An immunohydrogel for in situ therapy of bladder cancer and its preparation method

Abstract

This invention discloses an immunohydrogel for in situ treatment of bladder cancer and its preparation method. The immunohydrogel material provided by this invention comprises dopamine-modified oxidized mannodular glucan, thiolated hyaluronic acid, BCG, and arginine. After instillation, it forms an adhesive three-dimensional network structure in situ within the bladder, transforming the traditional "multiple instillation" model into a "single instillation, long-term effect" model. Simultaneously, the arginine in the system locally generates NO, enhancing T cell function through metabolism. The glucan component synergistically activates innate immunity such as dendritic cells, thereby systematically enhancing the BCG-triggered anti-tumor immune response, improving efficacy, and inducing long-term immune memory. This invention fundamentally improves the practicality of BCG therapy, significantly reducing the risk of tumor recurrence while decreasing the frequency of instillation and the dosage of BCG, providing a new generation of instillation immunotherapy strategy for the postoperative management of NMIBC.

Description

Technical Field

[0001] This invention belongs to the field of biomedical polymer materials technology, specifically relating to an immunomodulatory hydrogel that integrates "infusion-retention-treatment" functions for in situ instillation therapy after bladder cancer surgery and its preparation method. Background Technology

[0002] Bladder cancer, as one of the most common malignant tumors worldwide, poses a continuous threat to public health. According to GLOBOCAN 2020 statistics, there were approximately 573,000 new cases of bladder cancer globally that year, with about 213,000 deaths, bringing the total number of cases over five years to 1.721 million. Non-muscle-invasive bladder cancer (NMIBC) accounts for about 75% of initial cases, and transurethral resection of bladder tumor (TURBT) is the standard surgical procedure. However, the risk of tumor recurrence after surgery remains high; the recurrence rate within one year for high-risk NMIBC patients can exceed 50%, becoming a major challenge in clinical treatment.

[0003] Intravesical BCG instillation is currently a key immunotherapy for preventing recurrence after surgery in patients with intermediate- to high-risk non-malignant cystic bladder cancer (NMIBC). Although considered one of the most effective instillation regimens, BCG has a clinical efficacy rate of only about 50%, and some patients do not benefit from it due to insufficient immune response. Furthermore, this therapy faces several bottlenecks: BCG, as a biological agent, is expensive and its supply is unstable; it requires long-term, multiple instillations, increasing the economic and medical burden on patients; frequent instillations are often accompanied by local adverse reactions such as urinary frequency, dysuria, and hematuria, and in severe cases, even systemic BCG infection; patients need to hold their urine for extended periods and maintain specific body positions, severely impacting treatment tolerance and quality of life. Therefore, there is an urgent need to develop novel delivery strategies that can reduce BCG dosage and instillation frequency while improving efficacy and compliance.

[0004] In recent years, intravesical drug delivery technologies based on biomaterials have provided new avenues for optimizing BCG treatment. Injectable hydrogels, with their excellent biocompatibility and controllable physicochemical properties, are considered ideal bladder drug carriers. These materials can be instilled into the bladder in solution form, where they undergo in-situ gelation under physiological conditions, forming a three-dimensional network structure that adheres to the bladder wall, thereby prolonging drug retention time and achieving sustained release. Theoretically, a single instillation can achieve long-term therapeutic effects, potentially significantly improving the shortcomings of existing instillation methods. Currently, thermosensitive hydrogels (such as the Poloxamer series) and biomimetic adhesive hydrogels (such as materials based on catechol chemistry) have been used in intravesical small-molecule chemotherapy drug delivery research; however, dedicated hydrogel systems for live bacterial preparations like BCG are still relatively lacking. More importantly, traditional hydrogel designs mostly focus on physical retention functions and have not yet effectively addressed the issues of insufficient BCG immune activation and large individual patient response variability.

[0005] Meanwhile, interdisciplinary research in immunology and materials science suggests that some biomaterials possess intrinsic immunomodulatory potential. For example, dextran with specific structures can be recognized by innate immune cells and activate related signaling pathways, even inducing systemic anti-tumor immune memory; arginine, as a key substrate for T cell metabolism, releases NO locally in the tumor microenvironment, and supplementation helps reverse immunosuppression and enhance T cell function. However, how to organically combine these immunomodulatory properties with BCG delivery systems to construct an intelligent therapeutic platform that combines "long-acting retention" and "active immune activation" remains a gap in current research.

[0006] In summary, current bladder instillation therapy still has significant shortcomings in terms of drug retention, immune activation, and treatment tolerance. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide an immunohydrogel integrating "instillation-retention-treatment" functions for bladder cancer instillation therapy and its preparation method. It aims to achieve long-term sustained release of BCG in the bladder and enhanced immune response through the synergistic integration of materials and immune mechanisms, providing an innovative solution for the clinical management of NMIBC.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] In a first aspect, the present invention provides an immunohydrogel material for bladder cancer instillation therapy, the raw materials of which include: dopamine-modified oxidized mannan-PDA, thiolated hyaluronic acid (HASH), BCG (BCG) and arginine (L-Arg).

[0010] The raw materials are mixed to form a gel precursor solution, which is used as an infusion solution during treatment. The infusion solution is cross-linked to form an immunohydrogel. BCG and arginine are encapsulated in a three-dimensional hydrogel network structure formed by the Schiff base reaction between aldehyde and thiol-modified oxidized mannan and thiolized hyaluronic acid through the dopamine-modified oxidized mannan. The dopamine modification of oxidized mannan significantly enhances the hydrogel's resistance to erosion in a urine environment. The immunohydrogel material has strong structural stability and adhesion in a simulated urine environment.

[0011] Preferably, the mass ratio of the dopamine-modified oxidized mannan to the thiolated hyaluronic acid is 1:3-3:1, more preferably 1:2-2:1, and even more preferably 1:1.

[0012] Secondly, the present invention also provides a method for preparing the above-mentioned immunohydrogel material, comprising the following steps: preparing dopamine-modified oxidized mannodula and thiolated hyaluronic acid; dissolving and mixing dopamine-modified oxidized mannodula and thiolated hyaluronic acid respectively, adding BCG and arginine to obtain a hydrogel precursor solution; and crosslinking the hydrogel precursor solution to prepare the immunohydrogel material.

[0013] Preferably, the loading of BCG vaccine in the hydrogel precursor solution is 2 × 10⁻⁶. 7 -1×10 8 cfu / ml.

[0014] Preferably, the concentration of arginine in the hydrogel precursor solution is 2-6 mM;

[0015] Preferably, the concentration of the dopamine-modified oxidized mannan in the hydrogel precursor solution is 25-100 mg / ml, and the concentration of the thiol-modified hyaluronic acid in the hydrogel precursor solution is 25-100 mg / ml.

[0016] In practical use, the immunohydrogel material of this invention involves injecting the hydrogel precursor solution into the bladder, where a hydrogel containing BCG and arginine can be formed in situ through a Schiff base reaction.

[0017] Specifically, the preparation method of the dopamine-modified oxidized mannan includes the following steps:

[0018] (1) Oxidative modification: Mannan (GM) was dissolved in water, sodium periodate was added to carry out the oxidation reaction, ethylene glycol was added to terminate the reaction after the reaction was completed, and oxidized mannan (GM-CHO) was obtained after purification.

[0019] (2) Dopamine modification: The oxidized mannan obtained in step (1) is dissolved in water and reacted with dopamine. After the reaction is completed, it is purified and freeze-dried to obtain dopamine-modified oxidized mannan (GMO-PDA).

[0020] Specifically, in the above-mentioned oxidation modification step, the oxidation reaction is carried out under the condition of stirring overnight in the dark, the purification is carried out by dialysis, the molecular weight cutoff of the dialysis bag is 3000, and the oxidation degree of mannan is 25%-45%;

[0021] Specifically, in the above-mentioned dopamine modification step, the purification method is dialysis, followed by lyophilization to obtain the product, wherein the mass ratio of dopamine to oxidized mannan is 1:1-1:4.

[0022] In one specific embodiment, the degree of oxidation of mannan is 36.7%; the mass ratio of oxidized mannan (GMO) to dopamine (DA) is 1:1, and the dopamine modification reaction time is 24 hours.

[0023] Specifically, the method for preparing the thiol-modified hyaluronic acid includes:

[0024] (1) Carboxyl activation: Hyaluronic acid (HA) was dissolved in 2-morpholine ethanesulfonic acid buffer, and carbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS) were added to carry out the activation reaction to obtain the activated hyaluronic acid solution.

[0025] (2) Amide reaction: Cystamine dihydrochloride was added to the solution obtained in step (1) for reaction. After the reaction, the solution was purified to obtain hyaluronic acid-cystamine conjugate (HA-NHS).

[0026] (3) Disulfide bond reduction: Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) is added to the product obtained in step (2) to carry out the disulfide bond reduction reaction. After the reaction, the product is purified and lyophilized under acidic conditions to obtain thiolated hyaluronic acid (HASH).

[0027] In one specific embodiment, in the carboxyl activation step, the pH of the activation reaction system is maintained at approximately 5.5, and the reaction time is 2 hours; in the amidation step, the amidation reaction is carried out under inert gas protection, the pH of the reaction system is maintained at approximately 5.5, and purification is performed by dialysis, using dialysis bags with a molecular weight cutoff of 1,000 to 30,000; in the disulfide bond reduction step, the disulfide bond reduction reaction is carried out at room temperature, and the reaction time is 5 hours. The purification under acidic conditions is maintained at a pH of 3.5, and the purification method is dialysis, using dialysis bags with a molecular weight cutoff of 3,000 to 10,000.

[0028] Thirdly, the present invention also provides an immunohydrogel composition for bladder cancer instillation therapy, the components of which include dopamine-modified oxidized mannan, thiolated hyaluronic acid, BCG and arginine.

[0029] Preferably, the mass ratio of the dopamine-modified oxidized mannan to the thiolated hyaluronic acid is 1:3-3:1, more preferably 1:2-2:1, and even more preferably 1:1.

[0030] In practical use, the immunohydrogel composition of the present invention is prepared as a hydrogel precursor solution and instilled into the bladder. Through Schiff base reaction, an in situ immunohydrogel containing BCG and arginine can be formed.

[0031] Preferably, the loading of BCG in the hydrogel precursor solution is 2 × 10⁻⁶. 7 -1×10 8 cfu / ml.

[0032] Preferably, the concentration of arginine in the hydrogel precursor solution is 2-6 mM;

[0033] Preferably, the concentration of dopamine-modified oxidized mannan in the hydrogel precursor solution is 25-100 mg / ml, and the concentration of thiolated modified hyaluronic acid in the hydrogel precursor solution is 25-100 mg / ml.

[0034] Fourthly, the present invention also provides the application of the above-mentioned immunohydrogel material and immunohydrogel composition in the preparation of bladder cancer instillation therapy drugs, which are suitable for scenarios where drugs need to remain in the bladder for a long time and resist urine erosion.

[0035] The present invention has the following beneficial effects:

[0036] (1) In situ molding and long-term retention: The immunohydrogel material of the present invention is injectable during perfusion, which is easy to operate. After entering the bladder, it rapidly gels in the ROS environment through PDA self-polymerization and Schiff base reaction between GMO-PDA and HA-SH, forming a stable three-dimensional network structure in the tumor site. This effectively solves the problem of short retention time and easy flushing out by urine in traditional liquid preparations, and realizes long-term local release of drugs.

[0037] (2) Excellent anti-erosion performance: By introducing dopamine (PDA) modification on the polysaccharide backbone, the hydrogel is endowed with excellent adhesion and the ability to resist urine erosion, which significantly enhances its structural stability in the harsh bladder urine environment and ensures the durability of the "drug reservoir" function.

[0038] (3) Synergistic immune activation: This system innovatively loads the immune adjuvant BCG with the nitric oxide (NO) precursor arginine (L-Arg). BCG activates specific immune responses, while the NO produced locally by L-Arg can further improve T cell function and activate anti-tumor immune responses. The synergistic effect of the two can more effectively induce tumor cell apoptosis / necrosis and significantly reduce the tumor recurrence rate.

[0039] (4) High biocompatibility and safety: The selected matrix materials, mannan and hyaluronic acid, are both natural biological polysaccharides with good biocompatibility and degradability. The entire gelation process is mild and does not require the use of toxic crosslinking agents, thus ensuring high safety.

[0040] (5) Addressing Key Clinical Bottlenecks: This invention addresses the core issues in current bladder cancer instillation therapy, such as short drug retention time, low immune activation efficiency, and poor patient compliance, by providing a novel treatment strategy that combines long-acting sustained release with active immune regulation. This system achieves long-term immune regulation with a single instillation, significantly reducing BCG dosage and instillation frequency. While enhancing anti-tumor efficacy, it effectively improves treatment safety and patient quality of life, demonstrating significant clinical translational value and application prospects. Attached Figure Description

[0041] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0042] Figure 1 This is a schematic diagram of the design of the in situ immunohydrogel for intravesical instillation therapy according to the present invention;

[0043] Figure 2 a, Figure 2 b represents the nuclear magnetic resonance (NMR) test results and Fourier transform infrared (FTIR) test results of the GMO-PDA prepared in Preparation Example 1, respectively.

[0044] Figure 3 Images showing the morphology of the freeze-dried and dissolved GMO-PDA product prepared in Example 1;

[0045] Figure 4 a, Figure 4 b shows the nuclear magnetic resonance (NMR) test results and Fourier transform infrared (FTIR) test results of the HASH prepared in Example 2, respectively.

[0046] Figure 5 Images showing the morphology of the freeze-dried and dissolved HASH products prepared in Example 2;

[0047] Figure 6 Morphology of GMO-PDA@HASH gels formulated with different substrate ratios before and after gelation;

[0048] Figure 7 Microscopic porous structures of GMO-PDA@HASH gels formulated with different substrate ratios observed under scanning electron microscopy;

[0049] Figure 8 Figure 1 shows the results of the in vitro tissue adhesion test of GMO-PDA@HASH gel.

[0050] Figure 9Figure 1 shows the results of a simple in vitro tensile test of GMO-PDA and GMO-PDA@HASH gels.

[0051] Figure 10 The images show the morphology of the GMO-PDA@HASH@Arg gel before and after gelation.

[0052] Figure 11 This is a photograph of GMO-PDA@HASH@Arg gel loaded with BCG-gfp observed under confocal microscopy.

[0053] Figure 12 The figure shows the rheological test results of GMO-PDA@HASH@Arg gel after gelation;

[0054] Figure 13 Figure showing the underwater adhesion test results of GMO-PDA@HASH@Arg gel;

[0055] Figure 14 Figure showing the test results of the self-healing performance of GMO-PDA@HASH@Arg gel;

[0056] Figure 15 The graph shows the results of the injectability test of GMO-PDA@HASH@Arg gel;

[0057] Figure 16 The scanning electron microscope images show the microstructure of GMO-PDA@HASH@Arg gel after degradation at 0h, 24h, and 48h.

[0058] Figure 17 The graph shows the degradation results of GMO-PDA@HASH@Arg gel under different conditions;

[0059] Figure 18 Figure showing the results of the water erosion resistance test of GMO-PDA@HASH@Arg gel;

[0060] Figure 19 Figure showing the in vitro cell safety test results of GMO-PDA@HASH gel;

[0061] Figure 20 Figure showing the in vivo biocompatibility test results of GMO-PDA@HASH@Arg gel;

[0062] Figure 21 The image shows the results of the antitumor function test of GMO-PDA@HASH@Arg@BCG gel.

[0063] Figure 22 Flow cytometry results of the in vivo antitumor immunomodulatory effects of GMO-PDA@HASH@Arg@BCG gel. Detailed Implementation

[0064] To better explain the present invention, detailed descriptions of its embodiments are provided, and the main content of the invention is further clarified in conjunction with specific examples. However, the content of the present invention is not limited to the following embodiments. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0065] Please see Figure 1 The present invention provides an immunohydrogel material and composition for bladder cancer instillation therapy. Based on the in-situ construction of a three-dimensional network structure in the bladder by oxidized mannan and thiolated hyaluronic acid through a specific Schiff base reaction, during the cross-linking process, BCG and arginine (L-Arg) are efficiently encapsulated in the gel network, forming an immunohydrogel system that integrates "instillation-retention-treatment" functions.

[0066] The immunohydrogel material and composition of the present invention, after mixing the raw materials, form a gel precursor solution. Initially, it is a flowable solution upon instillation. Upon entering the bladder, it responds to ROS and rapidly undergoes a gelation transformation, forming a stable drug reservoir on the bladder wall through in-situ molding. This hydrogel system exhibits good mucosal adhesion and urine stability, effectively resisting urine flushing and achieving long-term retention and controlled release of BCG and L-Arg at the lesion site.

[0067] During sustained release, BCG acts as an immune activator, continuously stimulating local anti-tumor immune responses; L-Arg, on the other hand, can generate NO in situ, improving the tumor microenvironment and enhancing T cell function through metabolic regulation pathways. The two components in this hydrogel system create a synergistic immune effect, jointly enhancing anti-tumor efficacy and providing a highly promising new perfusion therapy strategy for clinical application.

[0068] Preparation Example 1: Synthesis of GMO-PDA

[0069] 5 g of mannodular glucan (GM) was dissolved in 250 mL of ultrapure water, and then 4 g of sodium periodate solution dissolved in 50 mL of ultrapure water was added. The mixture was stirred overnight in the dark to carry out the oxidation reaction. After the reaction was completed, 2 mL of ethylene glycol was added to terminate the reaction. The mixture was dialyzed for 4 days using a dialysis bag with a molecular weight cutoff of 3000 to obtain oxidized mannodular glucan (GM-CHO). The obtained GM-CHO was dissolved in ultrapure water at a mass ratio of 1:1 with dopamine (DA), mixed evenly, and stirred for 24 hours. After the reaction was completed, the mixture was dialyzed for 1 day and then lyophilized to obtain dopamine-modified oxidized mannodular glucan (GMO-PDA) solid powder. Figure 2As shown, the nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR) results demonstrate the successful synthesis of the matrix material GMO-PDA in the gel system. Figure 3 The morphology of the GMO-PDA final product after freeze-drying and after dissolution is shown.

[0070] Preparation Example 2: Synthesis of HASH

[0071] 1.95 g of 2-morpholinoethanesulfonic acid was dissolved in 150 mL of ultrapure water to prepare a buffer solution. 0.1 g of hyaluronic acid (HA) was added and fully dissolved. Then, 0.17 g of EDCI and 0.05 g of NHS were added sequentially, maintaining the pH at approximately 5.5, and the mixture was stirred for 2 hours. 0.2 g of cystamine dihydrochloride was added to the reaction solution, maintaining the pH at approximately 5.5. After bubbling with nitrogen, the mixture was stirred overnight under nitrogen protection. After the reaction was complete, the mixture was dialyzed for 2 days using a dialysis bag with a molecular weight cutoff of 1,000. 6 g of TCEP was added to the dialyzed product, and the mixture was stirred at room temperature for 5 hours. Subsequently, the mixture was dialyzed for 2 days at pH 3.5 using a dialysis bag with a molecular weight cutoff of 3,000. Finally, the product was lyophilized to obtain thiolated hyaluronic acid (HASH). Figure 4 As shown, the nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR) results demonstrate the successful synthesis of the matrix material HASH in the gel system. Figure 5 The morphology of the final HASH product after freeze-drying and after dissolution is shown.

[0072] Examples 1-3: Preparation of GMO-PDA@HASH Gel

[0073] The prepared GMO-PDA and HASH were dissolved in water to prepare GMO-PDA solution and HASH solution, both with a concentration of 100 mg / ml. After both were dissolved evenly, in Examples 1-3, the GMO-PDA solution and HASH solution were mixed evenly according to the mass ratio of GMO-PDA and HASH of 1:1, 2:1 and 1:2, respectively, to obtain a GMO-PDA@HASH gel precursor solution in a fluid state. The mixture was stirred for 20 min to form gels and obtain the GMO-PDA@HASH gel carrier. Figure 6 The morphology of the GMO-PDA@HASH gels prepared in Examples 1-3 before and after gelation is shown.

[0074] Example 4: Preparation of GMO-PDA@HASH@Arg Gel

[0075] The prepared GMO-PDA and HASH were dissolved in water to prepare GMO-PDA solution and HASH solution, respectively, with a concentration of 100 mg / ml. After both were dissolved evenly, the GMO-PDA solution and HASH solution were mixed evenly at a mass ratio of 1:1. L-Arg solution was added to a final concentration of 5.74 mM to obtain a fluid GMO-PDA@HASH@Arg gel precursor solution. The mixture was stirred thoroughly and reacted for 30 min to form a gel, thus obtaining the GMO-PDA@HASH@Arg gel carrier. Figure 10 The morphology of the GMO-PDA@HASH@Arg gel before and after gelation was shown, indicating that the gel system was successfully synthesized.

[0076] Example 5: Preparation of GMO-PDA@HASH@Arg@BCG gel

[0077] GMO-PDA and HASH were dissolved separately in water to prepare GMO-PDA and HASH solutions, each with a concentration of 100 mg / ml. After both solutions were thoroughly dissolved, they were mixed uniformly at a mass ratio of 1:1. L-Arg solution and BCG (Bacillus Calmette-Guérin) bacterial suspension were then added, with a final L-Arg concentration of 5.74 mM and a final BCG concentration of 6 × 10⁻⁶ mM. 7 A flowable GMO-PDA@HASH@Arg@BCG gel precursor solution was obtained by mixing cfu / ml and stirring thoroughly to obtain the GMO-PDA@HASH@Arg@BCG gel. Figure 11 This demonstrates the successful loading of BCG with GFP fluorescence onto GMO-PDA@HASH@Arg gel under confocal imaging.

[0078] Performance Testing

[0079] (1) Scanning electron microscopy was used to observe the microstructure of assemblies with different gel precursor ratios.

[0080] GMO-PDA@HASH gels with different substrate ratios (GMO-PDA:HASH = 1:1, 2:1, 1:2) prepared in Examples 1-3 were swollen in 1 ml of ultrapure water for 8 h, then dehydrated and frozen. After complete freezing, they were freeze-dried for 2 days, sectioned, and prepared for scanning electron microscopy. After gold sputtering, the samples were observed under a scanning electron microscope. The results are as follows. Figure 7 As shown.

[0081] Depend on Figure 7It can be observed that GMO-PDA and HASH gelled into a porous gel network structure through Schiff base bonding reaction, and the porous structure of the gel is more regular and dense when the mass ratio of GMO-PDA to HASH is 1:1.

[0082] (2) Verification of tissue adhesion of GMO-PDA@HASH gel in vitro

[0083] GMO-PDA@HASH gel was prepared according to the preparation conditions in Example 1. Subsequently, tissue adhesion tests were performed on mouse organs including heart, liver, spleen, lung, kidney, and bladder. The test results are as follows: Figure 8 As shown.

[0084] Depend on Figure 8 It can be observed that the GMO-PDA@HASH gel did not separate from the various organs of the mouse in a suspended state and remained tightly adhered, indicating that the gel system has good tissue adhesion.

[0085] (3) Simple tensile test of in vitro GMO-PDA and GMO-PDA@HASH gel

[0086] The GMO-PDA prepared in Preparation Example 1 was dissolved in water to a concentration of 200 mg / ml and stirred thoroughly to obtain GMO-PDA gel.

[0087] The prepared GMO-PDA gel and the GMO-PDA@HASH gel prepared in Example 1 were subjected to a simple in vitro tensile test, and the results are as follows. Figure 9 As shown.

[0088] Depend on Figure 9 It can be observed that GMO-PDA@HASH gel has a more obvious gel morphology and tensile toughness compared with gel made only from GMO-PDA precursor material, while GMO-PDA is significantly more solution-like.

[0089] (4) Confocal microscopy analysis of BCG loading in GMO-PDA@HASH@Arg gel carrier

[0090] Take 10 μL of the GMO-PDA@HASH@Arg@BCG gel precursor solution prepared in Example 5, drop it onto a glass slide, cover with a coverslip, and seal the sample at room temperature for confocal imaging. The results are as follows. Figure 11 As shown.

[0091] Depend on Figure 11 It can be observed that BCG active bacteria labeled with GFP fluorescence were successfully observed in GMO-PDA@HASH@Arg@BCG gel, indicating that BCG was successfully encapsulated in the hydrogel carrier formed by the system.

[0092] (5) Rheological testing to verify the mechanical strength of GMO-PDA@HASH@Arg gel

[0093] The GMO-PDA@HASH@Arg gel prepared in Example 4 was placed into a circular molding mold to prepare a gel sample with a diameter of 5 cm and a thickness of 2 mm. The sample was stored at 4 ℃ for instrumental testing. The rheological test results are as follows: Figure 12 As shown.

[0094] Depend on Figure 12 The results show that, in amplitude scanning mode, the storage modulus (G') and loss modulus (G'') vary with shear stress. The storage modulus (G') in the first half is significantly higher than the loss modulus (G''), confirming the formation of the gel system. Furthermore, within the LVER region, the value of G' is approximately 100 Pa, demonstrating that the GMO-PDA@HASH@Arg gel possesses moderate mechanical strength. It is strong enough to maintain its shape and resist certain external forces, but not excessively rigid or brittle. In bladder instillation applications, this strength ensures structural stability under urine flushing while also being flexible enough to adapt to bladder morphological changes.

[0095] (6) Validation of tissue adhesion of GMO-PDA@HASH@Arg gel in simulated urine

[0096] The GMO-PDA@HASH@Arg gel prepared in Example 4 was adhered to the wall of a fresh pig bladder and placed in pre-prepared ultrapure water with a pH of 5.5 to verify the gel's adhesion to the bladder in a simulated urine environment. The results are as follows. Figure 13 As shown.

[0097] Depend on Figure 13 It can be observed that the GMO-PDA@HASH@Arg gel maintains good adhesion in a simulated urine environment compared to air, and is not eroded or degraded by urine.

[0098] (7) Validation of the self-healing properties of GMO-PDA@HASH@Arg gel

[0099] Two 200 μL gel samples were prepared from the GMO-PDA@HASH@Arg gel prepared in Example 4. For ease of observation, one sample was stained with trypan blue. The two gel samples were then cut in half and cross-joined. After 30 minutes, the gels with different staining treatments were observed to see if they fused together, thus verifying its self-healing properties. The results are as follows: Figure 14 As shown.

[0100] Depend on Figure 14It can be observed that the gels with two different staining treatments completely fused together after being cross-joined and left for 30 minutes, and could not be separated, proving that the prepared gel system has good self-polymerization ability.

[0101] (8) Validation of the injectability of GMO-PDA@HASH@Arg gel

[0102] The GMO-PDA@HASH@Arg gel prepared in Example 4 was stained with trypan blue for easy observation. It was then transferred to a 1 ml syringe and allowed to stand for 30 minutes before being injected. The results are as follows: Figure 15 As shown.

[0103] Depend on Figure 15 It can be seen that as a 1 ml syringe is pushed, the trypan blue stained hydrogel can write words without interruption or spreading, proving that the gel system has good injectability.

[0104] (9) Verification of the degradability of GMO-PDA@HASH@Arg gel

[0105] The GMO-PDA@HASH@Arg gel prepared in Example 4 was divided into three groups of samples, with 200 μL prepared for each group. The three gel samples were placed in 1 ml of ultrapure water, artificial urine, and artificial urine + H2O2 environments, respectively, for degradation at 0 h, 24 h, and 48 h. After dehydration and freezing, the samples were freeze-dried for 2 days, sectioned, and prepared for scanning electron microscopy. After gold sputtering, the samples were observed under a scanning electron microscope. The scanning electron microscopy results are shown below. Figure 16 As shown, the degradation rate curves of the gel in different environments are plotted as follows. Figure 17 .

[0106] Depend on Figure 16 It can be observed that in an artificial urine environment, the porous network structure of the gel is significantly destroyed as the degradation time increases, indicating that the gel system has good degradability under physiological conditions in the human bladder. This also proves that the gel system will not affect the normal physiological function of the bladder in clinical applications.

[0107] Depend on Figure 17The degradation curves of the hydrogel under different conditions show that the hydrogel degrades very slowly in PBS. Even after more than 12 hours of degradation, its mass remains at 80%. This indicates that the basic structure of the hydrogel itself is very stable, and its three-dimensional network can effectively resist simple hydrolysis and physical erosion. The degradation curve of the hydrogel in urine highly overlaps with that in PBS, and is even slightly slower. This demonstrates that dopamine (PDA) modification helps the gel possess a certain degree of resistance to urine erosion, solving the problem of short retention time in the bladder for traditional formulations. In urine containing H2O2, the hydrogel underwent rapid and significant degradation, with its mass decreasing to less than 20% after approximately 30 hours, indicating that the hydrogel system exhibits ROS-responsive degradation characteristics.

[0108] (10) Verification of the erosion resistance of GMO-PDA@HASH@Arg gel

[0109] The GMO-PDA@HASH@Arg gel prepared in Example 4 was stained with trypan blue for easy observation. The stained gel was then adhered to the wall of a fresh pig bladder and rinsed with laboratory running water for 30 seconds to verify the gel's resistance to water erosion. The results are as follows. Figure 18 show.

[0110] Depend on Figure 18 It can be observed that after 30 seconds of flushing with running water, the position of the gel adhering to the pig bladder wall was almost completely different from that before flushing. This indicates that the gel has strong adhesion to bladder tissue and a certain degree of resistance to flushing with running water. This also shows that after the gel is injected into the bladder cavity and gels, it can resist urine flushing to a certain extent.

[0111] (11) In vitro biosafety verification of GMO-PDA@HASH gel

[0112] The GMO-PDA@HASH gel precursor solution prepared in Example 1 was added to a 96-well plate. The plates were divided into groups according to the total final concentrations of GMO-PDA and HASH: 0.5 mg / ml, 0.75 mg / ml, 1.00 mg / ml, and 1.25 mg / ml, respectively. The plates were then co-incubated with MB49 and T24 bladder cancer cell lines, with six replicates for each group, as detailed below:

[0113] The ratio of gel precursor solution to DMEM was 0.1 g / mL. -1Cells were incubated in 96-well plates for 24 h until they adhered to the plates. Different concentrations of hydrogel solutions (0.5 mg / ml, 0.75 mg / ml, 1.00 mg / ml, 1.25 mg / ml) were co-cultured with the cells for 24 h. The culture medium was then aspirated, and the cells were washed twice with 1×PBS. Subsequently, 10 μL of CCK-8 solution was added to 90 μL of cDMEM medium in each well, and the plates were incubated at 37 ℃ for 2 h. Then, 100 μL of the culture medium was transferred to fresh 96-well plates, and the absorbance was measured using a microplate reader. The results are shown below. Figure 19 As shown.

[0114] Depend on Figure 19 It was observed that as the gel concentration changed, the cell survival rate of the bladder cancer cell lines MB49 (left) and T24 (right) co-incubated with the gel showed a decreasing trend. However, in the group with the highest concentration of 1.25 mg / ml, although the cell survival rate was significantly lower than that of the control group, it was still maintained at about 80%, which shows that GMO-PDA@HASH gel has good cell compatibility.

[0115] (12) In vivo biosafety verification of GMO-PDA@HASH@Arg gel

[0116] The GMO-PDA@HASH@Arg gel precursor solution prepared in Example 4 was transferred to a 1 ml syringe. The syringe needle was replaced with a 24 G veterinary indwelling needle tip. After assembly, mice were anesthetized with isoflurane, and the syringe was inserted through the urethra and instilled into the bladder via in situ perfusion. The gel precursor solution was pushed in and then tied with a thin suture to prevent leakage when the syringe was withdrawn. The suture was untied after 30 minutes. The mice's behavior and weight were then observed daily. On the 5th day, the mice were euthanized, and tissues from the heart, liver, spleen, lung, and kidney were sent for HE sectioning for histological pathological testing. Blood samples were also collected for complete blood count and blood biochemistry analysis. The results are as follows: Figure 20 As shown.

[0117] Specifically, the mice were 6-8 w C57 female mice, and each mouse was perfused with 100 μL of GMO-PDA@HASH@Arg gel precursor solution.

[0118] Depend on Figure 20 Observations showed that, compared with the control group, pathological sections of the heart, liver, spleen, lung and kidney tissues of mice after gel perfusion showed no obvious lesions in the major organs of the mice, and the blood routine and blood biochemistry results were not different from those of the control group.

[0119] (13) In vivo antitumor validation of GMO-PDA@HASH@Arg@BCG gel

[0120] The GMO-PDA@HASH@Arg gel precursor solution prepared in Example 4 and the GMO-PDA@HASH@Arg@BCG gel precursor solution prepared in Example 5 were used to set up four groups: control, in situ perfusion of BCG alone, in situ perfusion of GMO-PDA@HASH@Arg gel alone, and in situ perfusion of GMO-PDA@HASH@Arg@BCG (Gel+BCG). Five C57 female mice were included in each group. First, 20 C57 female mice were uniformly subjected to MB49 bladder cancer cell line tumor-bearing treatment. The specific procedure was as follows: 100 μL of 5×10 5 One cell was transferred into a 1 ml syringe. The syringe needle was removed and replaced with a 24G veterinary indwelling needle tip. After assembly, the mice were anesthetized with isoflurane. The syringe was inserted through the urethra and infused into the bladder of the mice via in situ perfusion. 100 μL was infused into each mouse. After pushing the syringe in, it was tied with a thin suture to prevent the cells from flowing out when the syringe was pulled out. The suture was untied after 30 minutes. On the same day, in vivo imaging of the small animals was performed to observe the tumor bearing status of each mouse.

[0121] On day 3 after tumor implantation, using the same in situ perfusion method, 1×PBS, BCG, Gel, and Gel+BCG were infused into the bladders of C57 female mice in the corresponding groups to administer different treatments. On day 7, further small animal in vivo imaging was performed on the four groups of C57 female mice after different treatments. The growth and size of the bladder tumors in different groups of mice were observed by fluorescence. The results are as follows: Figure 21 As shown.

[0122] Depend on Figure 21Observations showed that the baseline images taken on the day of tumor development indicated that almost all mice developed bladder tumors on that day, and the similar fluorescence size reflected that the tumors grew uniformly. Some mice that did not develop tumors on the 7th day showed that they had also developed tumors in the fluorescence images taken on the 7th day. Meanwhile, in the in vivo imaging results on day 7, the fluorescence of the four groups of experimental mice showed a clear trend. Compared with the control group, the fluorescence values ​​of the BCG, Gel, and Gel+BCG groups were significantly lower, indicating that these three treatment groups all had a certain therapeutic effect on bladder tumors. The fluorescence values ​​of the BCG group and the Gel group were similar, proving that compared with the clinically commonly used BCG treatment, the "in situ infusion-retention-treatment" integrated immunohydrogel carrier designed in this invention also has a good anti-tumor effect. Moreover, the fluorescence value at the bladder of the mice in the Gel+BCG group was significantly lower than that of the other three groups. According to the fluorescence statistics, the fluorescence value of the Gel+BCG group was three stars lower than that of the control group, and compared with one star of BCG and two stars of Gel, it had a better anti-tumor effect. This also indicates that the combined effect of the immunohydrogel, Arg in situ release of NO and BCG-induced combined action effectively activated the anti-tumor immune response and induced in situ apoptosis / death of the tumor.

[0123] (14) Flow cytometry experiments were used to verify the in vivo antitumor immunomodulatory effects of GMO-PDA@HASH@Arg@BCG gel.

[0124] The GMO-PDA@HASH@Arg gel precursor solution prepared in Example 4 and the GMO-PDA@HASH@Arg@BCG gel precursor solution prepared in Example 5 were used to set up four groups: control, BCG perfusion alone, GMO-PDA@HASH@Arg gel (Gel) perfusion alone, and GMO-PDA@HASH@Arg@BCG gel (Gel+BCG) perfusion. Five C57 female mice were included in each group. Twenty C57 female mice were uniformly inoculated with MB49 bladder cancer cells. At 3, 10, 17, and 24 days post-tumor inoculation, 1×PBS, BCG, Gel, and Gel+BCG were perfused into the bladders of the corresponding group of tumor-bearing C57 female mice using the same in-situ perfusion method. After four treatments, at 27 days post-tumor inoculation... At the end of experiment d, mice were sacrificed and bladder tumor samples were collected for flow cytometry analysis, as follows: The tumor was carefully dissected, weighed, and photographed. It was then placed in ice-cold PBS. The tumor tissue was subsequently cut into fragments and digested in RPMI-1640 medium containing collagenase IV and DNase I at 37 °C for 30–45 min. The digested cell suspension was filtered through a 70 μm cell sieve and treated with erythrocyte lysis buffer to remove erythrocytes. Finally, the cells were resuspended in PBS and counted for later use. To comprehensively analyze immune cell subsets, the above single-cell suspension was stained as follows:

[0125] The prepared single-cell suspension was used to block non-specific binding with an Fc receptor blocker, followed by the addition of a mixture of fluorescently labeled antibodies targeting different immune cell markers. The cells were incubated at 4 °C in the dark for 30 min. After incubation, the cells were washed twice with PBS and resuspended in PBS containing 2% fetal bovine serum. After surface staining, the cells were fixed and perforated, followed by staining with anti-Foxp3 antibody. Data were then collected using flow cytometry. Cell debris was first excluded using forward and side scattering angles, and dead cells were excluded using a dead-live dye (DAPI). Data analysis was performed using FlowJo and Origin plotting software. Results are as follows: Figure 22 As shown.

[0126] Depend on Figure 22 Observations showed that, by comparing flow cytometry data between different groups, compared with the control group, the B+G group not only significantly increased the enrichment of CD45⁺, CD69⁺, CD4⁺ and CD8⁺ T cells in tumor tissue, but also significantly reduced the expression level of PD-1 in the CD4⁺ and CD8⁺ T cell populations. This indicates that the combination therapy can synergistically promote the activation and infiltration of immune cells and effectively reverse the exhaustion state of T cells.

[0127] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the above examples, and various exploratory changes can be made according to the inventive purpose of the present invention. Any changes, modifications, substitutions, combinations, or simplifications made based on the methods and techniques disclosed in the present invention should be considered equivalent substitutions, and all those within the spirit and principles of the present invention should be covered by the present invention.

Claims

1. An immunohydrogel material, characterized in that, The preparation raw materials include dopamine modified oxidized mannose dextran, thiolated hyaluronic acid, bacillus calmette-guerin and arginine.

2. The immunohydrogel material according to claim 1, characterized in that, The mass ratio of the dopamine modified oxidized mannose dextran and the thiolated hyaluronic acid is 1:3-3:

1.

3. A method for the preparation of the immunohydrogel material according to any one of claims 1-2, characterized in that, The method comprises the following steps: The dopamine modified oxidized mannose dextran and the thiolated hyaluronic acid are prepared. The dopamine modified oxidized mannose dextran and the thiolated hyaluronic acid are respectively dissolved and mixed, and the bacillus calmette-guerin and the arginine are added to obtain a hydrogel precursor solution. The hydrogel precursor solution is crosslinked to prepare the immunohydrogel material.

4. The method of claim 3, wherein the immunohydrogel material is prepared by mixing the antigen and the antigen-binding protein in a solution, and then adding the solution to the hydrogel material. The BCG is loaded at a concentration of 2 x 10 7 -1 x 10 8 cfu / ml; and the arginine is present in the hydrogel precursor solution at a concentration of 2-6 mM.

5. The method of claim 3, wherein the in situ immunohydrogel system is prepared by, The concentration of the dopamine modified oxidized mannose dextran in the hydrogel precursor solution is 25-100 mg / ml, and the concentration of the thiolated hyaluronic acid in the hydrogel precursor solution is 25-100 mg / ml.

6. The method of claim 3, wherein the immunohydrogel material is prepared by mixing the antigen and the antigen-binding protein in a solution. The preparation method of the dopamine modified oxidized mannose dextran comprises the following steps: oxidizing mannose dextran by using sodium periodate to obtain oxidized mannose dextran, and modifying the obtained oxidized mannose dextran by dopamine to obtain dopamine modified oxidized mannose dextran.

7. The method of claim 3, wherein the immunohydrogel material is prepared by the steps of: The preparation method of the thiolated hyaluronic acid comprises the following steps: activating carboxyl of hyaluronic acid, then carrying out amidation reaction of the activated hyaluronic acid and cystamine, and finally introducing thiol by reducing disulfide bond to obtain thiolated hyaluronic acid.

8. An immunohydrogel composition, characterized in that, The components include dopamine modified oxidized mannose dextran, thiolated hyaluronic acid, bacillus calmette-guerin and arginine.

9. The immunohydrogel composition according to claim 8, wherein, The mass ratio of the dopamine modified oxidized mannose dextran and the thiolated hyaluronic acid is 1:3-3:

1.

10. The use of any one of the immunohydrogel material of claims 1-2, the immunohydrogel material prepared by the preparation method of claims 3-7 and the immunohydrogel composition of claims 8-9 in the preparation of a bladder cancer in situ perfusion treatment drug.

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