, will inevitably be left behind through the implementation of drugs or biologics alone. It is therefore essential to develop emerging options for IBD treatment.

Mitochondria is deemed “powerhouse” of cell energy, which involved in numerous critical cellular functions, such as cell proliferation and differentiation, dynamical communication with other organelles, as well as mitochondrial ROS-mediated apoptosis [8]. However, numerous studies have shown that mitochondrial dysfunction plays an important role in multiple phenotypes of chronic inflammatory diseases, even involving cancer-related inducing factors and neurodegenerative diseases [9]. In pathological circumstances, imbalances in redox channels are due to mitochondrial dysfunction, which can lead to relatively high levels of reactive oxygen species (ROS) generation and induce organelles oxidative stress, thus exacerbating the development of mitochondria-derived ROS-induced diseases [10]. Therefore, the predominant fountainhead of ROS fluctuation depends on the mitochondrial status in cells and also is a driver of inflammation evolution [9, 11]. Moreover, the generation of abundant ROS in cells can crush the inherent antioxidant system. On the other hand, in high-ROS-environment, DNA damage, protein carbonylation, and lipid peroxidation could be induced, or even severely lead to intestinal epithelial damage, thus expanding gut permeability and recruiting immune cells [12]. Meanwhile, metabolic reprogramming of macrophages plays an important role in the pro-inflammatory phenotype in ROS baptism, thereby aggravating the release of inflammatory factors in inflammatory region, for example, pro-inflammatory cytokines: TNF-α, IL-1β and IL-6 [13]. More importantly, excessive ROS can also bring about intestinal flora dysbiosis through the dysregulation of inflammatory factors in immune cells [14]. Actually, regulating intracellular ROS by retelling mitochondrial functions or restoring ROS levels is already a promising strategy for treating inflammatory diseases. Huang et al. [15] fabricated an oral and metal-free melanin nanozymes with ROS scavenging ability for IBD therapy, which can effectively alleviate the symptoms of IBD. Song et al. [16] prepared double-targeted oral TACS diagnostic agent to accurately locate IBD lesions by noninvasive CT imaging and effectively treat IBD through regulating ROS levels. ROS is a kind of end-effector molecule which can directly produce inflammatory damage. However, there are many other end-effector molecules with similar effects, such as IL-6, IL-1β, TNF-α, etc. Therefore, ROS capture alone as a therapeutic method cannot completely block the occurrence of inflammatory damage [17, 18]. Given that high ROS levels in cells are often cited as an important culprit in the pathogenesis of IBD, nevertheless, the effectiveness of ROS-mediated therapeutic models in IBD remains limited.

Dysfunction of energy metabolism is associated with the pathogenesis of IBD [19]. Abnormal inflammatory responses, such as neutrophil infiltration and phagocytosis, urgently require a lot of energy supply. Also, due to inflammatory-induced abnormal angiogenesis, inflammatory tissue is in a state of ischemia and hypoxia, which can further exacerbate energy deficiency. As a result, this lack of energy leads to the destruction of mucosal epithelial barrier [20], i.e., the process of epithelial tissue renewal and repair is impeded [21]. In short, the recovery of damage epithelium from IBD is an energy-consuming process, and a new target for IBD treatment. Creatine (Cr) is a small metabolite that plays a central role in energy metabolism and mitochondrial functions. Cr is reversely phosphorylated by the ubiquitous mitochondrial creatine kinase (uMt-CK) to produce the high-energy compound phosphocreatine. Facilitated by intracellular adenosine diphosphate (ADP)/adenosine triphosphate (ATP) ratios, cytoplasmic isomers of uMt-CK subsequently then hydrolyze phosphocreatine, regenerating ATP [22]. Moreover, Cr supplementation was found to be protective in mice with colitis. Therefore, regulating energy metabolism is expected to be a promising strategy for IBD therapy, however, there are fewer reports of mechanisms of Cr-driven mitochondria-mediated IBD treatment.

Herein, we fabricated an oral creatine-modified selenium-based hyaluronic acid (HA) nanogel (HA_Se-Cr nanogel), effectively reversing mitochondrial dysfunction by eliminating ROS and up-gradating energy metabolism for efficient targeting treatment of IBD (Fig. 1). Selenium (Se) is an important trace element of the human body, and involved in numerous meaningful signal transduction pathways, especially in the maintenance of cell antioxidant system [23]. The study found that oral selenium-containing compounds or protein substances (e.g., selenomethionine and selenocysteine) are anti-inflammatory and maintain the intracellular balance of ROS by capturing oxygen/nitrogen radicals [23, 24]. However, selenium-containing agents are still less used in the treatment of enteritis, mainly due to nonspecific inflammatory targeting, single-model therapy and complex preparation processes. Additionally, Cr is recognized a rapid energy supplement, which can quickly produce ATP through phosphocreatine circulation for cell energy supply, accelerating the distribution of energy to recover cellular fatigue, as well as allowing for regulation of the body’s metabolism to participate in related diseases of adjuvant therapy, such as IBD [25, 26]. Although Cr-mediated IBD therapy is effective to some extent, its therapeutic efficacy is limited due to its lack of colitis targeting agglomeration, unknown dose regulation and patterned singularity on colitis treatment. Thus, the search for nano-mediated the combination of multimode therapeutic strategy has been approve based on the diversity attributes conferred by nano-materials, such as bio-targeting, multiple therapeutic substances [27]. Against this background, we customized ROS-responsive diselenide-bridged (Se-Se) nanogel that introduces CD44-targeted HA as the framework [28], and used Cr further modification of partially exposed amino functional groups (-NH₂) of nanogel (HA_Se-NH₂) to form a creatine-modified selenium-based HA nanogel for colitis treatment (Fig. 1 and Fig. S1, Support information). Oral HA_Se-Cr nanogel, by HA-CD44 interaction and electrostatic adsorption, displayed a high bio-targeting of enteritis in vivo [28], thus laying the foundation for enteritis treatment. In addition, synthetic nanogel could effectively consume ROS levels in the position of enteritis to maintain redox balance, alleviate immune responses, as well as improve the gut microbiome, thereby effectively inhibiting ROS-induced apoptosis and enhancing colonic epithelial repair. Meanwhile, Cr derivative was continuously released from HA_Se-Cr nanogel through ROS activation, which will effectively improve the distribution of energy, such as energy metabolism of mitochondrial regulation. Therefore, the recovery of metabolism dysfunction could effectively repair IBD symptoms such as mucosal barrier function. Collectively, oral Cr-modified diselenide-crosslinked HA nanogel (HA_Se-Cr) showed dual colon targeting characteristic and potential mitochondrial dysfunction reversion capacity via ROS elimination and energy metabolism upgradation for the prevention and management of IBD. Therefore, our study will provide an oral nanogel strategy for Cr-mediated energy metabolism restoration and ROS elimination in colitis, as a safe and effective treatment for IBD.

Fig. 1.

Schematic illustration of the synthesis of HA _Se -Cr nanogel, and the application of IBD treatment in DSS-colitis mice. (a) Demonstration of a facile synthesis of the HA_Se-Cr nanogel via the amide reaction in mild EDC/NHS system. (b) The applications of the HA_Se-Cr nanogel in the treatment of IBD. The prepared HA_Se-Cr nanogel showed a high inflamed targeting capacity by dual targeting strategy, i.e. electrostatic adsorption and HA-CD44 bond. Meanwhile, HA_Se-Cr nanogel efficiently ameliorates colitis by rebooting mitochondrial-mediated energy metabolism, as well as suppressing pro-inflammatory cytokines secretion, skewing M2 phenotype macrophages polarization, promoting epithelial barrier reparation and modulating of gut flora

Results and discussion

Abnormal mitochondrial morphology and energy metabolic disorders in CD patients

The center of energy in the majority of the eukaryotic cells, mitochondria, are involved in considerable metabolic energy, cell differentiation and mitochondria-evoked apoptosis pathways [8, 9]. Previous studies have shown that a positive correlation between the development of IBD and mitochondrial dysfunction (e.g., abnormal mitochondrial morphology, energy metabolic abnormality, mitochondria-related ROS leakage) [1], namely, the initial mode of tricarboxylic acid cycle (TCA) metabolic pathway transform into glycolysis direction, urging the TCA cycle to weaken, resulting in the generation of nitrogen or oxygen radicals, the formation of oxidative stress and participation in intestinal inflammation [29]. Thus, to deeply understand the direction of mitochondrial function and energy metabolism, the activity of key enzymes such as citrate synthase (CS), isocitrate dehydrogenase (ICDH), and α-ketoglutarate dehydrogenase (α-KGDH) in TCA [30, 31] were detected. These samples of IBD patients were randomly removed, including normal colon (health), inflammatory colon (CD patients) and tissue of normal colon (CD patients), and were separately determined on the activity of ICDH、CS、α-KGDH, as well as the patient’s CDAI score was recorded. As shown in Fig. 2a, b and c, the activity of ICDH, CS, and α-KGDH in the colon tissue of patients with CD were negatively associated with CDAI score of sufferers. Additionally, these associated enzymes presented significantly a low activity in inflammation than normal group, while the intestinal site of mucosal healing dramatically appeared a trend of partial recovery, but still lower than the normal control group (Fig. 2d, e and f), indicating that the energy-regulated enzymes were significantly associated with colitis. Moreover, we also characterized mitochondrial structure in colon tissue in different disease states using bio-TEM technique. Analysis results showed that the structure of inflammation-mediated mitochondria occurred obvious alterations, such as swelling, cross-fertilization and cristae degeneration [32], compared to normal mitochondria with rod-shaped or spherical, double-layer membrane structure and the lining folded into ridges (Fig. 2j). Conversely, in the upturn of intestinal inflammation, the morphology of mitochondria intuitively reversed and the mitochondrial spines re-emergence were observed, showing that mitochondrial defects are related to IBD active. Cr as the core of energy metabolism, effectively produces large amounts of ATP in cells. As shown in Fig S9c, when CaCO2 cell was treated with LPS, the level of intracellular Cr was slightly higher than in normal cells. Therefore, the single parameter of Cr may not be accurate as markers of IBD. Based on the above research data, mitochondrial dysfunction-mediated energy metabolic impairment can contribute to the development of intestinal inflammation, and provide the possibility that correcting energy metabolism may help to relieve intestinal inflammation.

Fig. 2.

Mitochondrial dysfunction associated with IBD patient. (a-c) The activity of α-ketoglutarate dehydrogenase (α-KGDH) (a), citric acid dehydrogenase (ICDH) (b) and citric acid synthase (CS) (c) were negatively correlated with the disease activity index (CDAI) of Crohn’s disease by the chemical luminescence method. (d-f) Differences of activity of ICDH (d), CS (e) and α-KGDH (f) in normal intestinal tissue, colitis and mucosal healing. (j) The bio-TEM images of mitochondrial morphology in normal intestinal, inflammatory and mucosal healing sites

Synthesis and characterization of multifunctional HASe-Cr nanogel

Hyaluronic acid (HA), as an inexpensive, bio-versatile and natural glycosaminoglycan biopolymer, is widely used in the biological field, especially in enteritis treatment due to its colon-targeted response capacity, bio-targeting attributes that interact with HA-CD44, as well as partial remission of inflammation [3, 28]. Thus, ROS-responsive HA_Se-NH₂ nanogel was prepared using selenocystamine as cross-linker according to the previously reported articles [28], as illustrated in Fig. 3a, S1. The as-prepared Se-Se-containing HA-based nanogel showed a rough surface sphere-like shape with about 85 nm using the transmission electron microscopy (TEM) measurement (Fig. 3b). Additionally, the particle size of aqueous solution containing HA_Se-NH₂ nanogel became larger compared with dehydrated state, about 170 nm, with polydispersity (PDI) value was 0.21 (Fig. 3d) via dynamic light scattering (DLS) test, indicating that nanogel had swelling effect in aqueous solution. Fourier transform infrared (FT-IR) spectra showed the characteristic peaks in HA_Se-NH₂ nanogel of -C-N- at 1564 cm^− 1 and 1304 cm^− 1, -Se-Se- at 728 cm^− 1 and 541 cm^− 1, respectively (Fig. 3f) [28, 32] indicating the successful preparation of HA_Se-NH₂ nanogel. The new deconvoluted Se 3d (53–57 eV) peak was showed by X-ray photoelectron spectroscopy (XPS) testing (Fig. 3h and i) [33], and a new absorption peak appeared at 366 nm in UV-vis spectra (Fig. 3g), compared with free HA, revealing the presence of selenium in the HA_Se-NH₂ nanogel. Importantly, the strong peak appeared at about 3400 cm^− 1 was attributed to partial amino groups exposure compared to free HA (Fig. 3f) [34], which provided a chemical reaction site for the subsequent grafting of Cr by the EDC/NHS catalytic system. Cr, is an energy power source that regulates the distribution of energy of the body and also plays an important role in the field of disease therapy, especially IBD [25, 26]. Therefore, the Cr-modified selenium-based HA nanogel (named as HA_Se-Cr nanogel) was fabricated using simple amidation reaction with EDC/NHS catalytic system under mild conditions. HA_Se-Cr nanogel had a spheroid morphology with particle size of about 160 nm by TEM measurement (Fig. 3c), and hydrodynamic particle diameter was approximately 361 nm, PDI value of 0.23 (DLS test) (Fig. 3d). Obviously, the variation of nano-diameter of HA_Se-Cr nanogel was showed owing to the swelling effect of the hydrophilic small molecule Cr, compared to HA_Se-NH₂ nanogel. Additionally, by zeta potential analysis, HA_Se-NH₂ nanogel had a high negative charge (-27.8 mV), much higher than that of Cr-modified nanogel (-19.8 mV) (Fig. 3e), implying that Cr was successfully decorated onto nanogel. Moreover, to verify the successful introduction of Cr component in HA_Se-NH₂ nanogel, we further performed XPS and UV-vis spectrometry experiments. Both XPS (Se 3d, 53–57 eV) (Fig. 3i) and UV-vis (Fig. 3j) results demonstrated that there were disturbed in the modification of Cr, compared to HA_Se-NH₂ nanogel. Furthermore, the content of Cr in HA_Se-Cr nanogel was calculated to be 5.43% by creatine content assay kit (BL889B, Biosharp) with corresponding mass standard curve (mol/g). In addition, the content of selenium in HA_Se-NH₂ nanogel and HA_Se-Cr nanogel was 12.31% and 8.71%, respectively, by ICP technology. To demonstrate the stability and ROS responsiveness of HA_Se-Cr nanogel, the fluctuations of particle diameters were collected in PBS and H₂O₂-containing solution. DLS provided real-time monitoring of the stability of PBS-mediated HA_Se-Cr nanogel, and displayed negligible changes of hydrodynamic size in 0 ~ 4 days of PBS culture (Fig. S3). Meanwhile, in view of oral administration, we also evaluated the effect of acidity on HA_Se-Cr nanogel. The process of disintegration of HA_Se-Cr nanogel was showed over time, indicating that strong acid (pH = 1.1) contributed to HA decomposition [3], but time of disintegration was longer, probably because of the hydrogen bond effect between Cr and HA (Fig. S4, 3a). Generally, the retention time of oral agent is short-lived in stomach, about 2 ~ 4 h [35], thus HA_Se-Cr nanogel with strong acid-prolonging biodegradation behavior was not affected in IBD treatment (Fig. S4). Further, the Se-Se-modified HA nanogel demonstrated a tendency to decompose over time in the presence of H₂O₂ solution (5 mM), mainly because Se-Se-containing nanogel was oxidized to SeOOH groups (Fig. 3j, S2), and further leaded to the occurrence of cross-linker fracture.

Fig. 3.

Synthesis and characterization of HA _Se-Cr nanogel. (a) Schematic illustration of creatine-modified selenium-based HA nanogel. (b, c) TEM image of HA_Se-NH₂ nanogel (b) and HA_Se-Cr nanogel (c). (d) The hydrodynamic size of different nanogel in water solution. (e) ζ-potential of nanogel. (f, g) FT-IR spectra (f) and UV-vis spectra (g) of HA, creatine, HA_Se-NH₂ and HA_Se-Cr nanogel. (h, i) XPS spectra (h) and Se element binding energy (i) of HA, HA_Se-NH₂ and HA_Se-Cr nanogel samples. (j) Changes of hydrodynamic size of HA_Se-Cr nanogel with or without H₂O₂ (5 mM) treating

Subsequently, we also evaluated the release of Cr from HA_Se-Cr nanogel in vitro. As shown in Fig. S5, HA_Se-Cr nanogel exhibited ROS-responsive Cr release curves. The cumulative release of Cr derivative from HA_Se-Cr nanogel demonstrated the time-dependent and sustained release in the conditions of high ROS in simulated enteritis (pH 5.0 with 10 mM H₂O₂), and after 29 h, the cumulative release of Cr derivative could reach about 64%. In contrast, under the normal intestine condition (pH 5.0), Cr derivative showed low leakage, only about 3% at 29 h. In conclusion, the HA_Se-Cr nanogel with ROS response had precise drug release properties at the site of inflammation, as well as low leakage effect of normal tissue, which allowed the precise treatment of IBD.

Furthermore, in order to investigate whether the release of Cr derivative from HA_Se-Cr nanogel has the ability to restart energy metabolism, the changes of ATP of LPS-treated CaCO2 cells with different concentrations of HA_Se-Cr nanogel were investigated. As shown in Fig S9, as the concentration of HA_Se-Cr nanogel increased, the intracellular APT level showed an increasing tendency of concentration dependence, indicating that Cr-containing hydrogel could effectively assist in restarting intracellular energy metabolism. Additionally, HA_Se-Cr nanogel exhibited better energy recovery in LPS CaCO2 cells compared to free small Cr molecule, which may be due mainly to the important role of the selenium bonds in nanogel in addition to the released Cr derivative (Fig S9).

Antioxidative activity and anti-inflammatory effect in vitro

Considering the bio-application of Se-Se containing HA-based nanogel in IBD treatment, the cytotoxicity of HA_Se-Cr nanogel was investigated on CaCO2 cells and RAW264.7 cells via CCK-8 assay. The obtained outcomes showed HA_Se-Cr nanogel had great biocompatibility and dose-dependent low cell toxicity (Fig. S5), indicating the potential biological applications. Indeed, the regulation of ROS equilibrium is crucial in IBD treatment to significantly ameliorate intracellular oxidative stress and intestinal ecology [14]. Thus, ROS scavenging ability of HA_Se-Cr nanogel in CaCO2 cells was investigated by simulating ROS environment with H₂O₂. We obviously found that in the presence of 30 mM H₂O₂, CaCO2 cells was decimated, with only 38.2% cell viability. Conversely, various concentrations of HA_Se-Cr nanogel treated CaCO2 cells still maintained high cellular viability (≥ 95% cellular viability) (Fig. S7). However, fascinatingly, the tendency of H₂O₂-treated cell death was suppressed as HA_Se-Cr nanogel concentration increased, indicating that HA_Se-Cr nanogel could effectively consume the content of H₂O₂ in culture medium, thereby protecting CaCO2 cells. Additionally, apart from H₂O₂ stimulation, lipopolysaccharide (LPS) acts as a pathogenic molecular pattern bacteria factor that activates immune system and is commonly used to form cellular oxidative stress states [15, 16]. As shown in Fig. 4b, S8a, the normal CaCO2 cells showed intracellular low ROS levels using ROS-responsive fluorescent dye probe (2,7-dichlorofluorescin diacetate, DCFH-DA). However, after LPS administration, the intracellular ROS levels were significantly expanded while LPS-induced of ROS fluorescence gradually disappeared after treatment with HA_Se-Cr nanogel, indicating that HA_Se-Cr nanogel had prominent intracellular ROS capture capacity, thereby adjusting intracellular oxidative stress to protect cells, mainly through the reduction reaction of Se-Se groups in nanogel.

Fig. 4.

Therapeutic effects of HA _Se-Cr nanogel in vitro. (a) Schematic diagram of mechanism of HA_Se-Cr nanogel-mediated enteritis treatment, including M1-like macrophage polarization adjustment, ROS scavenging, restoration of intestinal epithelial function barrier, as well as mitochondria-mediated related therapy on IBD. (b) Evaluation of intracellular ROS indicated by DCFH-DA after treatment of HA_Se-Cr nanogel. (c) Bio-TEM images of mitochondria changes. (d) Immunofluorescent staining of JC-1 in CaCO2 cells after different samples treatment for 12 h. (e) Differences of activity of ICDH, CS and α-KGDH in normal cell, LPS-treated CaCO2 cell and HA_Se-Cr nanogel-treated-CaCO2 cell with LPS treatment. (f) The changes of ATP in CaCO2 cells were measured by ELISA kit. (g) The production of MDA in CaCO2 cells was measured by ELISA kit. (h) The mRNA expression levels of OCLN, CLDN and ZO-1 in RAW 264.7 cells detected by RT-PCR. (i) The mRNA expression levels of TNF-α, IL-6, IL-1β and IL-10 in RAW 264.7 cells detected by RT-PCR. (j) The mRNA expression levels of Arg1 and iNOS in RAW 264.7 cells detected by RT-PCR. Statistical analysis was performed by unpaired t-test, *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001

Mitochondria, as the energy supply plant involved in cell physiology, is also involved in the regulation of a variety of diseases, such as IBD [8, 9]. Against the above-mentioned mitochondria relevant-background, the characteristic structure of mitochondria in LPS-induced CaCO2 cells was analyzed by bio-TEM technology. Compare to normal cells, the mitochondria in CaCO2 cells by LPS treating was severely damaged, including swelling morphology and incomplete mitochondrial membrane, while HA_Se-Cr nanogel could significantly restore the mitochondrial structure in intestinal epithelium (Fig. 4c). Subsequently, the mitochondrial membrane potential (MMP, ΔΨm), as an important factor to evaluate normal function of mitochondria, was detected by JC-1 fluorescence probe. Normally, JC-1 probe-stained normal cells shows red fluorescence due to the formation of aggregates of JC-1. On the contrary, when MMP is destroyed, green fluorescence in mitochondrial matrix is emited due to JC-1 probe cannot be aggregated (JC-1 monomers) [15]. LPS-treated CaCO2 cells showed a strong green signal while red fluorescence was remarkably observed after treatment with HA_Se-Cr nanogel, and changes of red/green fluorescence were consistent with normal cells, clarifying that HA_Se-Cr nanogel could effectively improve the integrity of MMP, i.e., mitochondrial repair in IBD (Fig. 4d, S8b). Additionally, we further investigated TCA cycle-associated the core of enzymes in the process of mitochondrial energy metabolism, such as CS, ICDH and α-KGDH. After treatment with HA_Se-Cr nanogel, LPS-induced CaCO2 cells were effectively restored at intracellular levels of related enzymes, even to normal cellular levels, compared to free LPS-treated CaCO2 cells (Fig. 4e). At the same time, the intracellular ATP levels of LPS treatment were also significantly reversed by HA_Se-Cr nanogel treating, such results were similar to the above-mentioned results (Fig. 4f). Malondialdehyde (MDA), moreover, is mainly derived from excessive ROS-mediated liposomal peroxides in inflammatory colitis, which can be sever as a reference for IBD treatment [36]. MDA levels decreased significantly to normal cell levels under HA_Se-Cr nanogel disposal. Other treatment groups, such as Cr and HA_Se-NH₂, also played a level of inhibition of MDA, compared to LPS group (Fig. 4g). Anyway, the above results illustrated that HA_Se-Cr nanogel, through their outstanding ROS scavenging ability, could effectively repair the oxidative stress damage of mitochondria to maintain normal mitochondrial functions, meanwhile, speedily restarting intracellular mitochondrial energy metabolism by Cr derivative release, thereby hopefully achieving mitochondria-mediated IBD therapy.

The mucosal mechanical barrier acts as an essential barrier against pathogens and harmful substances to maintain the ecological balance of gut, so its integrity is an important indicator in the treatment of colitis. The cell-associated tight junction proteins, like, Claudin (CLDN), Ocludin (OCLN), and Zonulin-1 (ZO-1), are regarded as a non-negligible role in maintaining the integrity of intestinal mucosal barrier and normal intestinal permeability. Therefore, we used LPS-induced CaCO2 cells in vitro to mimic the injury mucosal mechanical barrier, and the expression levels of CLDN, OCLN, and ZO-1 were detected using RT-PCR. As shown in Fig. 4h, by HA_Se-Cr nanogel treating, the expression levels of related mRNA (CLDN, OCLN, and ZO-1) could be well restored to normal level, while other treatment groups such as free Cr and HA_Se-NH₂ nanogel, to some extent, also played a reversible role compared to the LPS group, demonstrating that the proposed HA_Se-Cr nanogel had significant therapeutic efficacy of epithelial tissue reparation.

As we all know, abundant secretion of pro-inflammatory factors (TNF-α, IL-1β, and IL-6) build inflammatory micro-environments, which play an important role in IBD pathophysiology [13]. Thus, to assess the anti-inflammation capacity of HA_Se-Cr nanogel, the production of inflammatory cytokines in LPS-stimulated RAW264.7 cells were measured by RT-PCR analysis (Fig. 4i and j). A large number of pro-inflammatory factors, including TNF-α, IL-1β and IL-6, up-regulated under LPS stimulation, compared to normal RAW264.7 cells. Conversely, the yield of different pro-inflammatory factors produced by LPS-induced RAW264.7 cells were suppressed after HA_Se-Cr nanogel treatment, indicating its significant anti-inflammation capacity. Other treatment groups, including Cr and HA_Se-NH₂ nanogel, partial anti-inflammation effects were also displayed.

Gut biodistribution and biocompatibility valuation of HASe-Cr nanogel in DSS-colitis mice

Prior to the research of effective treatment of colitis, we first assessed the enrichment of HA_Se-Cr nanogel in colitis, which is a key index. Therefore, 2.5% dextran sulfate sodium salt (DSS)-induced colitis mice were established and oral HA_Se-Cr nanogel was labeled with fluorescent probe of IGC (named as HA_Se-Cr@ICG) before used. As shown in Fig. 5a, for normal mice, oral ICG-labeled HA_Se-Cr nanogel showed a routine process of gastrointestinal metabolism. After 24 h, strong fluorescence intensity was highest compared to 8 h after oral administration, while fluorescence signal in the gastrointestinal tract (GIT) almost disappeared over time to 48 h later, demonstrating that oral HA_Se-Cr nanogel lacked the strong enrichment ability in normal mice and was easily excused through gastrointestinal metabolism. Interestingly, oral HA_Se-Cr nanogel exhibited strong enrichment in site of inflammatory intestine from 8 h to 48 h, which was inconsistent with fluorescent signal changes in healthy group. In addition, quantitative analysis of fluorescence tracing in vivo found that the intensity of fluorescence in GIT was almost 2 times after 48 h compared to normal group (Fig. 5c). Therefore, such results demonstrated that HA_Se-Cr nanogel had highly specific targeting property in the inflammatory areas.

Fig. 5.

Biodistribution of HA _Se Cr nanogel in vivo. (a) Distribution of HA_Se-Cr nanogel in digestive tract after oral administration for 8, 24 and 48 h in health BABL/c mice and 2.5% DSS-induced colitis mice. (b) After 48 h of administration with ICG-labelled HA_Se-Cr nanogel, the fluorescence images of isolated organs of treated-mice. (c, d) The statistics of fluorescence intensity of digestive tract with different treatment times (c) and major organs (d), mainly including liver and GIT. Error bars represent means ± SD, n = 5. Statistical analysis was performed by unpaired t-test, *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001

Subsequently, 48 h-treated mice were euthanized and collected their organs, including GIT and vital organs (heart, liver, spleen, kidney and lung), and then evaluated via fluorescence imaging technology and quantitatively analyzed (Fig. 5b). There were no fluctuations of fluorescence for most organs (heart, spleen, kidney and lung), but the site of GIT of fluorescence signal appeared a greater difference, namely the fluorescence intensity of inflamed gut was far greater than normal mice, and the difference values were five times (Fig. 5d).

In theory, HA_Se-Cr nanogel with aggregation ability in enteritis site was mainly negative charge-bearing nanogel, which was easily captured by positively charged enteric mucin, thereby forming electrostatic adsorption effect. Simultaneously, due to the existence of HA, HA-based nanogel remarkably showed the capacity of mucosal adhesion and HA-CD44 interaction [3, 28] which further enhanced the adhesion of nanogel to the location of enteritis. Thus, the proposed dual-targeting strategy of oral HA_Se-Cr nanogel demonstrated significant enteritis enrichment capability and had potential value for colitis treatment.

Furthermore, the biosafety and effectiveness of HA_Se-Cr nanogel cannot be ignored in the biological field. Therefore, we chosen healthy C57BL/6 mice and randomly divided them into two groups (n = 6). Subsequently, the classified mice were given with equal volume (100 µL, 1 mg/mL) PBS (control group) and HA_Se-Cr nanogel (HA_Se-Cr group), respectively, and the weight of mice were tracked (Fig. S10a). Analysis of kidney functions, blood biochemistry, important tissue organs status (heart, liver, spleen, lung, kidney), and fluctuations in body weight showed that live toxicity and inflammation were not significant throughout the experiment (Fig. S10b, S10c, S10d). Subsequently, no changes such as necrosis occurred in main organs by hematoxylin and eosin (H&E) staining analysis, indicating the good biocompatibility (Fig. S11).

Therapeutic effect of HASe-Cr nanogel in colitis mice

Inspired by the up-and-coming results of both in vitro therapeutic effect (Fig. 4a) and dual bio-targeting capabilities (Fig. 5), we will further examine the therapeutic effects of HA_Se-Cr nanogel in DSS-colitis mice model. The experimental procedure was implemented according to the flowsheet (Fig. 6a), and recorded body weight and medical conditions in real time (like, defecation appearances, bleeding situation). 2.5% DSS-induced mice displayed a time-dependent reduction in body weight (Fig. 6b), and the disease activity index (DAI) scores obviously climbed over time, compared to normal group (Fig. 6c), inducting the successful establishment of DSS-induced colitis mice model. Nevertheless, body weight of DSS-colitis mice restored, as well as the DAI values were greatly alleviated, in the presence of HA_Se-Cr nanogel. Other treatment groups such as free Cr and HA_Se-NH₂ nanogel, mice weight and DAI scores also gained some improvement in comparison to DSS-treated group. Subsequently, all treated mice were sacrificed, followed by the related vital organs (GIT and spleen) were removed for subsequent inflammatory treatment evaluation. Analysis results showed HA_Se-Cr nanogel could retrieve colon shortening more advantageous than other treatment groups in DSS mice (Fig. 6d and f). Meanwhile, spleen index also acquired analogous conclusions (Fig. 6e, S12). Thus, these results showed that HA_Se-Cr nanogel had significant therapeutic effect on enteritis and reduced inflammatory burden. To further analyze the therapeutic effect, the intestinal tissue staining of various samples were performed (Fig. 6g). When the formation of intestinal lesions in mice using DSS intervention, the histological features of enteritis were showed shortening of intestinal villi and loss of crypt while nearly normal histological microstructure and less inflammatory cell infiltration were observed in HA_Se-Cr nanogel treated-DSS group, via H&E staining. Simultaneously, trace improvements in morphological structure of intestinal tissue were showed in Cr or HA_Se-NH₂ nanogel group. Colon fibrosis is a common complication of IBD, including CD and UC, as a result of chronic inflammation, which can be used as a target for diagnosis and treatment. Therefore, increased gut collagen production after DSS intervention was obviously observed using masson staining (Fig. 6g). However, the remission of collagen hyperplasia was significantly improved and returned to normal level in HA_Se-Cr nanogel treatment, compared to other treatment groups (Cr and HA_Se-NH₂ nanogel group). Additionally, the results of digital photographs after 10 days of different samples-treated DSS-induced mice indicated that HA_Se-Cr nanogel could significantly improve rectal bleeding, which further demonstrated that HA_Se-Cr nanogel had significant therapeutic effects (Fig. 6h). Anyway, these results illustrated that HA_Se-Cr nanogel had obvious therapeutic effect in DSS induced acute colitis mice, with strong potential.

Fig. 6.

Therapeutic effect of HA _Se -Cr nanogel in colitis model. (a) Diagram of HA_Se-Cr nanogel in treatment of DSS-colonic mice. (b) Weight fluctuations in mice. (c) Calculation of changes in DAI. (d) Digital photos of gastrointestinal tract in different treatment groups. (e, f) Spleen index (e) and colon length (f) of mice from various groups. (g) Histological scores in different treated-groups based on massons and H&E staining. Scale bar: 100 μm. (h). Digital photos of mice after 10 days of treatment of various samples. Statistical analysis was performed by unpaired t-test, *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001

Therapeutic mechanisms of HASe-Cr nanogel on IBD

In order to further theoretically understand oral HA_Se-Cr nanogel treat intestinal inflammation, the intestinal epithelial function barriers were assessed via immunofluorescence and RT-PCR in mice colon. Analysis results revealed that these associated tight junction proteins (OCLN, CLDN and ZO-1) down-regulated in DSS-induced mice and showed significantly higher levels in HA_Se-Cr nanogel therapy (Fig. 7a, S13), indicating the ability of HA_Se-Cr nanogel to reconstruct the intestinal epithelial barriers. In addition, other therapeutic groups (free Cr and HA_Se-NH₂ nanogel) showed a certain degree of upregulation, but their therapeutic effects were limited.

Fig. 7.

Therapeutic mechanisms of HA _Se-Cr nanogel on IBD. (a) Representative immunofluorescent pictures of intestinal mucosa repair (OCLN, CLDN and ZO-1) and macrophage polarization (Arg1 and iNOS). Scale bar: 100 μm. (b) The related expression of cytokines in mice through RT-PCR (pro-inflammatory cytokines: IL-6, IL-1β and TNF-α; anti-inflammatory factor: IL-10). (c) Expression of CTA-related enzymes activity, including ICDH, CS and α-KGDH. (d) The bio-TEM images of changes of intracellular mitochondrion. Scale bar: 500 μm. Statistical analysis was performed by unpaired t-test, *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001

As is well known, DSS administration can induce acute colitis and bias macrophages towards the M1 phenotype, i.e., high expression levels of iNOS and lower levels of Arg1. Compared to the colitis mice, HA_Se-Cr nanogel promoted Arg1 expression to return to normal while remarkably inhibited the expression of iNOS, suggesting that it could induce M1 phenotype macrophages polarization (Fig. 7a). Subsequently, the mRNA expression of inflammatory cytokines in the colonic tissue were measured, including IFN-α, IL-6, IL-1β and IL-10 (Fig. 7b), which played important indexes in the treatment of IBD. Compared to health group, these pro-inflammatory cytokines (IFN-α, IL-6, IL-β) greatly increased and anti-inflammatory factor of IL-10 showed maximal inhibition in DSS-treated mice. However, an opposite trend of cytokines secretion was collected with different samples treatment (free Cr, HA_Se-NH₂ and HA_Se-Cr nanogel), while therapeutic efficacy was best for HA_Se-Cr nanogel, further suggesting HA_Se-Cr nanogel could promote M1-like macrophage polarize to M2 phenotypic cells.

As we all known, the function of mitochondria in DSS management shows a disordered metabolism pathway, involving energy metabolism and abundant ROS pump-out, thereby resulting in apoptosis and abnormal expression of proteins. Thus, the morphology of mitochondria and the activity of TCA-related energy mitochondrial metabolism were detected. As shown in Fig. 7c and d, after the treatment of DSS mice with HA_Se-Cr nanogel, the intracellular mitochondria structure in intestinal epithelium returned to normal, and other treatment groups (Cr and HA_Se-NH₂ nanogel) were partially recovered. The results of these treatments were consistent with the above-mentioned outcomes. In additional, TUNEL staining showed that HA_Se-Cr nanogel could effectively inhibit cellular apoptosis and effectively restore the normalization of inflammatory gut (Fig. s14).

Intestinal microbiota improved through HASe-Cr nanogel

The intestinal flora barriers are recognized as an important “the Great Wall” to resist disease-related invasion, consisting of complex microorganisms whose metabolites are involved in regulating intestinal homeostasis. Massive studies confirmed that the diversity and composition of gut flora can be used as diagnostic indicators of IBD, as well as therapeutic tool by reversing microorganisms systems [37, 38] Therefore, to determine the status of intestinal flora of mice after treatment, we collected the treated-mice stool for 16RS RNA testing. As shown in Fig. 8a, a decrease α-diversity of intestinal flora in DSS-induced mice was showed, indicating that abundance and diversity of intestinal flora decreased in intestinal inflammatory mice. However, after intervention of Cr, HA_Se-NH₂ nanogel and HA_Se-Cr nanogel, the gut flora’s α-diversity showed partial recovery, with the most significant improvement of HA_Se-Cr nanogel treatment. Meanwhile, the β-diversity of bacterial community occurred changes in the existence of DSS-treated group, compared to PBS group. Conversely, through different samples treating, such as Cr, HA_Se-NH₂ nanogel and HA_Se-Cr nanogel, a shift tendency in intestinal flora from enteritis mice to PBS group was displayed (Fig. 8b). Bacteroidetes and firmicutes are considered as important cornerstones in the intestinal flora, and the ratio of both is closely related to intestinal homeostasis [39, 40]. As shown in Fig. 8c, dysregulation of the ratio of bacteroidetes to firmicutes were observed in DSS group, characterized by a large reduction in beneficial bacteria, thereby leading to disturbance in the composition of the gut flora [38, 41]. Moreover, Cr or HA_Se-NH₂ nanogel-treated group showed a low intervention effects in the associated flora (Fig. 8c, d and e). Luckily, after HA_Se-Cr nanogel treatment, the intestinal flora of mice gradually tended to be normal, in which the ratio of bacteroidetes and firmicutes reversed to normal [38]. In addition, the abundance of probiotics such as Lactobacillales, Rikenella, Lactobacillus, etc., are significantly increased, and these bacterial classes maintain intestinal homeostasis by metabolically participating in the regulation of intestinal mucosa immunity [42, 43]. Further, the abundance of Clostridiales also increased in HA_Se-Cr nanogel group, thereby ameliorating gut homeostasis by Clostridium cluster and Clostridium butyricum [41]. Therefore, the above data showed that HA_Se-Cr nanogel had the capability of increase intestinal flora diversity, regulate gut bacterial imbalances, upgrade gut biological barriers in mice, and help maintain homeostasis of intestine.

Fig. 8.

HA _Se -Cr nanogel corrects intestinal flora disorders. (a, b) α-diversity (Shannon index) (a) and β-of Diversity (Bray-Curtis Distance) (b) were faecal microbiomes of each group of mice. (c) The KEGG functional divergence among faecal microbiota in each group of mice were predicted based on 16 S rRNA sequencing. (d) Heat map (columns) of relative abundance of taxon (rows) at family levels for each mouse. The relative abundance of gut microbiota was expressed as relative percentages (n = 5). (e) GO biological process clustering analysis from each group

Conclusion

IBD can give rise abnormal mitochondrial indicators, especially CTA-relevant energy metabolism, thus, mitochondria is regarded as important target for mediated IBD therapy. However, there are fewer reports of mitochondria-mediated IBD treatment. Therefore, rebooting metabolism of mitochondrial energy was first proposed to treat IBD by depleting ROS double-sided factors and Cr-assisted energy adjustment. Herein, an oral Cr-modified selenium-based HA nanogel (HA_Se-Cr nanogel) was fabricated for efficient dual-targeting treatment of IBD in this study. More concretely, oral HA_Se-Cr nanogel showed dual targeted inflammatory function due to IBD lesion-specific positive charge and high expression of CD44. Meanwhile, ROS-driven degradation of HA-based nanogel containing Se-Se displayed continuous release of Cr derivative and exposure of Se components owing to high-yield ROS levels in the area of inflammation. As expected, HA_Se-Cr nanogel could effectively conclude pathology-related of IBD of amelioration, such as restoration of mitochondrial bio-functions, inhibition of M1-like macrophage polarization, gut mucosal reconstruction, microbial ecological repair, etc., thus achieving excellent treatment of IBD. Overall, the proposed strategy underlines great potentiality of clinical applications of HA_Se-Cr nanogel by restarting mitochondrial metabolic energy in IBD, providing a new pavement of mitochondria-mediated IBD treatment.

Experiment section

Materials, Cells and animals Low molecular weight sodium hyaluronate (HA, MW = 400,000, cosmetic grade) was provided by Shandong Freda Biopharm. Co., Ltd. 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (EDCl, 99%) and N-hydroxysuccinimide (NHS, 98%) were provided by J&K Chemical Co., Ltd. Creatine (Cr, 98%) was purchased from San Chemical technology (Shanghai) Co., Ltd. Selenocystamine dihydrochloride (SeDi, 98%), dextran sulfate sodium (DSS, Mw = 5 kDa) and indocyanine green (ICG, 95%) were purchased from Aladdin Industrial Corporation (Shanghai, China). 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) was purchased from Beyotime Chemical Reagent (Jiangsu, China). 30% H₂O₂ solution was acquired from Sinopharm Chemical Reagent Co., Ltd.

Intestinal epithelial cell (CaCO2 cells) and RAW264.7 cells were purchased from ATCC. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) containing 10% FBS (Gibco, USA) and 1% Penicillin-Streptomyc, and humid incubator (37℃, 5% CO₂).

All animals, 6–8 weeks, SPF environment, were fed in experimental animal center of Shanghai Jiaotong University (ICCUC No: 2020-09-XLM-03) and were strictly carried out guidelines from “National Research Council’s Guide for the Care and Use of Laboratory Animals” during the animal experimental process.

Patient samples analysis Tissue specimens from patients with Crohn’s diease (CD) were collected from Xinhua Hospital of Shanghai Jiaotong University, School of Medicine. Collection of samples were approved by the Ethics Committee at Xinhua Hospital (Approval No: XHEC-C2021-096-1), Shanghai Jiaotong University School of Medicine.

Subsequently, we investigated intestinal biopsy samples taken from CD patients were used to determine the enzyme activity of CS, ICDH, and α-KGDH, in which colon polyps were treated as control group. Meanwhile, the optimal tissue of CD patients was taken out inflammation and mucous membrane healing positions, respectively, prior to analysis. After recording the weight of diverse tissue samples, all experimental operations were carried out in ice conditions. Finally, the contents of enzymes, such as CS, ICDH, and α-KGDH, extracted from relevant tissue, were examined with the corresponding enzyme-active kits (BC1056, BC2165, and BC0715, Solarbio).

Synthesis of HA_Se-NH₂and HA_Se-Cr conjugates HA_Se-NH₂ was synthesized according to the previous literature [28]. EDCl (50 mg, 0.26 mM) and NHS (30 mg, 0.26 mM) catalytic system mixture solution containing HA (120 mg) and selenocystamine dihydrochloride (80 mg, 0.25 mM) were stirred in mild conditions. After 24 h of reaction, the purified product of HA_Se-NH₂ was obtained by dialysis (molecular weight cut-off: 10,000 Da) against deionized water and freeze drying.

Creatine (131 mg, 1 mM, Cr) was dissolved into 15 mL of catalytic solution containing EDCI (287 mg, 1.5 mM) and NHS (172.5 mg, 1.5 mM) and stirred for 2 h to activate the carboxyl group of Cr. Subsequently, 10 mL HA_Se-NH₂ (100 mg) solution was added and stirred at room temperature for 24 h. The obtained HA_Se-Cr conjugate was collected by dialysis (molecular weight cut-off: 10,000 Da) and freeze-drying.

Characterization The morphology of HA_Se-NH₂ nanogel and HA_Se-Cr nanogel were characterized by JEM-2100 F transmission electron microscope (TEM, JEOL, Tokyo, Japan). The mean diameter and zeta potential of the as-prepared nanogels were measured by the dynamic laser light scattering (DLS, ZEN 3600, Malvern Instruments). Fourier transform infrared spectroscopy (FT-IR) spectra were recorded on a Nicolet 6700 FTIR spectrometer in the 4000 –400 cm^− 1 region. X-ray photoelectron spectroscopy (XPS) analysis was performed using an ESCALAB 250 with a monochromated X-ray source (Al Kα hν = 1486.6 eV).

Cr release profiles in vitro The in vitro Cr release from the HA_Se-Cr nanogel was evaluated by dialysis method, in different simulated releasing media: pH 5.0 phosphate buffered saline (PBS), and pH 5.0 PBS with 10 mM H₂O₂ solution, respectively. The HA_Se-Cr nanogel (10 mg) dispersion in 10 mL of above releasing media was dialyzed (MWCO of 14,000) in 110 mL of the corresponding releasing solution with shaking at 110 rpm at 37 °C. After certain times, 5 mL of the releasing solution was taken out to measure the Cr concentration by creatine content assay kit (BL889B, Biosharp). Simultaneously, 5 mL of the corresponding releasing solution was added to ensure the volume of the releasing medium constant [44].

Cytotoxicity assay in vitro The cytotoxicity of HA_Se-Cr nanogel was evaluated using CCK-8 assay. Briefly, RAW264.7 cells and CaCO2 cells (1 × 10⁴ cells per well) were cultured in 96-well plates, respectively. After 12 h of cultivation, these cells were treated with different concentrations of HA_Se-Cr nanogel (from 0.36 to 5 µg/mL) for 24 h. Additionally, to further understand the protective cell ability of HA_Se-Cr nanogel, CaCO2 cells (1 × 10⁴ cells per well) were seeded into 96-well plates with or without 10 µg/mL HA_Se-Cr nanogel for 12 h. These cells were incubated for another 12 h in the presence of 30 mM H₂O₂, and then CCK-8 assay (Beyotime, China) was performed to obtain parameters for cellular viability.

Intercellular ROS and mitochondrial bio-function assessment RAW 264.7 cells (1 × 10⁴ cells per well) were seeded into 24-well plates and then incubated for 24 h. The HA_Se-Cr nanogel was dispersed in the medium at a concentration of 10 mg/mL and then incubated in LPS solution for 24 h. Subsequently, DCFH-DA (HY-D0940, MCE) probe was used to determine ROS producing in RAW 264.7 cells.

Based on the above, the mitochondrial membrane potential of HA_Se-Cr nanogel-treated RAW 264.7 cells was determined using JC-1 (C2006, Beyotime) staining. Typically, the presence of red fluorescence in cells indicates mitochondrial membrane potential is higher, conversely, green fluorescence indicates a low potential. Therefore, fluctuations in mitochondrial membrane potential are usually determined using the red-green fluorescence ratio.

Differences of intracellular ATP (ab113849, Abcam) and MDA (ab118970, Abcam) in different samples-treated LPS-pretreated CaCO2 cells were measured using ATP and MDA kit. In short, CaCO2 cells were treated with different samples. After 24 h of cultivation, the medium was wished three times with cold PBS and then lysed 10 min with 200 µL of lysis buffer. Subsequently, the supernatant that needs to be tested was collected via centrifuging (rpm = 12,000 rpm for 5 min). Finally, the ATP levels and MDA doses were detected using a fluorescence microplate reader.

RT-PCR analysis The expression levels of tightly linked proteins (OCLN, CLDN and ZO-1), interleukins (IL-6, TNF-α and IL-1β) and macrophage markers (iNOS and Arg1) in different samples-treated cells or colon tissue of mice colon often were detected by RT-PCR technology. Simply, total RNA of cells or tissue were extracted by Trizol (Takara, Japan), and then obtained the cDNA by using cDNA Synthesis Kit (Yeasen, China) transcription. The expression levels of related mRNA were quantified by RT-PCR (Hieff ^®qPCR SYBR Green Master Mix (11201ES03, Yeasen) according to primer sequences (Table S1).

DSS-induced IBD mice model The mice with colitis were obtained by 2.5% DSS treating. All C57BL/6 mice, 6–8 weeks, male, were randomly divided into five groups (5 mice in each group), i.e., health group (PBS, control) and DSS-induced colitis mice groups, including DSS, creation, HA_Se-NH₂ nanogel and HA_Se-Cr nanogel). These mice were daily orally administered with creation alone (5 mg/kg), HA_Se-NH₂ nanogel (5 mg/kg) and HA_Se-Cr nanogel (5 mg/kg, containing Cr 0.26 mg/kg), respectively. Over 9 days, the body weight of mice was recorded every day and the disease activity index (DAI) was calculated. At the end of all treatments, the mice were sacrificed, and spleens and colons tissue were harvested to perform H&E staining and immunohistochemical staining.

Biodistribution of HA_Se-Cr nanogel in mice Normal C57BL/6 mice and 2.5% DSS-induced C57BL/6 mice were orally given 100 µL ICG-labeled HA_Se-Cr nanogel, respectively. Then, fluorescence imaging (VISQE In vivo Smart-LF, South Korea) of mice was traced at the preset time (8 h, 24 h and 48 h). And after 48 h of administration, all mice were sacrificed, and the main organs were extracted, including heart, liver, spleen, lung, kidney and gastroenterology tract, and further analysis was performed.

Biocompatibility tests in vivo To assess the biocompatibility of HA_Se-Cr nanogel in vivo, health C57BL/6 mice were randomly divided into two groups (n = 6). HA_Se-Cr nanogel (100 µL, 1 mg/mL) was orally administered into mice. Over the course of the experiment, the weight of the mice was recorded every other day for a total of 10 days. In fifth and tenth days, respectively, two mice were randomly removed, and then blood samples of mice were collected by extracting eyeball for blood biochemistry and blood routine analysis (including aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine (CREA), ect.), and main organs (heart, brain, spleen, lung and kidney) were stained by H&E.

H&E staining and immunofluorescence Main organs and colons from the treated mice were fixed for 24 h in 4% paraformaldehyde, then embedded in paraffin, and sliced for immunofluorescence staining, masson staining and hematoxylin-eosin (H&E) staining for histological evaluation.

16 S rRNA sequencing The evaluation of microbial ecology in the intestine was performed by collecting stool from mice, and analyzed using 16 S rRNA sequencing method. All specific experimental operations followed the experimental guidelines [3].

Statistical analysis Each experiment results were performed at least three times. The data were presented as mean ± standard deviation, and statistical significance was represented by a value of p < 0.05. Statistical analysis was performed by student t test to assess the differences between groups.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

M. H.: Investigation, Formal analysis, Visualization, Data Curation, Writing-original draft preparation. M. P.: Investigation, Formal analysis, Visualization, Data Curation, Writing-original draft preparation, Writing-Review & Editing, Supervision, Project administration, Funding acquisition. C. J.: Funding acquisition, Data Curation. X. D.: Biocompatibility study, Conceptualization, Investigation. F. S.: Conceptualization, Methodology, Project administration. Y. Z: Animal study. F. Y.: Conceptualization, Methodology, Supervision, Writing-original draft preparation, Writing-Review & Editing, Supervision, Project administration. W. G.: Conceptualization, Methodology, Writing-original draft preparation, Writing-Review & Editing, Supervision, Project administration, Funding acquisition.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

All animal care and experimental protocols comply with the relevant laws, regulations and standards concerning animal welfare ethics. This project has been supervised and approved by Laboratory Animal Welfare and Ethics Committee of Shanghai Jiaotong University (ICCUC NO. 2020-09-XLM-03).

Competing interests

The authors declare no competing interests.