ROS-responsive oral microgel-in-situ intestinal bioadhesive patch system as well as preparation method and application of ROS-responsive oral microgel-in-situ intestinal bioadhesive patch system

Abstract

The invention discloses an ROS (reactive oxygen species) response type oral microgel-in-situ intestinal biological adhesion patch system. The patch system is prepared from the following raw materials: a polymer framework material, an ROS breakable crosslinking component, a covalent adhesion component, a negative electricity targeting component, an initiation system and a solvent, the ROS breakable crosslinking group is selected from monomers with double bonds at the two ends and an oxidative fracture bond in the middle; the covalent adhesion component is selected from monomers simultaneously containing double bonds and an X group, and the X group is selected from one or more of an N-hydroxysuccinimide group, an isocyanate group, an epoxy group and an aldehyde group; the negative electricity targeting component is selected from a monomer containing carboxyl. The system disclosed by the invention has the advantages that (1) oral administration can be realized; (2) the structure of the inflammation part is changed under the triggering of active oxygen; (3) converting the dispersed microgel into an integral bioadhesive patch in situ; (4) mucous membrane barrier reconstruction, ROS (reactive oxygen species) removal and local drug sustained release functions are realized; and (5) the composition can be stably retained at intestinal inflammation parts for not less than 24 hours.

Description

Technical Field

[0001] This invention relates to the technical field of biomedical materials and drug delivery, and in particular to a ROS-responsive oral microgel-in-situ intestinal bioadhesive patch system, its preparation method, and its application. Background Technology

[0002] Inflammatory bowel disease (IBD) includes ulcerative colitis and Crohn's disease. Its core pathological features include: (1) significantly elevated levels of reactive oxygen species (ROS) at the lesion site; (2) destruction of the intestinal mucus layer and damage to the intestinal epithelial barrier structure; and (3) persistent high expression of inflammatory factors and long-term abnormal activation of the immune response.

[0003] The main technical approaches for treating IBD in clinical practice currently include:

[0004] (1) Oral anti-inflammatory drugs and immunosuppressive drugs, such as 5-aminosalicylic acid, glucocorticoids, calcineurin inhibitors, etc. This type of regimen is mainly administered systemically. The drugs diffuse non-specifically in the gastrointestinal tract, resulting in low effective drug concentrations at the lesion site. Long-term use can easily cause serious systemic side effects.

[0005] (2) Biologics and small molecule targeted drugs are used to inhibit inflammatory signaling pathways. Although they have a certain degree of targeting, they are expensive, require long-term injection, and have problems with immune tolerance and secondary failure.

[0006] (3) Enema or rectal administration of hydrogels and mucosal protectants. These materials can form a local covering layer, but they are only applicable to the distal colon. Patient compliance is poor and they are difficult to use for long-term chronic disease management.

[0007] (4) Oral nano-drug delivery systems, such as nanoparticles, microspheres, polysaccharide coating systems, etc., can increase the intestinal retention time of some drugs, but under the actual intestinal peristalsis, mucus renewal and inflammatory fluid flushing, it is still difficult to form a stable and lasting physical barrier at the lesion site.

[0008] Based on existing technologies, the field of oral treatment and local mucosal protection for IBD has at least the following objective defects: (1) Current drug research mostly focuses on relieving inflammation and ignores the importance of intestinal barrier protection; (2) Existing oral drug delivery systems are difficult to form a stable and lasting physical barrier at the site of inflammation and cannot directly replace the damaged mucus layer structure; (3) Traditional gel systems usually require enema or local injection for drug delivery, resulting in poor patient compliance and making them difficult to use for long-term chronic disease management; (4) Existing oral nano-drug delivery systems mainly solve the "delivery" problem and cannot simultaneously achieve the synergistic function of "barrier reconstruction-anti-inflammatory treatment"; (5) There is a lack of selective response to ROS in the inflammatory microenvironment. Summary of the Invention

[0009] To address the shortcomings of existing technologies, this invention discloses a ROS-responsive oral microgel-in-situ intestinal bioadhesive patch system, which: (1) can be administered orally; (2) undergoes structural transformation at the site of inflammation triggered by reactive oxygen species; (3) transforms from a dispersed microgel in situ into a whole bioadhesive patch; (4) simultaneously possesses the functions of mucosal barrier reconstruction, ROS clearance and local drug sustained release; and (5) can remain stably in the site of intestinal inflammation for no less than 24 hours.

[0010] The specific technical solution is as follows:

[0011] A ROS-responsive oral microgel-in-situ intestinal bioadhesive patch system, comprising: a polymer framework material, a ROS-fragile crosslinking component, a covalent adhesive component, a negatively charged targeting component, an initiation system, and a solvent;

[0012] The polymeric framework material includes one or more of gelatin, collagen, histone, and chitosan;

[0013] The ROS-fragile crosslinking component is selected from monomers with double bonds at both ends and an oxidative cleavage bond in the middle, wherein the oxidative cleavage bond is selected from one or more of oxalate bonds, thioketal bonds, diselenide bonds, thioether bonds, and disulfide bonds.

[0014] The covalent adhesive component is selected from monomers containing both double bonds and X groups, wherein the X group is selected from one or more of N-hydroxysuccinimide groups, isocyanate groups, epoxy groups, and aldehyde groups;

[0015] The negatively charged targeting component is selected from monomers containing carboxyl groups.

[0016] This invention discloses an orally administered ROS-responsive microgel-in-situ intestinal bioadhesive patch system. The microgel passes through the gastrointestinal tract in a dispersed particle state in a non-inflammatory intestinal environment. After reaching the inflammatory lesion area, the originally dispersed microgel is transformed in situ into a continuous and integral bioadhesive patch structure (iPatch) under the action of high levels of reactive oxygen species (ROS).

[0017] The formation of the patch in this invention relies on the synergy of the following two mechanisms:

[0018] Swelling mechanism: In the inflammatory lesion area, high levels of ROS induce the breakage of ROS fracture-type cross-links inside the microgel, resulting in a rapid decrease in network cross-link density. The microgel swells in volume, and the polymer chain segments within a unit volume extend and entangle with each other, providing a physical structural basis for continuous film formation of the patch.

[0019] Adhesion mechanisms: (1) Electrostatic targeted adhesion: Negatively charged microgels electrostatically adsorb positively charged proteins enriched in the inflammatory area; (2) Covalent chemical fixation: The surface active groups of the microgel react with the amino groups exposed in the inflammatory tissue through covalent bonding to achieve covalent fixation;

[0020] The above-mentioned "swelling-driven-targeted adhesion" dual mechanism works synergistically. Swelling provides the structural dynamics basis for patch film formation, while targeted adhesion provides the long-term stable fixation ability of the patch on the surface of inflamed tissue. The two work together to enable the microgel to form an in-situ bioadhesive patch structure with "film-forming ability, high stability, and erosion resistance" at the inflammatory site.

[0021] In this invention, the polymeric framework material, the ROS-severable crosslinking component, the covalent adhesive component, and the negatively charged targeting component are all necessary and indispensable. Experiments have shown that without the addition of the polymeric framework material, the prepared microgel disintegrates after being immersed in an oxidizing environment for a period of time. If the added crosslinking component is a common crosslinking agent, it lacks the ability to trigger in-situ film formation via ROS.

[0022] In this invention:

[0023] Preferably, the ROS fracture-type crosslinking component is selected from one or more of polyethylene glycol oxalate diacrylamide (PEGCO), polyethylene glycol thioketal diacrylamide, polyethylene glycol selenide diacrylamide, polyethylene glycol disulfide diacrylamide, and polyethylene glycol thioether diacrylamide; more preferably, it is PEGCO.

[0024] Preferably, the covalent adhesive component is selected from one or more of N-hydroxysuccinimide acrylate, N-hydroxysuccinimide methacrylate, 2-isocyanate ethyl acrylate, and glycidyl acrylate; more preferably, it is N-hydroxysuccinimide acrylate.

[0025] Preferably, the negatively charged targeting component is selected from one or more of acrylic acid, methacrylic acid, maleic acid, fumaric acid, and itaconic acid; more preferably, it is acrylic acid.

[0026] Preferably, the polymeric framework material is selected from gelatin.

[0027] In this invention, the initiation system is selected from photoinitiation systems, thermal initiation systems, and redox initiation systems; specifically, all can be selected from common types in the field.

[0028] For example, the photoinitiating system is selected from one or more of Irgacure 2959, Irgacure 184, Irgacure 1173, lithium phenyl-2,4,6-trimethylbenzoylphosphonate, and α-ketoglutaric acid;

[0029] For example, the thermal initiation system is selected from one or more of ammonium persulfate, potassium persulfate, azobisisobutyronitrile, and azodimethylvalerate;

[0030] For example, the redox initiation system is selected from one or more of the following: ammonium persulfate / tetramethylethylenediamine system, potassium persulfate / ascorbic acid system, hydrogen peroxide / ascorbic acid system, and benzoyl peroxide / tertiary amine system;

[0031] Preferably, the initiation system is selected from photoinitiation systems.

[0032] In this invention, the solvent is selected from water or an aqueous buffer solution.

[0033] The aqueous buffer solution is selected from PBS buffer.

[0034] In this invention, the ROS-responsive oral microgel-in-situ intestinal bioadhesive patch system, based on a total mass of 100%, comprises the following raw materials:

[0035] Polymer framework materials: 2%~20%;

[0036] ROS fracture-compatible crosslinking component: 0.2%~20%;

[0037] Covalently bonded components: 0.2%~2%;

[0038] Negatively charged targeting components: 10%~50%;

[0039] Initiating system 0.02%~2%;

[0040] Solvent balance.

[0041] Preferably, the raw material composition includes:

[0042] Polymer framework materials: 2%~20%;

[0043] ROS fracture-compatible crosslinking component: 0.3%~3.0%;

[0044] Covalently bonded components: 0.2%~2%;

[0045] Negatively charged targeting components: 10%~50%;

[0046] Initiating system 0.02%~2%;

[0047] Solvent balance.

[0048] Further optimization reveals that the raw material composition includes:

[0049] Polymer framework materials 5%~10%;

[0050] ROS fracture-compatible crosslinking component: 0.3%~3.0%;

[0051] Covalently bonded components: 0.5%~1.5%;

[0052] Negatively charged targeting components: 15%~40%;

[0053] Initiating system 0.1%~1%;

[0054] Solvent balance.

[0055] Further optimized, the raw material composition includes:

[0056] Polymer framework materials 5%~10%;

[0057] ROS fracture-compatible crosslinking component 1.5%~3.0%;

[0058] Covalently bonded components: 0.5%~1.5%;

[0059] Negatively charged targeting components: 15%~40%;

[0060] Initiating system 0.1%~1%;

[0061] Solvent balance.

[0062] The optimal raw material composition includes:

[0063] Polymer framework materials account for 8%;

[0064] ROS fracture-resistant crosslinking component 1.5%;

[0065] Covalently bonded component: 0.8%;

[0066] Negatively charged targeting component 25%;

[0067] The triggering system was 0.4%;

[0068] Solvent balance.

[0069] With continuous optimization of the above raw material composition, the prepared microgels have a higher swelling ratio.

[0070] In this invention, drug loading can also be performed in a ROS-responsive oral microgel-in-situ intestinal bioadhesive patch system.

[0071] The raw materials also include hydrophilic anti-inflammatory drugs and / or hydrophobic immunosuppressive drugs;

[0072] Preferably, the hydrophilic anti-inflammatory drug includes one or more of 5-aminosalicylic acid, mesalazine, olsalazine sodium, sulfasalazine, hydrocortisone, prednisolone, dexamethasone sodium phosphate, diclofenac sodium, indomethacin sodium, and ibuprofen sodium.

[0073] Preferably, the hydrophobic immunosuppressive drug includes one or more of tacrolimus, cyclosporine A, sirolimus, everolimus, mycophenolate mofetil, and azathioprine.

[0074] Based on the mass of the ROS-responsive oral microgel-in-situ intestinal bioadhesive patch system, the mass fraction of the hydrophilic anti-inflammatory drug is 1-10%, and the mass fraction of the hydrophobic immunosuppressive drug is 0.1-1.0%; preferably, the mass fraction of the hydrophilic anti-inflammatory drug is 6.75%, and the mass fraction of the hydrophobic immunosuppressive drug is 0.59%.

[0075] The ROS-responsive oral microgel-in-situ intestinal bioadhesive patch system disclosed in this invention can simultaneously load at least one hydrophilic anti-inflammatory drug and at least one hydrophobic immunosuppressive drug, possessing a dual-mode drug loading and time-sequential release mechanism.

[0076] (1) Hydrophilic anti-inflammatory drugs are mainly released rapidly through diffusion after gel swelling, and are used to rapidly suppress inflammation in the acute phase;

[0077] (2) Hydrophobic immunosuppressive drugs exist in the form of nanoparticles or hydrophobic phase embedding, and rely mainly on the gradual unwinding of cross-linked networks to achieve slow and continuous release, which is used for long-term immune regulation in the later stage of inflammation.

[0078] This invention also discloses a method for preparing the ROS-responsive oral microgel-in-situ intestinal bioadhesion patch system, comprising:

[0079] (1) A gel precursor solution is prepared by uniformly mixing a polymer backbone material, a ROS fracture-type crosslinking component, a covalent adhesive component, a negatively charged targeting component, an initiation system, a selectively added hydrophobic immunosuppressive drug, and a solvent.

[0080] (2) The gel precursor solution is cross-linked after initiation to form a hydrogel;

[0081] (3) The hydrogel was washed and freeze-dried to obtain a crude product;

[0082] (4) The frozen-dried crude product is directly post-processed to obtain ROS-responsive microgel powder, or the frozen-dried crude product is soaked in a buffer solution containing hydrophilic anti-inflammatory drugs, taken out and frozen-dried again, and then post-processed to obtain ROS-responsive microgel powder.

[0083] When adding hydrophobic immunosuppressive drugs, it is preferable to pretreat the hydrophobic immunosuppressive drugs so that they are added in the form of hydrophobic drug-loaded nanoparticles.

[0084] Preferably, in step (4), the post-processing includes screening and grading.

[0085] The ROS-responsive microgel powder obtained after the post-processing has a particle size range of 10~200μm. Choosing this particle size range can balance intestinal permeability and patch formation efficiency at inflamed sites.

[0086] The present invention also discloses the application of the ROS-responsive oral microgel-in-situ intestinal bioadhesive patch system in the preparation of drugs for treating inflammatory bowel disease, wherein the ROS-responsive oral microgel-in-situ intestinal bioadhesive patch system is administered orally.

[0087] Preferably, the dosage is 1~100 mg / kg.

[0088] Compared with the prior art, the present invention has the following beneficial effects:

[0089] The ROS-responsive oral microgel-in-situ intestinal bioadhesive patch system disclosed in this invention is achieved through the synergistic effect of five key inventive improvements in the same system: "ROS-responsive cross-linking structure, swelling-driven film formation, electrostatic targeted adsorption, covalent fixation, and dual-mode drug delivery," resulting in the following multiple technical effects:

[0090] (i) In situ conversion into a patch to provide "band-aid" protection for damaged mucosa, blocking intestinal pathogens and inflammatory stimulation.

[0091] In the microgel of this invention, cross-linking bonds break due to increased ROS concentration in the inflammatory area, the cross-linking density of the gel network decreases, and the polymer chain segments rapidly extend and spatially entangle, transforming from dispersed particles into a continuous membrane structure. This "particle-to-membrane" structural transformation process allows the material to form a physical covering layer on the lesion surface that is highly similar in spatial structure to the natural mucus layer. This covering layer can simultaneously block: (1) the invasion of pathogenic microorganisms in the intestinal lumen; (2) the direct stimulation of toxins, digestive juices, and pro-inflammatory factors; and (3) the mechanical flushing caused by intestinal peristalsis. Thus, this invention does not only inhibit inflammation through drugs, but also breaks the vicious cycle of inflammation through in-situ barrier protection.

[0092] (ii) The ROS triggering mechanism endows the material with a highly selective response to inflammatory sites, avoiding retention in non-lesion areas.

[0093] ROS levels in inflamed tissues are significantly higher than in normal intestinal regions. This invention introduces ROS-crackable chemical bonds into the gel crosslinking network, enabling the swelling, structural delamination, and patch formation processes to be triggered only in high ROS environments. Under normal intestinal physiological conditions, this crosslinked structure remains stable, and the microgels are expelled from the body as dispersed particles with intestinal peristalsis. However, in inflamed areas, the crosslinking bonds are selectively cleaved by ROS, thereby initiating the patch formation process. This "lesion-only response" triggering mechanism avoids non-specific adhesion and retention in healthy tissues at the material's bulk level, significantly reducing the risk of interference with the normal mucosal barrier.

[0094] (III) The dual mechanism of "electrostatic targeted adsorption-covalent fixation" significantly improves the patch's resistance to detachment in the complex intestinal environment.

[0095] The surface of the inflamed area exhibits a distinct positive charge. This invention introduces a negatively charged polymer component with carboxyl groups, enabling the microgel to achieve rapid localization and adsorption in the inflamed area through electrostatic interactions. Simultaneously, its surface active groups react with the amino groups exposed on the inflamed tissue surface via covalent bonding, forming stable chemical covalent bonds. Electrostatic interaction provides "rapid targeting," while covalent bonding provides "long-term irreversible fixation." The synergistic effect of these two factors significantly improves the patch's stable retention under complex physiological conditions such as intestinal flushing, peristaltic shearing, and mucus turnover, ensuring the patch remains at the lesion site for at least 24 hours.

[0096] (iv) The dual-mode drug delivery mechanism matches the dynamic evolution of inflammation from the "acute phase to chronic phase" in a temporal dimension.

[0097] This invention constructs a dual-timescale drug delivery mechanism—rapid-release and sustained-release—by simultaneously loading a hydrophilic anti-inflammatory drug and a hydrophobic immunosuppressive drug into the same patch system. During the initial swelling phase of the patch, the hydrophilic drug is rapidly released via diffusion through its concentration gradient, quickly reducing local pro-inflammatory factor levels and achieving rapid control of acute inflammatory responses. The hydrophobic drug, on the other hand, exists as a nanocarrier or hydrophobic embedding, and its release relies on the gradual degradation and structural loosening of the gel network, resulting in a longer release period for sustained inhibition of abnormal immune activation. This drug delivery mechanism simultaneously covers both the "inflammatory outbreak phase" and the "immune remodeling phase" in terms of time, avoiding inflammatory rebound caused by single, short-acting medications.

[0098] (v) Low systemic exposure and high local enrichment significantly improve treatment safety and drug efficacy.

[0099] Because this invention employs an "inflammation-triggering-local patch-long-term retention" drug delivery modality, the drug is primarily concentrated and acts on the inflamed area, creating a high effective drug concentration gradient locally, while significantly reducing the proportion of drug entering systemic circulation. Compared to traditional oral or injectable administration methods, this modality achieves the same or better inflammation control while significantly reducing systemic exposure levels. This reduces the hepatotoxicity and nephrotoxicity, infection risk, and other systemic adverse reactions associated with hormones and immunosuppressants, achieving truly precise treatment with "low dose, high local concentration, and low toxicity." Attached Figure Description

[0100] Figure 1 The Fourier Transform Infrared (FT-IR) spectrum of the polyethylene glycol oxalate diacrylamide prepared in Example 1 is shown below.

[0101] Figure 2 The curves showing the swelling ratio of the product prepared in Example 1 as a function of time after immersion in PBS buffer or H2O2 aqueous solution are shown.

[0102] Figure 3 The swelling ratio of the product prepared in Example 2 after immersion in PBS buffer or H2O2 aqueous solution varies with time.

[0103] Figure 4 The swelling ratio of the product prepared in Example 3 after immersion in PBS buffer or H2O2 aqueous solution varies with time.

[0104] Figure 5 The swelling ratio of the product prepared in Comparative Example 3 after immersion in PBS buffer or H2O2 aqueous solution varies with time.

[0105] Figure 6 Scanning electron microscope (SEM) images of the products prepared in Example 4 and Comparative Example 1 before and after swelling in a non-oxidizing environment (PBS) and an oxidizing environment (H2O2), respectively.

[0106] Figure 7 The image shows a scanning electron microscope (SEM) image of the product prepared in Example 1.

[0107] Figure 8 The infrared spectrum (FT-IR) of the product prepared in Example 1;

[0108] Figure 9 The figure shows the zeta potential of the product prepared in Example 1. The figure also shows the zeta potential of the product after it was immersed in simulated gastric juice or simulated intestinal juice.

[0109] Figure 10The figures show the drug release curves of olsalazine sodium (OSA) and tacrolimus (TAC) in the product prepared in Example 4. The inset shows the slope of the drug release curves of OSA in the first 2 hours and TAC in the first 12 hours.

[0110] Figure 11 RosGel prepared in Example 1 and the gel prepared in Comparative Example 1 were used to block Escherichia coli in an oxidizing environment (H2O2). Comparison chart of different situations;

[0111] Figure 12 Fluorescence images and statistical analysis of reactive oxygen species (ROS) in cells after applying RosGel prepared in Example 1 and Gel prepared in Comparative Example 1 under an oxidizing environment (H2O2);

[0112] Figure 13 Fluorescence (a) and SEM (b) images of the mouse intestines were taken 24 hours after the mice were administered RosGel prepared in Example 1. Detailed Implementation

[0113] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially. The features and performance of the present invention will be further described in detail below with reference to the embodiments.

[0114] Example 1

[0115] (1) Synthesis of ROS-severable crosslinking components: 0.5 mmol of acrylamide-polyethylene glycol-hydroxyl (acrylamide-PEG-OH) (purchased from Xi'an Kaixin Biotechnology Co., Ltd.), 0.25 mmol of oxaloyl chloride (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.), and 0.5 mmol of triethylamine (purchased from Sinopharm Chemical Reagent Co., Ltd.) were dissolved in 15 mL of dichloromethane (purchased from Sinopharm Chemical Reagent Co., Ltd.) and reacted overnight at -20 ℃. After the reaction, the organic solvent was removed by rotary evaporation, and the residue was reconstituted with deionized water, purified by dialysis under light-protected conditions, and then lyophilized to obtain polyethylene glycol oxalate diacrylamide, denoted as PEGCO, with the following structural formula:

[0116] .

[0117] The transmission infrared (FT-IR) spectrum of PEGCO is shown below. Figure 1 As shown, 1544 cm -1The characteristic peak at 1616 cm⁻¹ corresponds to C=O in acrylamide. -1 and 1742 cm -1 The characteristic peaks at these locations correspond to the C=O in the carbon-carbon double bond and the oxalate group, respectively.

[0118] (2) Mix the following components to form a gel precursor solution: 50 mg gelatin (7% by mass, purchased from Yuanye Biotechnology), negatively charged targeting component: 150 mg acrylic acid (AAc, 21% by mass, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.), covalent adhesive component: 5 mg N-hydroxysuccinimide acrylate (AAc-NHS, 0.7% by mass, purchased from Maclean's reagent), photoinitiator: 2.5 mg α-ketoglutarate (0.4% by mass, purchased from Sigma-Aldrich), ROS breakable crosslinking component: 10 mg PEGCO (1.4% by mass), and solvent: 485 mg deionized water (balance).

[0119] (3) The above-mentioned gel precursor solution was sonicated for 10 minutes and vortexed for 30 seconds, then placed in a quartz mold of the corresponding volume and irradiated for 300 seconds under ultraviolet light at 365 nm and 120 mW / cm² to induce a cross-linking reaction and form an integral hydrogel. The obtained gel was washed three times with 15 mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) buffer, then freeze-dried, mechanically pulverized, and sieved and graded to obtain dry ROS-responsive microgel powder, denoted as RosGel. The particle size of the obtained RosGel was tested to be 10~200 μm.

[0120] Comparative Example 1

[0121] The preparation process is basically the same as in Example 1, with the only difference being:

[0122] In step (2), PEGCO in the gel precursor solution is replaced with an equal mass of N,N′-methylenebisacrylamide, and the resulting product is denoted as Gel.

[0123] Comparative Example 2

[0124] The preparation process is basically the same as in Example 1, with the only difference being:

[0125] In step (2), PEGCO was not added when preparing the gel precursor solution.

[0126] Experimental observations revealed that the gel in this comparative example could not be formed and instead appeared as a viscous liquid.

[0127] Comparative Example 3

[0128] The preparation process is basically the same as in Example 1, with the only difference being:

[0129] In step (2), the mass fraction of PEGCO in the gel precursor solution is replaced with 0.1%.

[0130] Comparative Example 4

[0131] The preparation process is basically the same as in Example 1, with the only difference being:

[0132] In step (2), gelatin was not added when preparing the gel precursor solution.

[0133] Example 2

[0134] The preparation process is basically the same as in Example 1, with the only difference being:

[0135] In step (2), the mass fraction of PEGCO in the gel precursor solution is replaced with 0.3%.

[0136] Example 3

[0137] The preparation process is basically the same as in Example 1, with the only difference being:

[0138] In step (2), the mass fraction of PEGCO in the gel precursor solution is replaced with 3.0%.

[0139] Example 4

[0140] (1) Pretreatment of hydrophobic immunosuppressive drugs: 50 mg PLGA was dissolved in 1 mL dichloromethane, 25 mg tacrolimus (TAC, purchased from Selleck Chemicals) was added, and then 5 mL of 1% polyvinyl alcohol 1788 (PVA, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) aqueous solution was added and ultrasonically emulsified for 60 seconds. The resulting colostrum was poured into 15 mL of 0.5% PVA solution and stirred to evaporate the organic solvent for 6 hours. The colostrum was centrifuged at 7000 rpm for 3 minutes and washed 3 times. The colostrum was then lyophilized to obtain drug-loaded nanoparticles.

[0141] (2) The gel precursor solution prepared in step (2) of Example 1 was used, and 10 mg of the drug-loaded nanoparticles prepared in step (1) were added to obtain the drug-loaded gel precursor solution;

[0142] (3) The above-mentioned drug-loaded gel precursor solution was sonicated for 10 minutes and vortexed for 30 seconds, then placed in a quartz mold of the corresponding volume size and irradiated under ultraviolet light at 365 nm and 120 mW / cm² for 300 seconds to induce a cross-linking reaction and form an integral hydrogel. The resulting gel was washed three times with 15 mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) buffer and then freeze-dried to obtain a dry gel.

[0143] (4) The dry gel was immersed in HEPES buffer containing 10 mg / mL of the hydrophilic anti-inflammatory drug oxalazine sodium (OSA, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) for 2 hours to allow the hydrophilic drug to diffuse into the gel network, followed by lyophilization. Subsequently, it was mechanically pulverized and then sieved and graded to obtain drug-loaded dry ROS-responsive microgel powder, denoted as […]. After testing, the results were obtained. The particle size is 10~200 μm. The mass fraction of hydrophilic anti-inflammatory drugs was 6.75%, and the mass fraction of hydrophobic immunosuppressive drugs was 0.59%.

[0144] Performance testing:

[0145] 1. Swelling ratio

[0146] Ten mg of the products prepared in Examples 1-3 and Comparative Examples 3-4 were immersed in 50 mL of phosphate-buffered saline (PBS, purchased from Sinopharm Chemical Reagent Co., Ltd.) or 1 mM hydrogen peroxide (purchased from Sinopharm Chemical Reagent Co., Ltd.) aqueous solution. Gels were weighed at different time points, and the swelling ratio was calculated. The results are as follows: Figures 2-5 As shown.

[0147] The formula for calculating the swelling ratio is as follows:

[0148] .

[0149] Experiments showed that the gel skeleton of the product prepared in Comparative Example 4 disintegrated and dissolved in the solution after being soaked in 1 mM hydrogen peroxide (H2O2) aqueous solution for 2 hours. No curve of its swelling ratio changing with time was provided.

[0150] In Comparative Example 3, the dry microgel powder prepared with 0.1% PEGCO by mass did not exhibit ROS-responsive swelling properties. However, the dry microgel powders prepared in Examples 1-3 showed higher swelling ratios in 1 mM hydrogen peroxide aqueous solution than in PBS buffer solution at different time points, demonstrating ROS-responsive swelling ability. Further comparison of Examples 1-3 shows that the dry microgel powders prepared in Examples 1 and 3 exhibited higher swelling ratios.

[0151] 2. Swelling behavior

[0152] To 5 mg (Example 4) and Gel (Comparative Example 1) powder were each added with 50 μL of PBS buffer or 50 μL of 1 mM H2O2 aqueous solution, incubated for 0.5 hours and then dried. The film formation was evaluated by SEM imaging.

[0153] See results Figure 6 Under PBS buffer conditions, Both gel and granules exist as dispersed particles and do not form a continuous film structure; in a 1 mM H₂O₂ aqueous solution, gel still maintains a predominantly discrete structure, while... Significant particle fusion occurred, forming a continuous film-like structure. Furthermore, the attached diagram shows a continuous gel cross-section structure with observable drug-loaded particles, indicating... It has a significant ability to trigger in-situ film formation via ROS.

[0154] 3. Physical and chemical properties

[0155] The morphology and structure of RosGel prepared in Example 1 were observed using scanning electron microscopy (SEM), as follows: Figure 7 As shown. Observation revealed that the RosGel prepared in Example 1 consisted of micron-sized particles.

[0156] The functional groups and chemical composition of RosGel prepared in Example 1 were analyzed using Fourier transform infrared spectroscopy (FT-IR), such as... Figure 8 As shown. The infrared spectrum shows 1720 cm⁻¹. -1 The characteristic peak corresponds to the carboxyl group structure in acrylic acid, at 1166 cm⁻¹. -1 and 1264 cm -1 The characteristic peaks correspond to the structure of NHS active ester, proving that the carboxyl group and the NHS active ester adhesive group have been successfully introduced into the gel system.

[0157] The surface charge and its changes in the RosGel prepared in Example 1 were analyzed using a Zetasizer, such as... Figure 9 As shown, the surface charge of RosGel was directly tested (referred to as untreated). After soaking it in simulated gastric fluid (pH 1.2) for 4 hours (referred to as gastric pH), it was then soaked in simulated intestinal fluid (pH 7.4) for another 4 hours (referred to as intestinal pH). Observations revealed that the RosGel gel carried a negative charge, and this charge was stable, remaining negative throughout without charge reversal.

[0158] 4. Drug release behavior

[0159] To investigate the drug release behavior of olsalazine sodium (OSA) and tacrolimus (TAC), (Example 4) The powders were packaged into dialysis bags with a molecular weight cutoff of 3000 Da. Phosphate-buffered saline (PBS) and PBS containing 10% (v / v) ethanol were used as dispersion media, respectively, and the samples were incubated at 37 °C and 100 rpm in a shaker. Samples were collected at predetermined time points. The release of OSA was detected at 354 nm using ultraviolet spectrophotometry (Metash UV-8000), and the release of TAC was detected at 220 nm using HPLC-UV. The results are as follows: Figure 10 As shown.

[0160] The results showed that OSA was rapidly released within 24 hours, exhibiting rapid release, while TAC was continuously and slowly released over 96 hours, exhibiting sustained release, demonstrating... It achieves a dual-mode drug delivery of "hydrophilic rapid release + hydrophobic sustained release".

[0161] 5. Gel barrier against Escherichia coli in in vitro cellular environments ( ) ability

[0162] To evaluate the physical barrier effect of RosGel prepared in Example 1 and the gel prepared in Comparative Example 1 on oxidatively damaged Escherichia coli on cell surfaces, NCM460 cells were seeded in the upper chamber of a 24-well Transwell and cultured to 100% confluence. Subsequently, 1.5 mM hydrogen peroxide aqueous solution was added to the culture medium to induce oxidative stress (H2O2 group). RosGel or gel was then added to the upper chamber, with untreated normal cells serving as the negative control (NC). After 8 hours of incubation, 200 μL of E. coli suspension was added to the upper chamber, and 600 μL of culture medium was added to the lower chamber. Incubation continued for 1 hour, and the lower chamber culture medium was then collected for plate culture and imaging analysis.

[0163] The results are as follows Figure 11 As shown, in the normal control group (NC), the cell barrier was normal and effectively blocked. In the first group, almost no colonies formed in the lower chamber; after H2O2 treatment, the number of colonies in the lower chamber increased significantly, indicating that oxidative damage destroyed the cell barrier; the inhibitory effect of adding gel on bacterial penetration was limited; while the number of colonies in the lower chamber of the RosGel group was significantly reduced, approaching the NC level, indicating that RosGel can effectively rebuild the physical barrier under oxidative damage conditions and has a certain repair effect on damaged cells, significantly blocking bacterial penetration. penetrate.

[0164] 6. Hydrogen peroxide scavenging behavior of gels in in vitro cellular environments

[0165] NCM460 cells were seeded in 24-well plates and cultured to 70-80% confluence. The following treatment conditions were then applied and incubated for 4 hours, with 600 µL added to each well: 400 µM H2O2; 400 µM H2O2 + 250 µg / mL Gel; 400 µM H2O2 + 250 µg / mL RosGel; and the untreated control group (NC).

[0166] For fluorescence microscopy, 200 µL of 10 µM DCFH-DA (purchased from Sigma-Aldrich) and 1 µg / mL Hoechst (purchased from Sigma-Aldrich) were used to label intracellular ROS and cell nuclei, respectively. After incubation for 30 minutes, the cells were observed (Figure (a)). The fluorescence intensity was then quantitatively analyzed using ImageJ (Figure (b)).

[0167] The results are as follows Figure 12 As shown, compared with the control group (NC), H2O2 treatment significantly induced a large increase in intracellular ROS; the gel group had almost no effect on alleviating ROS; while RosGel treatment significantly reduced intracellular ROS levels, approaching normal levels. Quantitative results further confirm that RosGel has significant ROS scavenging and oxidative stress relief capabilities.

[0168] 7. Verification of the formation and stable retention of the microgel in situ as a patch in the inflamed intestine after oral administration.

[0169] To establish an acute colitis model, mice were administered 3.0% sodium dextran sulfate (DSS, purchased from Sigma-Aldrich) in their drinking water for 7 consecutive days. One day after DSS administration, mice began daily gavage treatment with 200 µL of PBS buffer or 200 µL of 4 mg / mL RosGel. All animals (5 mice per cage) had free access to standard rodent food and drinking water. All experiments used 6-8 week old male mice weighing 20-22 g.

[0170] Inflammation model mice were randomly selected and administered 200 µL of RosGel labeled with the red fluorescent dye rhodamine b via gavage. Twenty-four hours after administration, the distal colon of the mice was harvested, sectioned, and photographed using laser confocal microscopy, or fixed and photographed using SEM.

[0171] The results are as follows Figure 13 As shown in the figure, blue fluorescence of DAPI represents the cell nucleus, and red fluorescence represents RosGel. Fluorescence image (a) and SEM image (b) show that the patch remains stably in the intestinal inflammatory site for no less than 24 hours.

[0172] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. The specific examples used above to illustrate the present invention are only for the purpose of helping to understand the present invention and are not intended to limit the present invention. Those skilled in the art to which this invention pertains can make several simple deductions, modifications, substitutions, or combinations based on the concept of the present invention. These deductions, modifications, substitutions, or combinations also fall within the scope of the claims of the present invention.

Claims

1. A ROS-responsive oral microgel-in-situ intestinal bioadhesion patch system, characterized in that, Raw materials include: Polymer framework materials, ROS fracture-compatible crosslinking components, covalent adhesive components, negatively charged targeting components, initiation systems, and solvents; The polymeric framework material includes one or more of gelatin, collagen, histone, and chitosan; The ROS-fragile crosslinking component is selected from monomers with double bonds at both ends and an oxidative cleavage bond in the middle, wherein the oxidative cleavage bond is selected from one or more of oxalate bonds, thioketal bonds, diselenide bonds, thioether bonds, and disulfide bonds. The covalent adhesive component is selected from monomers containing both double bonds and X groups, wherein the X group is selected from one or more of N-hydroxysuccinimide groups, isocyanate groups, epoxy groups, and aldehyde groups; The negatively charged targeting component is selected from monomers containing carboxyl groups.

2. The ROS-responsive oral microgel-in-situ intestinal bioadhesion patch system according to claim 1, characterized in that: The initiation system is selected from photoinitiation systems, thermal initiation systems, and redox initiation systems; The solvent is selected from water or an aqueous buffer solution.

3. The ROS-responsive oral microgel-in-situ intestinal bioadhesion patch system according to claim 1, characterized in that: The ROS fracture-type crosslinking component is selected from one or more of polyethylene glycol oxalate diacrylamide, polyethylene glycol thioketal diacrylamide, polyethylene glycol selenide diacrylamide, polyethylene glycol disulfide diacrylamide, and polyethylene glycol thioether diacrylamide. The covalent adhesive component is selected from one or more of N-hydroxysuccinimide acrylate, N-hydroxysuccinimide methacrylate, 2-isocyanate ethyl acrylate, and glycidyl acrylate; The negatively charged targeting component is selected from one or more of acrylic acid, methacrylic acid, maleic acid, fumaric acid, and itaconic acid.

4. The ROS-responsive oral microgel-in-situ intestinal bioadhesion patch system according to claim 2, characterized in that: The photoinitiating system is selected from one or more of Irgacure 2959, Irgacure 184, Irgacure 1173, lithium phenyl-2,4,6-trimethylbenzoylphosphonate, and α-ketoglutaric acid; The thermal initiation system is selected from one or more of ammonium persulfate, potassium persulfate, azobisisobutyronitrile, and azodimethylvalerate. The redox initiation system is selected from one or more of the following: ammonium persulfate / tetramethylethylenediamine system, potassium persulfate / ascorbic acid system, hydrogen peroxide / ascorbic acid system, and benzoyl peroxide / tertiary amine system. The aqueous buffer solution is selected from PBS buffer.

5. The ROS-responsive oral microgel-in-situ intestinal bioadhesion patch system according to claim 1, characterized in that, Based on a total mass of 100%, the raw material composition includes: Polymer framework materials: 2%~20%; ROS fracture-compatible crosslinking component: 0.2%~20%; Covalently bonded components: 0.2%~2%; Negatively charged targeting components: 10%~50%; Initiating system 0.02%~2%; Solvent balance.

6. The ROS-responsive oral microgel-in-situ intestinal bioadhesion patch system according to claim 1, characterized in that: The raw materials also include hydrophilic anti-inflammatory drugs and / or hydrophobic immunosuppressive drugs; The hydrophilic anti-inflammatory drugs include one or more of the following: 5-aminosalicylic acid, mesalazine, olsalazine sodium, sulfasalazine, hydrocortisone, prednisolone, dexamethasone sodium phosphate, diclofenac sodium, indomethacin sodium, and ibuprofen sodium. The hydrophobic immunosuppressive drugs include one or more of tacrolimus, cyclosporine A, sirolimus, everolimus, mycophenolate mofetil, and azathioprine; Based on the mass of the ROS-responsive oral microgel-in-situ intestinal bioadhesive patch system, the mass fraction of the hydrophilic anti-inflammatory drug is 1-10%, and the mass fraction of the hydrophobic immunosuppressive drug is 0.1-1.0%.

7. A method for preparing a ROS-responsive oral microgel-in-situ intestinal bioadhesive patch system according to any one of claims 1 to 6, characterized in that, include: (1) A gel precursor solution is prepared by uniformly mixing a polymer backbone material, a ROS fracture-type crosslinking component, a covalent adhesive component, a negatively charged targeting component, an initiation system, a selectively added hydrophobic immunosuppressive drug, and a solvent. (2) The gel precursor solution is cross-linked after initiation to form a hydrogel; (3) The hydrogel was washed and freeze-dried to obtain a crude product; (4) The frozen-dried crude product is directly post-processed to obtain ROS-responsive microgel powder, or the frozen-dried crude product is soaked in a buffer solution containing hydrophilic anti-inflammatory drugs, taken out and frozen-dried again, and then post-processed to obtain ROS-responsive microgel powder.

8. The preparation method of the ROS-responsive oral microgel-in-situ intestinal bioadhesive patch system according to claim 7, characterized in that: In step (4), the post-processing includes screening and grading. The particle size range of the ROS-responsive microgel powder obtained after the post-processing is 10~200 μm.

9. The use of the ROS-responsive oral microgel-in-situ intestinal bioadhesive patch system according to any one of claims 1 to 6 in the preparation of a medicament for treating inflammatory bowel disease, characterized in that, The ROS-responsive oral microgel-in-situ intestinal bioadhesive patch system is administered orally.

10. The application of the ROS-responsive oral microgel-in-situ intestinal bioadhesive patch system according to claim 9 in the preparation of a medicament for treating inflammatory bowel disease, characterized in that, The dosage is 1~100 mg / kg.

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