ROS responsive hydrogel delivery system and preparation method and application thereof

Abstract

The invention discloses an ROS responsive hydrogel delivery system as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The hydrogel delivery system is prepared from sulfur-doped ginger carbon dots (SGCDs) and an ROS (reactive oxygen species) responsive sulfhydrylation hyaluronic acid (HS) hydrogel matrix, the SGCDs have excellent antioxidant and anti-inflammatory activity, and can be used for clearing ROS and simulating SOD and CAT enzyme functions; the HS hydrogel matrix triggers gelling in an inflammation part high ROS environment, and targeted drug release and colitis part retention are achieved. Through the synergistic effect of the SGCDs and the HS hydrogel matrix, the delivery system can efficiently remove ROS, promote polarization of macrophages from M1 type to M2 type, up-regulate an anti-inflammatory factor IL-10, down-regulate proinflammatory factors TNF-alpha, IL-1beta and IL-6, repair Occludin and remodel the intestinal flora homeostasis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an oral ROS-responsive hydrogel delivery system and its preparation method, as well as its application in the treatment of inflammatory bowel disease with anti-inflammatory, antioxidant, and gut microbiota regulation. Background Technology

[0002] Inflammatory bowel disease (IBD), primarily including ulcerative colitis and Crohn's disease, is a group of chronic, relapsing inflammatory bowel diseases with complex etiologies. Over the past few decades, the global incidence and prevalence of IBD have continued to rise, becoming an increasingly serious global public health problem.

[0003] Currently, clinical treatment of IBD primarily relies on 5-aminosalicylic acid, glucocorticoids, and immunosuppressants. In recent years, the introduction of biologics and small molecule drugs has provided more options for moderate to severe patients; however, these treatment regimens still face significant challenges. First, poor drug targeting is a major problem. After traditional oral administration, most active ingredients are absorbed or degraded in the upper gastrointestinal tract, making it difficult to achieve effective therapeutic concentrations at the site of colonic inflammation. Second, significant systemic toxicity is a concern. Long-term use of glucocorticoids can lead to complications such as central obesity and diabetes; immunosuppressants carry the risk of bone marrow suppression; and biologics may cause infections and allergic reactions. Furthermore, poor long-term efficacy and drug tolerance are prominent issues. Epidemiological data show that approximately 30% of patients receiving anti-TNF-α treatment experience primary non-response, leading to recurrent disease flare-ups.

[0004] From a pathological perspective, IBD is the result of the interaction of multiple factors, including genetic susceptibility, environmental factors, abnormal responses of the intestinal mucosal immune system, and gut microbiota dysbiosis. Among these, intestinal mucosal barrier dysfunction and gut microbiota dysbiosis are considered core pathological links. In the IBD state, excessive activation of immune cells produces a large amount of reactive oxygen species (ROS), leading to a severe oxidative stress microenvironment in the intestinal tract. Studies have shown that ROS levels in the colonic mucosa of IBD patients can reach 10-100 times that of normal individuals. This excessive ROS not only directly attacks intestinal epithelial cells, disrupting the tight junctions between cells, but also inhibits the function of goblet cells, resulting in thinning or even loss of the protective mucus layer.

[0005] This impaired barrier function creates conditions for gut microbiota and their antigens to invade the submucosa. These foreign components are recognized by innate immune cells, prompting the release of large amounts of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6, forming a vicious cycle of "inflammation-barrier disruption-microbiota translocation-intensified inflammation." Simultaneously, the abnormal immune inflammatory response also leads to decreased microbiota diversity, a reduction in beneficial bacteria, and an increase in the abundance of potentially pathogenic bacteria; the dysbiosis, in turn, exacerbates the inflammatory process.

[0006] In recent years, the development of nanotechnology has provided new opportunities for the treatment of IBD. Carbon dots (CDs), as an emerging nanomaterial, possess excellent biocompatibility and unique enzyme-like activity. Sulfur (S) doping can effectively enhance the antioxidant properties of CDs, directly scavenging free radicals through a hydrogen atom transfer mechanism. Regarding carbon source selection, natural plants such as ginger, rich in antioxidant components, are ideal carbon sources for synthesizing bioactive CDs.

[0007] However, oral delivery of nanomedicines faces numerous challenges. Hydrogels, with their excellent biocompatibility and three-dimensional network structure, have become ideal drug carriers. Smart responsive hydrogels can sense microenvironmental signals at the lesion site, enabling on-demand drug release. The high concentration of ROS in IBD lesion areas provides unique conditions for designing responsive delivery systems. Thiol-modified natural polysaccharides can achieve in-situ gelation at the inflamed colon, not only prolonging the retention time of the formulation and forming a physical barrier to protect the mucosa, but also controlling drug release behavior.

[0008] In summary, developing an intelligent delivery system capable of oral administration, targeting the site of colonic inflammation, and integrating multiple functions such as antioxidation, immune modulation, barrier repair, and microbiome regulation is of great significance for the treatment of IBD. This invention constructs a composite therapeutic system based on sulfur-doped ginger carbon dots and ROS-responsive hydrogels, aiming to provide an innovative treatment solution for IBD through multi-target synergistic intervention. Summary of the Invention

[0009] The purpose of this invention is to construct an oral ROS-responsive hydrogel delivery system that can target the site of colonic inflammation and achieve intelligent release of sulfur-doped ginger carbon dots (SGCDs). Through the multidimensional synergistic effects of clearing ROS, regulating immunity, repairing the intestinal barrier, and reshaping the microbiota homeostasis, it can effectively treat inflammatory bowel disease.

[0010] The technical solution adopted in this invention is as follows: The oral ROS-responsive hydrogel delivery system of this invention is composed of sulfur-doped ginger carbon dots (SGCDs) and ROS-responsive thiolized hyaluronic acid (HS) hydrogel matrix.

[0011] Preferably, the SGCDs are prepared by a hydrothermal method using ginger juice as the carbon source and β-mercaptoethylamine as the sulfur source. The SGCDs have superoxide dismutase (SOD) and catalase (CAT)-like activities, an average particle size of approximately 3 nm, and produce maximum fluorescence emission at an excitation wavelength of 370 nm.

[0012] Preferably, the ROS-responsive thiolized hyaluronic acid (HS) hydrogel matrix is ​​prepared by amidation reaction of hyaluronic acid and cystamine dihydrochloride, followed by reduction, with a molar ratio of 1:2 to 1:6. The HS hydrogel matrix undergoes disulfide cross-linking to form a gel under ROS conditions, and its gelation time is related to the H2O2 concentration and the degree of thiol substitution. Specifically, the HS prepared at a molar ratio of 1:6 yields... 1:6 The gelation time is approximately 1 hour under 100 μM H2O2 conditions.

[0013] The precursor solution of this oral ROS-responsive hydrogel can undergo in-situ oxidative cross-linking in the high ROS environment of the colonic inflammation site to form a three-dimensional network structure gel.

[0014] The present invention discloses a method for preparing an oral ROS-responsive hydrogel delivery system, comprising the following steps: Step 1: Prepare raw materials; Preparation of sulfur-doped ginger carbon dots (SGCDs): Ginger was peeled and juiced, the supernatant was collected after initial filtration and centrifugation, and the lyophilized product was denoted as Gin. Gin was mixed with β-mercaptoethylamine and subjected to a hydrothermal reaction. After the reaction was completed, the mixture was purified by dialysis and then lyophilized to obtain SGCDs powder. Preparation of thiolized hyaluronic acid (HS): Hyaluronic acid (HA) was dissolved in 2-morpholinoethanesulfonic acid (MES) buffer, activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), followed by the addition of cystamine dihydrochloride and reaction under nitrogen protection. After purification, tris(2-carboxyethyl)phosphine (TCEP) was added for reduction, followed by dialyzing and lyophilization to obtain solid HS.

[0015] Step 2: Prepare HS@SGCDs composite hydrogel; The prepared SGCDs and HS were dissolved separately and then mixed in proportion to obtain the HS@SGCDs precursor solution.

[0016] Preferably, in step 1, the hydrothermal reaction temperature is 100℃-180℃, the reaction time is 2-10 hours, and Gin and β-mercaptoethylamine are mixed at a mass ratio of 90:0-90:30.

[0017] More preferably, in step 1, the hydrothermal reaction temperature is 140°C, the reaction time is 8 hours, and the mass ratio of Gin to β-mercaptoethylamine is 90:20.

[0018] Preferably, in step 1, the feed ratio of HA to cystamine dihydrochloride is HA:cystamine = 1:2-1:6 by molar ratio, the reaction is carried out at 30℃-40℃ under nitrogen protection, the pH of the reaction system is 2.0-4.0, and the dialysis time is 24-72 hours.

[0019] Preferably, in step 2, the final concentrations of SGCDs and HS in the precursor solution are 10 mg / mL-15 mg / mL and 10 mg / mL-15 mg / mL, respectively.

[0020] More preferably, in step 2, the final concentrations of SGCDs and HS in the precursor solution are 10 mg / mL and 15 mg / mL, respectively.

[0021] This invention also provides the application of the aforementioned ROS-responsive hydrogel delivery system in the preparation of drugs for treating acute inflammatory bowel disease. This hydrogel delivery system, administered orally, gels at the site of colonic inflammation in response to ROS, achieving targeted release of sulfur-doped ginger carbon dots. It synergistically treats inflammatory bowel disease through multiple pathways, including antioxidant and anti-inflammatory effects, regulation of macrophage polarization, repair of the intestinal barrier, and remodeling of gut microbiota homeostasis.

[0022] The beneficial effects of this invention are: (1) The SGCDs prepared by this invention have uniform nanoscale size (about 3 nm), excellent fluorescence performance, excellent free radical scavenging ability and superoxide dismutase (SOD) and catalase (CAT) enzyme activities.

[0023] (2) The HS hydrogel matrix prepared by the present invention has good ROS responsiveness, can be specifically gelled at the site of inflammation to form a physical barrier, prolong the drug retention time, and achieve controlled release of SGCDs.

[0024] (3) Through the synergistic effect of SGCDs and HS hydrogel matrix, this delivery system can efficiently clear ROS, promote macrophage polarization from M1 to M2, upregulate anti-inflammatory factor IL-10, downregulate pro-inflammatory factors TNF-α, IL-1β and IL-6, repair intestinal tight junction protein (ZO-1, Occludin), and reshape intestinal flora homeostasis.

[0025] (4) In the DSS-induced mouse model of acute IBD, HS 1:6 @SGCDs have demonstrated superior therapeutic effects compared to the commercially available drug sulfasalazine (SASP), significantly improving the disease activity index (DAI), colon length, and histopathological damage, providing a novel synergistic treatment strategy for IBD involving "ROS clearance, immune regulation, barrier repair, and gut microbiota homeostasis." Attached Figure Description

[0026] Figure 1 Comparison of the scavenging rates of (A) DPPH, (B) ABTS and (C) ·OH by Gin and GCDs prepared at different temperatures for Example 1.

[0027] Figure 2Comparison of the scavenging rates of (A) DPPH, (B) ABTS and (C) ·OH with different feed ratios of Gin and β-mercaptoethylamine prepared in Example 1.

[0028] Figure 3 The comparison of the scavenging rates of SGCDs prepared in Example 1 for (A) DPPH, (B) ABTS and (C) ·OH under different heating times.

[0029] Figure 4 The transmission electron microscope image of the SGCDs prepared in Example 1 shows that they have a uniform particle size of approximately 3 nm.

[0030] Figure 5 The XPS spectrum of the SGCDs prepared in Example 1 confirms successful S doping.

[0031] Figure 6 The Gin, GCDs, and SGCDs prepared in Example 1 are paired with O2 - Comparison of clearance rates.

[0032] Figure 7 A comparison of the H2O2 scavenging rates of Gin, GCDs, and SGCDs prepared in Example 1.

[0033] Figure 8 The 1H NMR spectrum of the HS prepared in Example 2 is shown.

[0034] Figure 9 The gelation time diagram of HS prepared in Example 2 under different concentrations of H2O2 is shown.

[0035] Figure 10 Scanning electron microscope (SEM) images of the HS hydrogel prepared in Example 2; wherein, (a) HA, (b) HS 1:2 (c)HS 1:4 , (d)HS 1:6 .

[0036] Figure 11 HS prepared in Example 3 1:6 In vitro release curves of @SGCDs.

[0037] Figure 12 HS prepared in Example 3 1:6 Adhesion strength test results for @SGCDs.

[0038] Figure 13 HS prepared in Example 3 1:6 Confocal microscopy image of the effect of @SGCDs on ROS scavenging in RAW 264.7 cells.

[0039] Figure 14 HS prepared in Example 31:6 Flow cytometry analysis of @SGCDs promoting macrophage M1 to M2 phenotypic polarization.

[0040] Figure 15 HS prepared in Example 3 1:6 Effects of @SGCDs on body weight changes (A) and DAI score (B) in a mouse IBD model.

[0041] Figure 16 HS prepared in Example 3 1:6 @SGCDs were used for ultra-microvascular imaging and two-dimensional ultrasound images of the mouse colon; among them, G1 was the control group, G2 was the IBD group, G3 was the SGCDs group, and G4 was the HS group. 1:6 Groups G5 and G6 are SASP groups. 1:6 @SGCDs group.

[0042] Figure 17 HS prepared in Example 3 1:6 Effects of @SGCDs on the expression of pro-inflammatory and anti-inflammatory factors in colonic tissue; where A: MPO, B: TNF-α, C: IL-1β; D: IL-6; E: IL-10.

[0043] Figure 18 HS prepared in Example 3 1:6 Effects of @SGCDs on the expression of intestinal tight junction proteins ZO-1 and Occludin.

[0044] Figure 19 HS prepared in Example 3 1:6 The effect of @SGCDs on the relative abundance of gut microbiota at the phylum (A) and family (B) levels. Detailed Implementation

[0045] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by this invention.

[0046] Example 1

[0047] Preparation and characterization of sulfur-doped ginger carbon dots (SGCDs): Fresh ginger was peeled and juiced, filtered through cheesecloth, and the supernatant was collected by centrifugation. 30 mL of this juice was freeze-dried and weighed to obtain 900 mg of freeze-dried ginger product (Gin). The ginger content was then quantified using this ratio. Gin was reacted at 100℃-180℃ for 8 hours to obtain GCDs. The antioxidant properties of different concentrations of Gin and GCDs against DPPH, ABTS, and ·OH were compared, and the optimal temperature was determined to be 140℃. Figure 1 Sulfur-doped ginger carbon dots (SGCDs) were prepared by reacting Gin with β-mercaptoethylamine at different feed ratios (90:0 to 90:30) at 140℃. The antioxidant properties of SGCDs prepared at different feed ratios and concentrations were then screened for DPPH, ABTS, and ·OH. The results showed that the SGCDs prepared at a feed ratio of 90:20 exhibited the strongest antioxidant properties. Figure 2 Gin was reacted with β-mercaptoethylamine at a feed ratio of 90:20 at 140℃. The effect of different heating times (2-10 hours) on the antioxidant properties of SGCDs prepared at different concentrations was investigated. The results showed that the antioxidant properties were strongest at a heating time of 8 hours. Figure 3 ).

[0048] The morphology of SGCDs was characterized using high-resolution transmission electron microscopy (TEM). Figure 4 SGCDs were dispersed in ultrapure water to prepare a suspension of 0.1 mg / mL, which was then added dropwise onto a copper grid and allowed to dry naturally before observation. The results showed that the SGCDs were highly uniform, nearly spherical, well-dispersed, and exhibited no significant aggregation, with an average particle size of 3 nm. X-ray photoelectron spectroscopy (XPS) analysis revealed that the elemental contents of C, N, O, and S in the SGCDs were 61.45%, 10.87%, 21.27%, and 6.41%, respectively. The characteristic peaks of the S2p orbital at 163.1 eV and 164.4 eV confirmed the successful formation of CSC bonds. Figure 5 Its enzyme-like catalytic activity was further evaluated, such as... Figures 6-7 As shown, SGCDs exhibited excellent SOD and CAT enzyme activities at different concentrations.

[0049] Example 2

[0050] Synthesis and performance evaluation of thiolated hyaluronic acid (HS): Accurately weigh 400 mg of hyaluronic acid (HA, molecular weight 800,000-1,500,000) and dissolve it in 80 mL of MES buffer (0.1 M). Stir magnetically until completely dissolved. Add 766 mg of EDC and 345 mg of NHS sequentially, and activate at room temperature for 40 minutes. Then add cystamine dihydrochloride (HA to cystamine molar ratio of 1:2, 1:4, and 1:6), purge with nitrogen, and react in a 37°C water bath for 4 hours. After the reaction, transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 8000-14000 Da and dialyze with ultrapure water for 48 hours. Add 1.0 g of TCEP to the dialyzed solution, purge with nitrogen again, and react at 37°C for 4 hours for reduction. Finally, dialyze the reaction solution in hydrochloric acid solution at pH 3.5 for 48 hours and freeze-dry to obtain a white solid HS.

[0051] By nuclear magnetic resonance hydrogen spectrum ( 1 The structure of the product was confirmed by 1H NMR. The sample was dissolved in D2O and measured on a 500MHz NMR spectrometer. In addition to the characteristic peak of the HA skeleton (methyl peak at δ=1.93ppm), characteristic signals at δ=2.58ppm and δ=2.82ppm, belonging to the methylene proton in the -CH2-CH2-S- structure, were observed, confirming the successful grafting of the thiol group. Figure 8 ).

[0052] The ROS-responsive gelation behavior of the systems was determined using the inverted tube method. Under 100 μM H₂O₂ conditions, all three materials crosslinked and formed hydrogels in approximately 1 hour. When the H₂O₂ concentration was reduced to 1 μM and 10 μM, the gelation process slowed down, but all samples still gelled within 8 hours. At any given H₂O₂ concentration, the ROS-responsive gelation behavior of the HS… 1:6 The gelation rate of all samples was the fastest, indicating a high degree of thiol substitution. As a control, under conditions without H2O2, the gelation time of all samples exceeded 24 hours, further confirming that H2O2-triggered disulfide crosslinking is the main mechanism of gel formation. Figure 9 The microstructure of the sample was observed using SEM (Sequencing Microscopy). Figure 10 ), and unmodified HA and HS were observed by SEM. 1:2 HS 1:4 and HS 1:6 All four samples formed typical three-dimensional porous network structures, which are traces of pores left by the sublimation of ice crystals after freeze-drying. However, there are significant differences among the four in pore size, pore wall thickness, and network regularity. The network structure formed by the unmodified HA is relatively loose, with larger and unevenly distributed pores and relatively thinner pore walls. Compared with HS... 1:2 and HS 1:4 In comparison, HS1:6 The resulting three-dimensional network is the densest and most regular, with significantly reduced pore size and thickened pore walls, exhibiting a more refined and stable porous structure. This morphological evolution indicates that the crosslinking density of the hydrogel increases significantly with increasing thiol substitution degree. Ultimately, HS was selected. 1:6 As a gel matrix.

[0053] Example 3

[0054] HS 1:6 Construction and performance study of @SGCDs composite system: Accurately weigh 150mg HS 1:6 The solid was dissolved in 6 mL of PBS (pH 7.4) to prepare a stock solution of 25 mg / mL. Separately, 100 mg of SGCDs was accurately weighed and dissolved in 4 mL of ultrapure water to prepare a stock solution of 25 mg / mL. The two solutions were mixed in a 2:3 ratio and vortexed for 5 minutes to obtain a homogeneous HS solution. 1:6 @SGCDs precursor solution. The mixture was reacted into a gel under 100 μM H₂O₂ conditions and placed in 15 mL of simulated colonic fluid (SCF, pH 7.4) for release experiments. Samples of 3 mL were taken at predetermined time points (0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 hours), with an equal volume of fresh release medium added simultaneously. After filtration through a 0.22 μm filter, the absorbance was measured at 340 nm, and the cumulative release rate of SGCDs was calculated.

[0055] Release curve shows ( Figure 11 The in vitro release profiles of SGCDs showed that in the initial rapid release phase (0-2 hours), the cumulative release rate rapidly reached approximately 60%, mainly attributed to the rapid diffusion of SGCDs physically adsorbed in the pores of the hydrogel network surface. Subsequently, the release entered a sustained-release phase (2-10 hours), with the release rate slowing significantly, ultimately reaching approximately 80% cumulative release within 10 hours. 1:2 / 1:4 / 1:6 The adhesion strength test results of @SGCDs show that ( Figure 12 Of the three systems, HS 1:6 @SGCDs exhibited stronger adhesion to colonic tissue, with an adhesion force of (3.23±0.25) kPa. Therefore, HS was selected. 1:6 @SGCDs were used as the final formulation group.

[0056] Example 4

[0057] Cellular experimental studies: (1) Intracellular ROS scavenging experiment: RAW264.7 cells were seeded in 24-well plates. An inflammation model was established by stimulation with 0.5 μg / mL LPS for 4 hours. Cells were then treated with GCDs, SGCDs, and HS-containing enzymes, respectively. 1:6 and HS 1:6 @SGCDs were treated with culture medium for 2 hours. 20 μM DCFH-DA probe was added, and the mixture was incubated at 37°C in the dark for 30 minutes. After washing with PBS, the samples were immediately observed under a confocal microscope. The results showed ( Figure 13 ), HS 1:6 The @SGCDs treatment group showed the weakest fluorescence intensity and the strongest ROS scavenging rate, significantly outperforming all other groups.

[0058] (2) Macrophage phenotypic polarization experiment: RAW264.7 cells were stimulated with 0.5 μg / mL LPS for 24 hours to induce M1 polarization, followed by treatment with different formulations for 24 hours. Cells were collected, stained with CD86-FITC and CD206-PE antibodies, and analyzed by flow cytometry. The results showed ( Figure 14 ), HS 1:6 The proportion of CD86+ M1 macrophages in the @SGCDs treatment group decreased to 7.93%, while the proportion of CD206+ M2 macrophages increased to 30.4%, indicating the most significant phenotypic transformation effect.

[0059] Example 5

[0060] Evaluation of therapeutic effects in animals: (1) Animal model establishment and drug administration: Thirty-six healthy male C57BL / 6 mice (18-22g, 7-9 weeks old) were acclimatized for one week under standard laboratory conditions. They were randomly divided into 6 groups (n=6): normal control group, IBD model group, SGCDs group (80mg / kg), and HS group. 1:6 Group (100 mg / kg), sulfasalazine (SASP) group (SASP, 100 mg / kg) and HS 1:6 @SGCDs group (SGCDs 80mg / kg as standard). Except for the normal control group, mice in the other groups had free access to sterile water containing 3% (w / v) DSS to establish an acute IBD model for 7 consecutive days. Starting from the first day of modeling, the mice were administered the drug by gavage every morning. The normal control group and the model group were given an equal volume of physiological saline for 10 consecutive days.

[0061] (2) Efficacy evaluation indicators: Mouse weight, fecal characteristics, and fecal blood loss were recorded daily, and the Disease Activity Index (DAI) was calculated. At the end of the experiment, mice were anesthetized and euthanized, and colon tissue was harvested to measure length and calculate spleen coefficient. A portion of the colon tissue was fixed with 4% paraformaldehyde for histopathological examination; another portion was cryopreserved at -80°C for subsequent molecular biological assays.

[0062] (3) Results analysis: Compared with the normal control group, the IBD model group mice showed a significant decrease in body weight ( Figure 15 The DAI score was significantly elevated, the colon length shortened to 4.95±0.74 cm, and the spleen coefficient increased. In each treatment group, HS... 1:6 The @SGCDs group showed the best treatment results, the fastest weight recovery, the lowest DAI score, and colon length recovery to 7.30±0.46cm, close to the normal level.

[0063] There were significant differences in the microcirculation status of the colon wall in the mice of different groups. Figure 16 ). According to HS 1:6 After treatment with @SGCDs, the colonic blood flow signal in IBD mice was significantly enhanced, and vascular elasticity and blood flow were effectively restored, approaching normal levels, demonstrating the remarkable efficacy of this preparation in repairing intestinal microcirculation. (Two-dimensional ultrasound image) Figure 16 The images show that the colon thickness in the IBD model group mice was significantly thinner than that in the healthy control group, which may reflect pathological changes such as mucosal atrophy, fibrosis, and tissue remodeling caused by inflammation. HS 1:6 The intestinal wall thickness recovery was most significant in the @SGCDs treatment group, with its structure most closely resembling that of the normal control group, demonstrating remarkable efficacy in reversing intestinal structural damage. Further investigation of HS was conducted using ELISA. 1:6 The mechanism of action of @SGCDs. Results showed that this complex system inhibited the expression of pro-inflammatory factors such as TNF-α, IL-1β, and IL-6, and promoted the secretion of anti-inflammatory factors such as IL-10. Figure 17 Simultaneously, it can upregulate the expression of tight junction proteins ZO-1 and Occludin. Figure 18 ), and repair the intestinal barrier function.

[0064] (4) Intestinal flora analysis: Fecal samples were collected from each group of mice, and 16S rRNA sequencing was performed. At the phylum level, the Firmicutes / Bacteroidetes ratio tended to normalize; at the family level, the relative abundance of beneficial bacteria such as Trichophyceae significantly increased, while the abundance of opportunistic pathogens such as Tanneraceae decreased. Figure 19 ).

[0065] In summary, the HS prepared by this invention 1:6The @SGCDs composite system exhibits excellent ROS responsiveness, biocompatibility, and multiple therapeutic functions, demonstrating significant advantages in the treatment of inflammatory bowel disease and showing important clinical application prospects.

Claims

1. A ROS-responsive hydrogel delivery system, characterized in that, It is composed of sulfur-doped ginger carbon dots and a ROS-responsive thiolized hyaluronic acid hydrogel matrix, wherein the sulfur-doped ginger carbon dots have superoxide dismutase-like and catalase-like activities.

2. The ROS-responsive hydrogel delivery system according to claim 1, characterized in that, The sulfur-doped ginger carbon dots are prepared by hydrothermal method using ginger juice as the carbon source and β-mercaptoethylamine as the sulfur source; the average particle size is 3 nm, and the maximum fluorescence emission is produced at an excitation wavelength of 370 nm.

3. The ROS-responsive hydrogel delivery system according to claim 1, characterized in that, The ROS-responsive thiolized hyaluronic acid hydrogel matrix is ​​prepared by amidation reaction of hyaluronic acid and cystamine dihydrochloride followed by reduction, with a molar ratio of 1:2 to 1:

6. The ROS-responsive thiolized hyaluronic acid hydrogel matrix undergoes disulfide cross-linking to form a gel under ROS environment. The gelation time is related to the H2O2 concentration and the degree of thiol substitution. The hydrogel matrix prepared with a molar ratio of 1:6 has a gelation time of 1 hour under 100 μM H2O2 conditions.

4. A method for preparing the ROS-responsive hydrogel delivery system according to any one of claims 1-3, characterized in that, Includes the following steps: Preparation of sulfur-doped ginger carbon dots: Ginger juice was mixed with β-mercaptoethylamine and subjected to hydrothermal reaction. After the reaction was completed, the mixture was purified by dialysis and freeze-dried to obtain sulfur-doped ginger carbon dots. Preparation of thiolized hyaluronic acid: Hyaluronic acid was dissolved in MES buffer, activated with EDC and NHS, and then cystamine dihydrochloride was added. The reaction was carried out under nitrogen protection, reduced by TCEP, dialyzed, and lyophilized to obtain thiolized hyaluronic acid. Preparation of HS@SGCDs composite hydrogel: The sulfur-doped ginger carbon dots and thiolized hyaluronic acid were dissolved separately and then mixed in proportion to obtain HS@SGCDs precursor solution.

5. The preparation method according to claim 4, characterized in that, The hydrothermal reaction temperature is 100℃-180℃, the reaction time is 2-10 hours, and the mass ratio of ginger juice to β-mercaptoethylamine is 90:0-90:

30.

6. The preparation method according to claim 4, characterized in that, The molar ratio of hyaluronic acid to cystamine dihydrochloride is hyaluronic acid:cystamine = 1:2-1:6, the pH of the reaction system is 2.0-4.0, and the dialysis time is 24-72 hours.

7. The preparation method according to claim 4, characterized in that, The final concentrations of sulfur-doped ginger carbon dots and thiolated hyaluronic acid in the precursor solution were 10 mg / mL-15 mg / mL and 10 mg / mL-15 mg / mL, respectively.

8. The use of the ROS-responsive hydrogel delivery system according to any one of claims 1-3 in the preparation of a medicament for treating acute inflammatory bowel disease.

9. The application according to claim 8, characterized in that, This hydrogel system, administered orally, gels in response to ROS at sites of colonic inflammation, enabling targeted release of sulfur-doped ginger carbon dots. It synergistically treats inflammatory bowel disease through multiple pathways, including anti-oxidation, anti-inflammation, regulation of macrophage polarization, repair of the intestinal barrier, and reshaping of gut microbiota homeostasis.

Images