Sulfhydrylated hyaluronic acid drug-loaded hydrogel preparation as well as preparation method and application thereof

Abstract

The invention belongs to the field of pharmaceutical preparations, and particularly relates to a sulfhydrylated hyaluronic acid drug-loaded hydrogel preparation and a preparation method and application thereof, and the preparation method comprises the following steps: preparing isorhamnetin nanocrystalline ISO-NCs by adopting an anti-solvent precipitation method; the preparation method comprises the following steps: constructing thiolated hyaluronic acid HA-SH responded by GSH; and uniformly embedding the ISO-NCs into the HA-SH hydrogel, so as to prepare the sulfhydrylated hyaluronic acid drug-loaded hydrogel preparation ISO-NCs-coated Gel, and preparing the sulfhydrylated hyaluronic acid drug-loaded hydrogel preparation ISO-NCs-coated Gel. The drug-loaded hydrogel preparation constructed by the invention can be used for targeted therapy of colon cancer.

Description

A thiol-modified hyaluronic acid drug-loaded hydrogel formulation, its preparation method and application Technical Field

[0001] This invention belongs to the field of pharmaceutical formulation technology, specifically relating to a thiolized hyaluronic acid drug-loaded hydrogel formulation, its preparation method, and its application. Background Technology

[0002] Colorectal cancer (CRC) is one of the most common malignant tumors of the digestive tract. Inflammatory bowel disease-related or long-term chronic intestinal inflammation can promote tumor development and progression. Its core pathological processes include: long-term high expression of inflammatory factors, redox homeostasis imbalance, damage to the tight junctions of the intestinal epithelium, and impairment of the mucus barrier, leading to increased intestinal barrier permeability, microbiota / toxin translocation, and persistent immune activation. Clinical and basic research suggests that intestinal inflammation and barrier damage can affect central nervous system function, inducing or exacerbating neuropsychiatric symptoms such as depression and anxiety. Current treatments mainly consist of surgery, radiotherapy, chemotherapy, and targeted / immunotherapy, but systemic administration still carries a high risk of adverse reactions and recurrence. Furthermore, there is a lack of treatment options that can simultaneously inhibit tumor progression and improve tumor-related mood disorders.

[0003] The tumor microenvironment of chronic CRC typically exhibits chronic inflammation, abnormal intestinal barrier function, and a state of abnormal reduction (high GSH). High GSH can act as a "reduction trigger," driving the breakage or degradation of carriers containing disulfide bonds / thioethers, thereby achieving local responsive release. Therefore, applying a strategy of "inflammatory localization + high GSH response" to oral colon-targeted delivery holds promise for achieving high drug concentrations at the lesion site and reducing systemic exposure.

[0004] Existing technological approaches include:

[0005] (a) Oral colon-targeted: pH-sensitive coating (Eudragit, etc.), time-controlled release, enzyme response (colonic flora degrading polysaccharides) are commonly used. However, there are release deviations due to individual differences, inflammation, and pH / transport conditions. The release position / time of oral colon-targeted systems is easily affected by gastrointestinal pH, peristalsis, degree of inflammation, and individual differences, leading to premature or insufficient drug release.

[0006] (b) Mucosal adhesion and local retention: Chitosan, carbomer, polysaccharide gels, etc. can improve adhesion and retention, but their adhesion strength / anti-flushing ability is limited, and they lack lesion-specific triggering. Conventional adhesive carriers have limited retention time in the environment of strong colonic flushing and mucus renewal, and it is difficult to maintain local drug concentration.

[0007] (c) Nanoparticles: Nanoparticles, liposomes, nanocrystals, etc. can improve the solubility and cellular uptake of poorly soluble drugs, but they are easily diluted by gastrointestinal fluid and cleared by mucus in the oral route, making them easy to be diluted / cleared and difficult to accumulate in the inflammatory tumor area for a long time, resulting in insufficient effective delivery.

[0008] (d) Intelligent response release: Red oxygen response, ROS response and pH response systems have been studied for tumor treatment, but there are still few systems that integrate multiple functions such as "oral colon targeting + mucosal adhesion + inflammation targeting + GSH response degradation / drug release".

[0009] (e) Using polymers containing disulfide bonds to achieve reduction-response release.

[0010] However, the above-mentioned approaches often cannot simultaneously address: ① selective adhesion and enrichment of inflammatory lesions; ② rapid and controllable triggering of release in the high-GSH tumor microenvironment of CRC; ③ reducing systemic exposure while simultaneously addressing intestinal barrier repair and mood disorder improvement. Most systems focus only on a single endpoint of anti-tumor or anti-inflammatory treatment, lacking the comprehensive intervention capability targeting the interconnected pathology of "intestinal barrier damage—inflammation—tumor—mood disorder". Summary of the Invention

[0011] To address the shortcomings of the prior art, the present invention aims to provide an orally administered, colon-targeted, multifunctional delivery platform.

[0012] In a first aspect, the present invention provides a method for preparing a thiolized hyaluronic acid drug-loaded hydrogel formulation, comprising the steps of: preparing isorhamnetin nanocrystals (ISO-NCs) using an antisolvent precipitation method; constructing GSH-responsive thiolized hyaluronic acid (HA-SH); and uniformly embedding the ISO-NCs into the HA-SH hydrogel to prepare a thiolized hyaluronic acid drug-loaded hydrogel (ISO-NCs@Gel).

[0013] The preparation method of this invention first involves preparing isorhamnetin (ISO) into nanocrystals (ISO-NCs) using an antisolvent precipitation method. Secondly, considering the characteristics of mucosal barrier disruption and abnormal glutathione (GSH) enrichment in the colorectal tumor microenvironment (TME), a hydrogel based on thiolized hyaluronic acid (HA) (HA-SH) is constructed. Due to the introduction of thiol groups, this adhesive hydrogel is responsive to GSH. Subsequently, by uniformly embedding ISO-NCs into this hydrogel, a composite delivery system ISO-NCs@Gel is prepared.

[0014] Preferably, the preparation method of the ISO-NCs includes the following steps: injecting the isorhamnetin organic phase into the aqueous phase under stirring conditions, and homogenizing under high pressure to obtain the ISO-NCs dispersion, wherein DMSO is used as a good solvent in the isorhamnetin organic phase, and the ratio of isorhamnetin to DMSO is 20 mg to 1 ml.

[0015] Further preferably, the aqueous phase also contains the stabilizer poloxamer 407 (POL 407) to obtain a more stable nanocrystalline dispersion with a finer particle size.

[0016] Preferably, the construction of the GSH-responsive cystamine and thiolated hyaluronic acid HA-SH includes the following steps: 1) Preparation of thiolated hyaluronic acid HA-Cys; a) Dissolving hyaluronic acid HA in MES buffer at pH=6 to obtain an HA solution; b) Adding EDC·HCl and NHS to the HA solution under stirring at room temperature, such that the molar ratio of HA to the carboxyl group in the repeating unit is 1:1-2:1-2, and then stirring the reaction system under N2 protection and light protection to form an activated ester intermediate; c) Adding cystamine sulfate to the activated system, such that the molar ratio of cystamine to -COOH in HA is 1:1-2, and stirring the reaction system under room temperature, N2 protection and light protection to undergo an amidation reaction, generating cystamine and thiolated hyaluronic acid HA-Cys; d) After the reaction, the solution was dialyzed to obtain purified HA-Cys; 2) HA-Cys solution was prepared using the purified HA-Cys obtained above, the pH value was adjusted to 4.5-5.5, excess TCEP was added, and the reaction was carried out under the conditions of room temperature, N2 protection and protection from light, with stirring to reduce and break the disulfide bonds of the side chain of HA-Cys into free thiol groups; 3) Mild oxidation (such as air oxidation or the addition of a trace amount of oxidant) was used to oxidize some of the free thiol groups into disulfide bonds, forming a cross-linked network that can be reduced and broken, to obtain GSH-responsive cystamine and thiolized hyaluronic acid HA-SH.

[0017] Preferably, "uniformly embedding the ISO-NCs into the HA-SH hydrogel" is achieved through the following steps: uniformly mixing the ISO-NCs dispersion with HA-SH to obtain a mixture, followed by mild oxidation to crosslink it into a gel, resulting in ISO-NCs@Gel. More preferably, the final concentration of HA-SH in the mixture is 3 wt%. The mild oxidation can be air oxidation or oxidation with the addition of a trace amount of oxidant.

[0018] A second aspect of the present invention also provides a thiolized hyaluronic acid drug-loaded hydrogel formulation obtained by the aforementioned preparation method. This formulation comprises an ISO nanocrystal (ISO-NCs) carrier and a thiolized hyaluronic acid hydrogel (HA-SH). On one hand, isorhamnetin is prepared into nanocrystals to improve the solubility, stability, and cellular uptake of ISO. On the other hand, hyaluronic acid possesses good biocompatibility and the potential to interact with inflammation / tumor-related receptors (such as CD44); thiol groups (-SH) can form disulfide bonds / thiol exchange with mucins, enhancing intestinal mucosal adhesion; simultaneously, the gel network contains disulfide bonds that can be reduced by GSH, enabling GSH-responsive degradation and drug release. The combination of these two elements forms a multifunctional delivery platform, improving therapeutic efficacy.

[0019] The formulation can be made into gel blocks / gel microparticles / gel capsule fillers, etc.

[0020] In a third aspect, the present invention also provides a medicament comprising the aforementioned thiolated hyaluronic acid drug-loaded hydrogel formulation, wherein the medicament is available for oral administration.

[0021] A fourth aspect of the invention also provides the use of the above-described medicament in the preparation of colorectal cancer drugs. In particular, it is used to improve neuropsychiatric and behavioral abnormalities such as depression and anxiety associated with the disease in inflammation-related colorectal cancer / colitis-related CRC.

[0022] The beneficial effects of the present invention are as follows: The present invention provides an orally administered hydrogel preparation that can be used for targeted therapy of colon cancer, enabling ISO to achieve the following at the inflammation-related CRC lesion: (1) strong adhesion and long retention: firmly adheres to the surface of the inflamed colonic mucosa, resists the flushing of intestinal contents, and prolongs local retention.

[0023] (2) Precise release from the inflammation / tumor microenvironment: Rapid degradation and release of drug-loaded nanocrystals under the high GSH conditions of the CRC tumor microenvironment, enabling on-demand drug delivery.

[0024] (3) Improve the availability of poorly soluble drugs: improve solubility, stability and cellular uptake through ISO nanocrystallization.

[0025] (4) Comprehensive treatment: While suppressing the tumor burden, it relieves intestinal inflammation, repairs tight junctions and mucus barrier, thereby improving depression / anxiety-like behaviors associated with colon cancer pathology. Attached Figure Description

[0026] Figure 1 is a flowchart of the preparation process of the thiolized hyaluronic acid drug-loaded hydrogel formulation of this invention; Figure 2 shows the representative SEM images, particle size distribution, short-term particle size stability, and PDI distribution of ISO-NCs of this invention; Figure 3 shows the scanning electron microscope images of ISO-NCs@Gel of this invention; Figure 4 shows the nuclear magnetic resonance spectra and Fourier transform infrared spectra of hyaluronic acid, cystamine, and thiolized hyaluronic acid of this invention; Figure 5 shows the biodistribution, inflammation targeting ability, and glutathione (GSH) responsiveness of ISO-NCs@Gel of this invention; Figure 6 shows the GSH-responsive degradation and drug release curves of ISO-NCs@Gel of this invention; Figure 7 shows the animal experimental results of this invention (inflammation score, barrier index, tumor burden); Figure 8 shows the behavioral evaluation results of this invention. Detailed Implementation

[0027] Referring to Figure 1, the preparation method of this invention first uses an antisolvent precipitation method to prepare isorhamnetin (ISO) into nanocrystals (ISO-NCs). Secondly, considering the characteristics of mucosal barrier disruption and abnormal glutathione (GSH) enrichment in the colorectal tumor microenvironment (TME), a hydrogel based on thiolized hyaluronic acid (HA) (HA-SH) was constructed. Due to the introduction of thiol groups, this adhesive hydrogel is responsive to GSH. Subsequently, ISO-NCs are uniformly embedded in the hydrogel through physical stirring to prepare the composite delivery system ISO-NCs@Gel.

[0028] The hydrogel formulation of this invention comprises ISO nanocrystals and thiolized hyaluronic acid hydrogel, and the hydrogel network contains a disulfide bond cross-linking structure that can be reduced and broken by GSH. ISO nanocrystals can improve the solubility and stability of poorly soluble drugs, promote interaction and uptake with tumor / inflammatory cells, thereby exerting anti-inflammatory, barrier repair, and anti-tumor effects. The thiol groups in HA-SH can undergo disulfide bond exchange / covalent binding with mucin, enhancing adhesion and anti-erosion ability on inflamed colonic mucosa, achieving long-term retention of the gel through thiol adhesion and inflammation targeting, resulting in prolonged retention of colonic lesions. Furthermore, the high GSH reductiveity of the CRC tumor microenvironment triggers disulfide bond breaking, leading to rapid gel degradation, increased porosity, and release of ISO-NCs, achieving rapid release at the lesion site. This rapid gel degradation and on-demand release significantly improves local effective drug exposure and reduces systemic exposure. This combined strategy can simultaneously alleviate intestinal inflammation, repair tight junctions and the mucus barrier, and reduce tumor burden, further contributing to the relief of depressive / anxiety-like behaviors associated with intestinal pathology.

[0029] ISO-NCs are prepared by anti-solvent precipitation, whereby a uniform and stable nanocrystal is obtained by dispersing the organic phase in an aqueous phase. In a preferred embodiment, the following steps are included: injecting the isorhamnetin organic phase into the aqueous phase under stirring conditions, and homogenizing under high pressure to obtain the ISO-NCs dispersion, wherein DMSO is used as a good solvent in the isorhamnetin organic phase, and the ratio of isorhamnetin to DMSO is 20 mg to 1 ml.

[0030] Furthermore, the aqueous phase also contains a stabilizer. Preferably, poloxamer 407 (POL 407) can be selected as the stabilizer to obtain a more stable nanocrystalline dispersion with a finer particle size. In a preferred embodiment, the amount of POL 407 used is 1-2 mg / ml, more preferably 1.5 mg / ml.

[0031] The preferred reaction route for HA thiolation is activation-amidation with EDC·HCl and NHS. In a preferred embodiment, the construction of the GSH-responsive thiolated hyaluronic acid HA-SH includes the following steps: 1) Preparation of thiolated hyaluronic acid HA-Cys; a) Dissolving hyaluronic acid HA in MES buffer at pH=6 to obtain an HA solution; b) Adding EDC·HCl and NHS to the HA solution under stirring at room temperature, such that the molar ratio of HA to the carboxyl group in the repeating unit is 1:1-2:1-2, and then stirring the reaction system under N2 protection and light protection to form an activated ester intermediate; c) Adding cystamine sulfate to the activated system, such that the molar ratio of cystamine to -COOH in HA is 1:1-2, and stirring the reaction system under room temperature, N2 protection and light protection to undergo an amidation reaction, generating cystamine and thiolated hyaluronic acid HA-Cys; d) After the reaction, the solution was dialyzed to obtain purified HA-Cys; 2) HA-Cys solution was prepared using the purified HA-Cys obtained above, the pH value was adjusted to 4.5-5.5, excess TCEP was added, and the reaction was carried out under the conditions of room temperature, N2 protection and protection from light, with stirring to reduce and break the disulfide bonds of the side chain of HA-Cys into free thiol groups; 3) Mild oxidation (such as air oxidation or the addition of a trace amount of oxidant) was used to oxidize some of the free thiol groups into disulfide bonds, forming a cross-linked network that can be reduced and broken, to obtain GSH-responsive thiolized hyaluronic acid HA-SH.

[0032] The loading of nanocrystals and hyaluronic acid networks is selected through cross-linking after mixing. In a preferred embodiment, the ISO-NCs dispersion is uniformly mixed with HA-SH to obtain a mixture, which is then subjected to mild oxidation to cross-link into a gel, resulting in ISO-NCs@Gel. In a preferred embodiment, the final concentration of HA-SH in the mixture is 3 wt%.

[0033] The mild oxidation in the preparation method of this invention can be air oxidation or oxidation with the addition of a trace amount of oxidant.

[0034] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to embodiments.

[0035] Example 1: Preparation of ISO nanocrystals (ISO-NCs) ISO nanocrystals were prepared by antisolvent precipitation, specifically including the following steps: a) 20 mg of ISO was dissolved in 1 ml of DMSO (DMSO was used as a good solvent); b) Using antisolvent precipitation, the ISO organic phase was injected into an aqueous phase containing 1.5 mg / ml stabilizer (Poloxamer 407, POL 407) under stirring at 1000 rpm / min; c) The suspension was refined by high-pressure homogenization, and isorhamnetin nanocrystals (ISO-NCs) were successfully prepared.

[0036] The obtained ISO-NCs exhibit a spherical morphology (Fig. 2A). Dynamic light scattering (DLS) analysis results show that their average particle size is 158.4 nm, with a narrow particle size distribution (Fig. 2B), while also possessing good uniformity and short-term stability (Fig. 2C and Fig. 2D).

[0037] Example 2 Preparation of GHS-responsive thiolated hyaluronic acid (HA-SH) (1) Preparation of thiolated hyaluronic acid (HA-SH) a) Dissolve hyaluronic acid HA in MES buffer (pH=6) to obtain a HA solution with a mass concentration of 1wt%, and mix under magnetic stirring; b) Under stirring conditions at room temperature, add EDC·HCl and NHS to the above HA solution so that the molar ratio of EDC·HCl and NHS to the carboxyl group (-COOH) in the repeating unit of HA is 1:1.5:1.5; place the reaction system under light-protected conditions and introduce N2 to replace the air for 15 min to obtain an inert atmosphere, and continue to react for 3 h under N2 protection, light-protected conditions, and stirring at 300 rpm / min to form an activated ester intermediate; c) Cystamine sulfate was added to the activation system to achieve a molar ratio of -COOH to cystamine of 1:1.5. The reaction was continued for 4 hours under N2 protection and light-protected conditions with stirring at 300 rpm / min to induce amidation and obtain thiolized HA (HA-Cys). d) After the reaction was completed, the reaction solution was transferred to a dialysis bag (molecular weight cutoff 3.5 kDa) and dialyzed with deionized water for 12 hours. The dialysate was replaced every 4 hours during dialysis to remove unreacted small molecules and byproducts. The dialysate was kept in the dark to obtain purified HA-Cys.

[0038] (2) Construction of GSH-responsive thiolated hyaluronic acid (HA-SH) a) The pH of the HA-Cys solution was adjusted to approximately 5, and excess TCEP was added. The reaction was carried out at room temperature, under N2 protection, and in the dark, with stirring at 300 rpm / min for 4 h to reduce and break the disulfide bonds in the side chain into free thiol groups, thus obtaining the HA-SH solution; b) Some thiol groups were oxidized to disulfide bonds by air oxidation to form a reducible cross-linked network; c) After the reaction was completed, the reaction solution was transferred to a dialysis bag (molecular weight cutoff 3.5 kDa), and dialyzed with hydrochloric acid solution at pH=4 for 24 h. The dialysate was replaced every 6 h during dialysis to remove unreacted TCEP and its byproducts; after purification, the solution was frozen and lyophilized to obtain thiolated hyaluronic acid (HA-SH) solid. It was stored under light-protected, low-temperature, and N2 protection to reduce thiol oxidation.

[0039] Figure 4 shows the NMR and Fourier transform infrared (FTIR) spectra of hyaluronic acid (HA), cystamine (Cys), and thiolated hyaluronic acid (HA-SH) from Example 2. The proton NMR (¹H NMR) spectrum of HA-SH shows characteristic methylene peaks associated with cystamine at δ = 2.52 and 2.79 ppm, indicating that cystamine has been successfully grafted onto the HA backbone. Simultaneously, the Fourier transform infrared (FTIR) spectrum further confirms the formation of HA-SH: at 3450 cm⁻¹... -1 The O–H stretching vibration peak was observed at 1638 cm⁻¹. -1 A C=O stretching vibration peak appears at 1560 cm⁻¹, and the peak is observed at 1560 cm⁻¹. -1 An absorption band associated with amide N–H was detected at 2661 cm⁻¹. Furthermore, an absorption band related to amide N–H was also detected at 2661 cm⁻¹. -1 and 1313 cm -1 Weak absorption peaks corresponding to the S–H and C–S bonds were detected at the respective positions. These results indicate that the thiol group was successfully introduced into the HA backbone via an amide bond.

[0040] Example 3: Preparation of ISO-NCs@Gel. The ISO-NCs dispersion obtained in Example 1 was uniformly mixed with the HA-SH solid obtained in Example 2 to achieve a final HA-SH concentration of 3 wt%. Subsequently, air oxidation was performed to crosslink the HA-SH into a gel, yielding ISO-NCs@Gel.

[0041] Figure 3 shows a scanning electron microscope image of ISO-NCs@Gel. It can be seen that the cross-linked ISO-NCs@Gel system exhibits a three-dimensional network structure with obvious pores, which is a typical feature of hydrogel systems.

[0042] Validation Example: A mouse colitis-associated colorectal cancer model induced by azomethane / sodium dextran sulfate (AOM / DSS) was established. Male C57BL / 6J mice, aged 6–8 weeks and weighing 20–25 g, were housed in a specific pathogen-free (SPF) animal room (temperature 25 ± 2 ℃, humidity 55 ± 5%, 12 h light / 12 h dark cycle) with free access to food and water. After one week of acclimatization, all mice except the normal control group received a single intraperitoneal injection of AOM (10 mg / kg) to induce colonic cancer; simultaneously, the normal control group received an equal volume of physiological saline. Subsequently, starting one week after AOM administration, chronic colitis was induced by adding 2.5% DSS to the drinking water for 7 consecutive days, followed by 14 days of normal drinking water. This 21-day DSS cycle (1 week of DSS + 2 weeks of normal drinking water) was repeated 4 times (total duration 12 weeks).

[0043] Experiment 1 Cy5@ISO-NCs group: ISO-NCs obtained in Example 1 were orally administered and fluorescently labeled with Cy5.

[0044] Cy5@ISO-NCs@Gel group: The ISO-NCs@Gel obtained in Example 3 was orally administered and fluorescently labeled with Cy5.

[0045] Figure 5 shows the biodistribution, inflammation targeting ability, and glutathione (GSH) responsiveness of ISO-NCs@Gel. In terms of gastrointestinal residence time and overall signal persistence, the Cy5@ISO-NCs group showed a rapid decrease in fluorescence signal after administration, with significant decay after 6 h, and the fluorescence signal becoming virtually undetectable at 12 h and 24 h. In contrast, the Cy5@ISO-NCs@Gel group exhibited a more persistent fluorescence signal in the gastrointestinal tract, indicating that the hydrogel carrier can significantly prolong the residence time of nanocrystals in the gastrointestinal tract and enhance local retention. Gastrointestinal migration and colonic enrichment trends: In vitro gastrointestinal fluorescence imaging showed that the formulation passed through the stomach within the first 2 h after administration; within the first 6 h after administration, it was mainly located in the ileum and cecum, then gradually migrated towards the colon and maintained a strong fluorescence signal in the colonic region. Significant gastrointestinal fluorescence was still detectable in the experimental group at 12 h and 24 h, suggesting strong resistance to digestive fluid degradation and clearance, and persistent retention / enrichment characteristics in the colon. Major organ accumulation and systemic exposure: The control group showed strong fluorescence signals in the liver and lungs, indicating that ISO-NCs were more likely to enter the systemic circulation and distribute to non-target organs; while the experimental group showed only weak fluorescence signals in major organs such as the heart, liver, spleen, lungs, and kidneys, with no obvious organ-specific accumulation. Furthermore, blood fluorescence detection results showed that the blood fluorescence intensity of the control group was significantly higher than that of the experimental group at 12 h and 24 h, indicating that only a small amount of nanocrystals entered the systemic circulation in the experimental group, and systemic exposure was significantly reduced. In vitro experiments showed that the formulation had the strongest adhesion to mucin; and the higher the GSH level, the weaker the adhesion, consistent with its design logic of being able to disintegrate / release the drug in a tumor-reducing environment.

[0046] Experiment 2 involved immersing ISO-NCs@Gel in PBS and PBS containing different concentrations of GSH (to simulate the reducing conditions of the tumor microenvironment). Weights were measured at corresponding time points to compare the swelling and degradation of each group, thus assessing their sensitivity to GSH.

[0047] To observe the glutathione (GSH)-responsive drug release of ISO-NCs@Gel, experiments were conducted in PBS containing different concentrations of GSH (to simulate the reducing conditions of the tumor microenvironment). ISO-NCs@Gel samples were placed in PBS containing 0 mM (control) and higher concentrations (experimental group) of GSH and incubated at 37 ℃ with gentle shaking. After 24 h, a certain volume of sample solution was removed from the release medium and replenished with an equal volume of fresh solution. The release amount of ISO was measured at 370 nm using UV-Vis spectrophotometry, and the release curves of each group were compared to evaluate its sensitivity to GSH.

[0048] Figure 6 shows the GSH-responsive degradation and drug release curves of ISO-NCs@Gel. The HA-SH hydrogel exhibited a significant GSH concentration-dependent degradation under GSH conditions ranging from 0.01 to 10 mM: the higher the GSH concentration, the faster the disulfide bonds were reduced and broken, the lower the crosslinking density, and the faster the network structure disintegrated. Degradation began at approximately 48 h at 0.05 mM and was completely degraded within 24 h at 0.3 mM, while remaining stable in GSH-free PBS. Due to the hydrophilicity of HA, the hydrogel initially swelled rapidly (approximately 6-fold increase in weight) under all conditions, followed by accelerated degradation and drug release under high GSH conditions; furthermore, the cumulative release rate of ISO-NCs@Gel increased from 88.51% to 97.59% after 24 h at 0.3 mM GSH. Therefore, the results indicate that under the typical high GSH conditions of the tumor microenvironment, the HA-SH hydrogel exhibits accelerated degradation, which is beneficial for drug release.

[0049] Experiment 3: Immunohistochemical (IHC) staining was used to detect specific proteins in colon tissue sections. Paraffin-embedded colon tissue sections (5 μm) were dewaxed and then rehydrated. Antigen retrieval was performed by heating the sections in sodium citrate buffer to expose the epitopes, followed by cooling to room temperature. The sections were then treated with 3% H2O2 for 15 min to block endogenous peroxidase activity, and then washed with PBS. Subsequently, the sections were blocked with 5% BSA or normal serum for 1 h at room temperature to reduce non-specific binding. A primary antibody against MPO was added to the sections, and the sections were incubated overnight at 4 °C. The next day, after washing, the sections were incubated with HRP-conjugated secondary antibody at room temperature for 1 h. After washing again, DAB chromogenic substrate solution was added to produce a brown staining signal at the antibody binding sites. The reaction was terminated with water once the desired staining intensity was reached. The cell nuclei were then counterstained with hematoxylin, followed by dehydration, clearing, and mounting. When the stained sections were observed under an optical microscope, the IHC positive signal appeared brown on the blue background stained with hematoxylin.

[0050] AB-PAS staining: Paraffin-embedded intestinal tissue sections (5 μm) were dewaxed with xylene, rehydrated to water using graded ethanol, and then stained in 1% Alcian Blue solution at pH 2.5 for 15–30 min, followed by thorough rinsing with water. Oxidation was then performed with 0.5–1% periodic acid for 5–10 min, followed by rinsing and reaction with Schiff's reagent in the dark for 10–20 min, followed by thorough rinsing with running water. The cell nuclei were counterstained with hematoxylin for 0.5–2 min, and after blue staining, the sections were dehydrated with graded ethanol, cleared with xylene, and mounted with neutral mounting medium. The staining results were observed and recorded under an optical microscope. Acidic mucus appeared blue, neutral mucus / glycogen appeared reddish-purple, double-positive mucus appeared blue-purple, and the cell nuclei appeared blue.

[0051] Immunofluorescence staining was used to visualize the expression and localization of specific protein markers in cells after different treatments. Cells were seeded in confocal culture dishes and, after reaching confluence, were treated according to the experimental design. After treatment, cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. They were then permeabilized with 0.5% Triton X-100 for 20 min and washed again with PBS. Blocking with 1% bovine serum albumin (BSA) for 30 min was used to prevent non-specific antibody binding. The cells were then incubated overnight at 4 °C with the corresponding primary antibody ZO-1 / Occludin. The next day, after washing with PBS, the cells were incubated with the corresponding fluorescent dye-conjugated secondary antibody at room temperature for 1 h in the dark. Finally, the cell nuclei were counterstained with DAPI. Samples were mounted with anti-quenching mounting medium and observed using a confocal laser scanning microscope (CLSM) to analyze the localization and expression levels of the target proteins.

[0052] The levels of TNF-α, IL-6, IL-1β, and IL-10 in serum were quantitatively detected using a commercial sandwich ELISA kit (Jianglai Bio, China) according to the manufacturer's instructions. The simplified procedure is as follows: Standards and serum samples diluted as required were added to 96-well plates pre-coated with capture antibodies and incubated under the conditions specified in the kit. After washing, the corresponding detection antibodies and enzyme conjugates were added sequentially, followed by the addition of TMB substrate for color development. The reaction was terminated using the stop solution provided in the kit, and the absorbance was measured at 450 nm using a microplate reader. Cytokine concentrations were calculated by interpolation using a standard curve fitted with a four-parameter Logistic (4-PL) regression method and corrected for dilution. Samples exceeding the upper limit of quantitation were retested after further dilution. All samples were tested in triplicate, and results are expressed in pg / mL.

[0053] The experiment was conducted in the following groups: Cotrol group: No modeling treatment was performed; patients consumed normal food and water, and were given an equal volume of physiological saline by gavage during the drug administration period. Model group: In the first week of the experiment, patients received a single intraperitoneal injection of AOM (10 mg / kg). In the second week (one week after AOM administration), 2.5% DSS was added to drinking water for 7 consecutive days, followed by 14 days of normal drinking water. The above 21-day DSS cycle (1 week of DSS + 2 weeks of normal drinking water) was repeated 4 times (total duration 12 weeks). Positive group: The modeling treatment was the same as the Model group. In the seventh week of the experiment, drug administration began, with 5-fluorouracil (5-FU) 10 mg / kg administered intraperitoneally every other day.

[0054] ISO group: The modeling treatment was the same as the Model group. In week 7 of the experiment, the drug treatment was started: ISO-L (10 mg / kg), ISO-M (40 mg / kg), ISO-H (80 mg / kg), by gavage once a day.

[0055] ISO-NCs: The modeling treatment was the same as the Model group. In week 7 of the experiment, the treatment was started with ISO-NCs (ISO equivalent dose, 10 mg / kg), administered by gavage once a day.

[0056] ISO-NCs@Gel: The modeling treatment was the same as the Model group. In week 7 of the experiment, the treatment was started with ISO-NCs@Gel (ISO equivalent dose, 10 mg / kg), administered by gavage once a day.

[0057] Gel group: The modeling treatment was the same as the Model group. In week 7 of the experiment, the drug treatment began. Gel (without drugs, blank gel) was administered by gavage once a day.

[0058] Eight mice per group.

[0059] Figure 7 shows the inflammation score, barrier indices, and tumor burden in the animal experiments. In the colorectal cancer model group, significant aggregation of MPO-positive cells was observed, indicating extensive neutrophil infiltration and a strong chronic inflammatory response. Compared with the model group, all treatment groups reduced neutrophil infiltration to varying degrees, with the ISO-NCs@Gel group showing the most significant effect, indicating that this hydrogel delivery system can effectively inhibit neutrophil recruitment, thereby alleviating inflammation-related tissue damage. The study also assessed the integrity of the colonic epithelial barrier by detecting the tight junction proteins ZO-1 and Occludin using immunofluorescence. The results showed that the model group exhibited significant epithelial continuity disruption; while ISO-NCs@Gel treatment significantly restored the tight junction structure and arrangement. Serum cytokine detection further indicated significant systemic inflammatory activation in the model group: TNF-α, IL-6, and IL-1β (key pro-inflammatory mediators of tumor-associated chronic inflammation) levels were significantly elevated; after treatment, all three levels decreased significantly, suggesting that treatment can effectively inhibit inflammation-driven pathological activation and reduce tumor burden.

[0060] Experiment 4 assessed the effects of different treatments on the neurological function and emotional state of mice. Several behavioral tests were conducted in a quiet room with controlled lighting. Mice were allowed at least 30 minutes to acclimatize to the testing environment before each test.

[0061] 1) Sucrose Preference Test (SPT): Each mouse was provided with two drinking bottles: one containing a 1% (w / v) sucrose solution and the other containing plain water. The total test duration was 4 hours. Mice were pre-acclimated to drinking the 1% sucrose solution before the test. The intake of sucrose solution and plain water was recorded during the test. Sucrose preference (reflecting anhedonia, i.e., reduced interest and pleasure) was calculated as the percentage of sucrose solution intake relative to total fluid intake.

[0062] 2) Open Field Test (OFT): Mice were placed in the center of an open field box (60 cm × 60 cm × 40 cm) and allowed to explore freely for 5 minutes. A video tracking system was used to record the total distance the mice walked and the time spent in the central and peripheral areas. Reduced exploration or avoidance of the central area was generally interpreted as increased anxiety-like behavior.

[0063] 3) Suspended Tail Test (TST): Secure the mouse's tail with tape and suspend it, allowing it to hang freely approximately 50 cm above the ground. Observe for 5 minutes and record the immobility time (the time the mouse stops struggling and remains still). The longer the immobility time, the more pronounced the behavioral despair or depression-like behavior.

[0064] 4) Forced Swimming Test (FST): Mice are placed alone in a transparent cylindrical container filled with water (water temperature 25 ℃, water depth sufficient to prevent the mouse from touching the bottom or escaping) for 5 minutes. The total immobility time is recorded (floating without struggling, only making movements necessary to keep the head above water). Increased immobility time is usually considered a manifestation of depressive-like behavior.

[0065] 5) Novel Object Recognition (NOR) Test: First, mice were acclimatized to an empty testing area. Then, in the training phase, two identical objects were placed in the area for the mice to explore. In the testing phase (after a certain holding interval), one of the familiar objects was replaced with a new object of similar size. The time spent exploring the new object and the familiar object was recorded. The discrimination index was calculated as an indicator of recognition memory (a higher percentage of time spent exploring the new object indicates good memory function and curiosity). The experiment was conducted in the following groups: Cotrol group: No modeling treatment was performed; mice received normal food and water; during the drug administration period, an equal volume of physiological saline was administered via gavage.

[0066] Model group: In week 1 of the experiment, AOM (10 mg / kg) was administered intraperitoneally once. In week 2 (one week after AOM administration), 2.5% DSS was added to drinking water for 7 consecutive days, followed by 14 days of normal drinking water. The above 21-day DSS cycle (1 week of DSS + 2 weeks of normal drinking water) was repeated 4 times (total duration 12 weeks).

[0067] Positive group: The modeling treatment was the same as that of the Model group. In week 7 of the experiment, the drug treatment began, 5-fluorouracil (5-FU) 10 mg / kg, intraperitoneally, once every other day.

[0068] ISO group: The modeling treatment was the same as the Model group. In week 7 of the experiment, the drug treatment was started: ISO-L (10 mg / kg), ISO-M (40 mg / kg), ISO-H (80 mg / kg), by gavage once a day.

[0069] ISO-NCs: The modeling treatment was the same as the Model group. In week 7 of the experiment, the treatment was started with ISO-NCs (ISO equivalent dose, 10 mg / kg), administered by gavage once a day.

[0070] ISO-NCs@Gel: The modeling treatment was the same as the Model group. In week 7 of the experiment, the treatment was started with ISO-NCs@Gel (ISO equivalent dose, 10 mg / kg), administered by gavage once a day.

[0071] Gel group: The modeling treatment was the same as the Model group. In week 7 of the experiment, the drug treatment began. Gel (without drugs, blank gel) was administered by gavage once a day.

[0072] Eight mice per group.

[0073] Figure 8 shows the behavioral evaluation results of the mice. It can be seen that, based on multiple behavioral results, the model group exhibited typical depressive-like phenotypes: in the sucrose preference test, sucrose preference significantly decreased to approximately 43.9%, indicating anhedonia; in the tail suspension test and forced swimming test, immobility time was significantly prolonged, reflecting increased hopelessness-like behavior; in the new object recognition test, the recognition index decreased, indicating impaired cognition and novelty exploration motivation; in the opening test, total distance traveled, speed, and time spent in the central area were all reduced, demonstrating decreased activity and exploration abilities. Compared with the model group, both ISO-NCs and ISO-NCs@Gel improved the above-mentioned depression-related behaviors to varying degrees, with ISO-NCs@Gel showing more significant improvement (e.g., sucrose preference increased to approximately 75.7%, better than ISO-NCs' approximately 70.7%, and the reduction in TST / FST immobility time was more pronounced).

[0074] It is evident that the ISO-NCs@Gel formulation of this invention has the following effects: (1) reducing the degree of colonic inflammation and alleviating tissue damage; (2) restoring the structure of tight junction proteins and improving intestinal barrier permeability; (3) promoting the recovery of mucus barrier function; (4) reducing tumor burden indicators such as tumor number; (5) exhibiting good biocompatibility and in vivo safety; and (6) alleviating depression / anxiety-like behavioral indicators in animals due to the improvement of intestinal inflammation and barrier function. It can be used in the preparation of drugs for colorectal cancer, especially for inflammation-related colorectal cancer / colitis-related CRC, to improve neuropsychiatric and behavioral abnormalities such as depression and anxiety associated with this disease.

[0075] The ISO-NCs@Gel formulations of this invention can be made into gel blocks / gel microparticles / gel capsule fillers, etc.

[0076] Furthermore, the ISO-NCs@Gel formulation of this invention can also be combined with other excipients to prepare drugs.

[0077] In the ISO-NCs@Gel formulation of this invention, HA is a commonly used clinical biomaterial, and thiolation and cross-linking can be completed under mild aqueous conditions; ISO is a natural flavonoid small molecule, and the overall system exhibits good biocompatibility. The preparation process mainly involves solution mixing, homogenization, dialysis, and gelation, and is feasible for scale-up production.

[0078] This invention is not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for this invention. Without departing from the spirit and essence of this invention, those skilled in the art can easily make other modifications and variations, but these corresponding modifications and variations should all fall within the protection scope claimed by this invention.

[0079] The above description is only a part of the embodiments of the present invention, and is not intended to limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the content of the present invention specification should be included within the protection scope of the present invention.

Claims

1. A method for preparing a thiol-modified hyaluronic acid drug-loaded hydrogel formulation, characterized in that, The steps include: preparing isorhamnetin nanocrystals (ISO-NCs) using an antisolvent precipitation method; constructing GSH-responsive thiolated hyaluronic acid (HA-SH); and uniformly embedding the ISO-NCs into the HA-SH hydrogel to prepare thiolated hyaluronic acid drug-loaded hydrogels (ISO-NCs@Gel).

2. The preparation method according to claim 1, characterized in that, The preparation method of the ISO-NCs includes the following steps: injecting the isorhamnetin organic phase into the aqueous phase under stirring conditions, and homogenizing under high pressure to obtain the ISO-NCs dispersion, wherein DMSO is used as a good solvent in the isorhamnetin organic phase, and the ratio of isorhamnetin to DMSO is 20 mg to 1 ml.

3. The preparation method according to claim 2, characterized in that, The aqueous phase also contains the stabilizer poloxamer 407.

4. The preparation method according to claim 1, characterized in that, The construction of the GSH-responsive thiolized hyaluronic acid HA-SH includes the following steps: 1) Preparation of thiolized hyaluronic acid HA-Cys; a) Dissolving hyaluronic acid HA in MES buffer at pH=6 to obtain an HA solution; b) Adding EDC·HCl and NHS to the HA solution under stirring at room temperature, such that the molar ratio of HA to the carboxyl group in the repeating unit of HA is 1:1-2:1-2, and then stirring the reaction system under N2 protection and light protection to form an activated ester intermediate; c) Adding cystamine sulfate to the activated system, such that the molar ratio of cystamine to -COOH in HA is 1:1-2, and stirring the reaction system under room temperature, N2 protection and light protection to undergo an amidation reaction, generating thiolized hyaluronic acid HA-Cys; d) After the reaction was completed, the solution was dialyzed to obtain purified HA-Cys; 2) HA-Cys solution was prepared using the purified HA-Cys obtained above, the pH value was adjusted to 4.5-5.5, excess TCEP was added, and the reaction was carried out under the conditions of room temperature, N2 protection and protection from light, with stirring to reduce and break the disulfide bonds of the side chain of HA-Cys into free thiol groups; 3) Mild oxidation was used to oxidize some of the free thiol groups into disulfide bonds, forming a crosslinked network that can be reduced and broken, to obtain GSH-responsive thiolized hyaluronic acid HA-SH.

5. The preparation method according to claim 1, characterized in that, "The ISO-NCs are uniformly embedded in the HA-SH hydrogel" is achieved through the following steps: the ISO-NCs dispersion is uniformly mixed with HA-SH to obtain a mixture, and then gently oxidized to crosslink it into a gel to obtain ISO-NCs@Gel.

6. The preparation method according to claim 5, characterized in that, The final concentration of HA-SH in the mixture is 3 wt%.

7. The thiolized hyaluronic acid drug-loaded hydrogel formulation obtained by the preparation method according to any one of claims 1-6.

8. A drug, characterized in that, Including the thiolized hyaluronic acid drug-loaded hydrogel formulation as described in claim 7.

9. The medicament according to claim 8, characterized in that, The drug is administered orally.

10. The use of the medicament according to claim 8 or 9 in the preparation of a colorectal cancer medicament.

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