A dendritic polypeptide nanogel for electrostatic adsorption of hydrogen sulfide on montmorillonite and a preparation method thereof

Abstract

The application belongs to the technical field of biological polymers, and particularly relates to a kind of montmorillonite on electrostatic adsorption load hydrogen sulfide dendritic polypeptide nanogel and its preparation method.The present application is polyhedral oligomeric silsesquioxane POSS as the core three generation lysine dendrimer as branched unit POSS-Lys-G3, by crosslinking agent 3,3'-dithiodipropionic acid di(N-hydroxy succinimide) ester DSP Chemical crosslinking to obtain dendritic polypeptide nanogel carrier;In its network structure, load diallyl trisulfide DATS molecule, on its surface, wrap montmorillonite.The beneficial effects of the present application are: can be specific targeting to the damaged mucosa of large intestine and can also release H2S in response to GSH level in physiological state, reduce multiple pro-inflammatory pathways to achieve effective anti-inflammatory effect, and can activate hemin oxygenase expression to resist oxidative stress, have significant in vivo and in vitro anti-inflammatory, and have good biological safety performance.

Description

Technical Field

[0001] This invention relates to the field of biopolymer technology, and in particular to a dendritic polypeptide nanogel that electrostatically adsorbs hydrogen sulfide on montmorillonite and its preparation method. Background Technology

[0002] Inflammatory bowel disease (IBD) is a group of persistent inflammatory diseases of the digestive tract with unknown pathogens, including ulcerative colitis and Crohn's disease. Its main symptoms include abdominal pain, bleeding, and decreased bowel motility. It can also induce colon cancer, posing a significant risk to patients' health and quality of life, seriously endangering human health and quality of life. Due to the complexity and extremely high incidence of IBD, it has extremely adverse consequences for patients' daily lives and physical condition, while also adding a heavy financial burden to medical and social welfare systems worldwide.

[0003] The occurrence of colitis is mainly accompanied by apoptosis of colonic epithelial cells and damage to the colonic barrier function, which allows bacteria and endotoxins in the intestine to break through the intestinal barrier and enter the blood vessels or lymphatic system, inducing an inflammatory response in the body. This leads to the large-scale recruitment of immune cells to the site of colonic inflammation, and the continuous secretion of large amounts of chemokines, pro-inflammatory factors and ROS at the damaged mucosa site, keeping the mucosa in a state of inflammation and oxidative stress, making it difficult to heal.

[0004] Immune responses are a key factor in inducing the exacerbation of colitis. The intestinal mucosal immune system includes intestinal epithelial cells, macrophages, and lymphocytes expressing Toll receptors. These cells maintain intestinal homeostasis by generating immune responses to exogenous antigens. Therefore, abnormalities in their immunomodulatory functions directly lead to damage to the intestinal barrier and immune dysfunction. Macrophages play a crucial role in maintaining the intestinal immune environment, regulating cell apoptosis and proliferation to maintain the dynamic balance of the tissue environment. Macrophages are mainly classified into two phenotypes: classically activated M1 macrophages and selectively activated M2 macrophages. M1 macrophages are pro-inflammatory cells, primarily induced by bacterial lipopolysaccharide (LPS) or pro-inflammatory factors (TNF-α, IL-1β, etc.). M1 macrophages produce large amounts of pro-inflammatory factors (TNF-α, IL-1β, etc.), causing tissue damage and further exacerbating inflammation. M2 macrophages are anti-inflammatory cells, producing large amounts of anti-inflammatory factors to alleviate the inflammatory response and promote tissue healing. Current research has found that during colitis, a large number of intestinal epithelial cells undergo apoptosis, leading to loss of intestinal barrier function. This results in the large-scale recruitment of macrophages in the intestinal tissue, and the macrophages undergo a large-scale transformation to the M1 type under the influence of intestinal bacteria and toxins. This disrupts intestinal homeostasis and creates an inflammatory environment in the gut, characterized by: 1) the large-scale secretion of pro-inflammatory factors, such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α); 2) an increase in the concentration of matrix metalloproteinases (MMPs). MMPs are expressed at low levels in a normal colonic environment and can cleave and remodel the extracellular matrix. In a colonic inflammatory environment, MMPs expressed at high levels degrade proteoglycans, laminin and fibronectin required for the formation of the intestinal barrier, which further leads to damage to the colonic barrier; 3) The polarization of M1 and M2 macrophages is disturbed. Current research shows that in an intestinal inflammatory environment, due to the disorder of intestinal immune function, macrophages are over-activated by inflammation, resulting in M1 macrophages becoming dominant. The intestinal environment is always in a pro-inflammatory state, and the conversion of macrophages to M2 type is blocked, making it difficult to alleviate the intestinal inflammatory state.

[0005] Oxidative stress is also a significant factor inducing further exacerbation of colitis. Reactive oxygen species (ROS) are a series of oxygen-containing, reactive chemical substances, including hydrogen peroxide, singlet oxygen, hydroxyl radicals, and superoxide anions. In a normal colonic environment, the concentration of ROS is low, and the entire environment is in a state of redox equilibrium. Current research has found a large accumulation of ROS in sites of colitis. Studies have shown that when intestinal tissue is continuously exposed to harmful stimuli (such as pathogenic bacterial infection or exogenous harmful substances), it will be stimulated to secrete ROS, leading to an inflammatory response. Simultaneously, during inflammation, the large-scale aggregation and infiltration of immune cells such as macrophages in the colon further increases ROS production, further increasing the concentration of ROS in the intestine, exacerbating the inflammatory response, and forming an inflammatory cascade that amplifies and persists the inflammation. It can also increase mucosal permeability and damage the intestinal epithelial barrier function by inducing apoptosis of intestinal epithelial cells.

[0006] How to effectively reduce inflammation, alleviate oxidative stress, and repair the damaged colonic barrier are urgent problems to be solved in the treatment of colitis. Currently, salicylates, steroids, and immunomodulators are mainly used in clinical practice to treat colitis, but these drugs all have their own shortcomings.

[0007] 1) Salicylate: 5-Aminosalicylic acid (5-ASA) has always played a crucial role in the relief and treatment of IBD. For early and mid-stage colitis, topical salicylate administration is the preferred initial treatment method with good therapeutic effects, and it also has some preventive effects against colorectal cancer. However, there are issues with nephrotoxicity due to prolonged administration and decreased patient compliance due to frequent administration.

[0008] 2) Steroids: Steroids are more effective than 5-ASA in treating colitis that has progressed to the intermediate or even late stages, and can effectively alleviate the condition. However, they have serious side effects such as weight gain, blood sugar disorders, and peptic ulcers, making them difficult to treat patients long-term.

[0009] 3) Biologics: Various biologics, such as TNF-α inhibitors (IPX, ADA), IL-23 inhibitors (ustekinumab), and JAK inhibitors (CP-690), have been used to treat colitis. Biologics are relatively safe drugs with low side effects and can promote the healing of damaged colonic mucosa while relieving colitis. However, they have disadvantages such as a high relapse rate after treatment, secondary failure due to ineffective re-treatment, and high cost.

[0010] In summary, current medications for treating colitis generally have drawbacks such as significant side effects, difficulty in long-term use, and poor targeting efficacy. Therefore, the development of more effective and safer IBD treatment drugs or strategies is urgently needed.

[0011] Due to its excellent anti-inflammatory, antioxidant, and intestinal barrier protective functions, H2S holds promise as a novel treatment strategy for IBD. However, its gaseous nature makes precise delivery and controlled release into diseased tissue difficult. To address this, various H2S donor molecules have been developed, but most face practical problems such as poor controllable release and the inability to release H2S for extended periods. For example, NaHS and Na2S are extremely unstable under physiological conditions, rapidly releasing large amounts of H2S upon contact with water, potentially causing cytotoxicity. While JK1 and JK2 can respond to the acidic environment of the colon by releasing H2S, the release rate is also rapid, failing to achieve long-term sustained release.

[0012] Diallyl trisulfide (DATS) is an oil-soluble organosulfur compound isolated from garlic and a unique H2S donor. Compared to other donor molecules, DATS has several unique advantages: 1) It only slowly releases H2S in the presence of bio-thiols (such as glutathione GSH), thus its release behavior is more controllable and it has long-lasting sustained-release properties; 2) 1 mol of DATS can theoretically release 3 mol of H2S, thus significantly reducing the amount of DATS required. In conclusion, DATS shows great potential in the treatment of colitis, but due to its poor water solubility, the development of an ideal DATS delivery system is essential. Summary of the Invention

[0013] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a dendritic polypeptide nanogel with hydrogen sulfide electrostatic adsorption on montmorillonite and its preparation method, so as to solve the above problems.

[0014] The technical solution of this invention is achieved as follows: a dendritic polypeptide nanogel with hydrogen sulfide electrostatic adsorption on montmorillonite, comprising a dendritic polypeptide nanogel carrier, wherein the dendritic polypeptide nanogel uses a third-generation lysine dendritic macromolecule with polyhedral oligomeric silsesquioxane (POSS) as the core as the branching unit POSS-Lys-G3, and blank nanogel PDNs obtained by chemical crosslinking with the crosslinking agent 3,3'-dithiodipropionate di(N-hydroxysuccinimide) ester (DSP), further comprising: diallyl trisulfide (DATS) molecules are loaded into the network structure of dendritic polypeptide nanogels. Montmorillonite (MMT) is coated onto the surface of a dendritic peptide nanogel.

[0015] Furthermore, montmorillonite (MMT) is encapsulated around the nanogel through electrostatic adsorption.

[0016] Furthermore, the molar ratio of POSS-Lys-G3 and DSP crosslinking is 1:8.

[0017] Furthermore, a method for preparing a dendritic polypeptide nanogel with hydrogen sulfide electrostatic adsorption on montmorillonite, comprising the following steps: loading H2S diallyl trisulfide (DATS) molecules into the network structure of the dendritic polypeptide nanogel. S1. Add the methanol solution containing DATS dropwise to the methanol solution containing 50mg PDNs, stir evenly, and then sonicate for 10min. S2. Add the methanol solution dropwise to deionized water at a volume ratio of 1:20, and stir vigorously at 1500 r / min for 24 h at room temperature. S3. Place the reaction solution in a dialysis bag (1kD) and dialyze it in deionized water. Centrifuge to remove residual methanol and free DATS from the reaction solution. S4. The solution obtained in S3 was freeze-dried to obtain the slightly yellow cotton-like material DATS@PDNs.

[0018] Furthermore, the step of coating the surface of the dendritic peptide nanogel with montmorillonite (MMT) is as follows: a. First, while gently stirring for 48 hours, add 3g of MMT powder to 100mL of water to form a mixture of MMT and excipients; b. Centrifuge the mixture at 5000 rpm for 2 minutes to remove excipients; c. Add 50 mL of NaOH solution (pH 9-10) to dissolve MMT under vigorous stirring, and then use ultrasound to exfoliate MMT into a monolayer structure. d. The obtained solution was freeze-dried to obtain a white flocculent solid product; e. Dissolve the MMT and DATS@PDNs from step d in deionized water to obtain a suspension of MMT and a pure aqueous solution of DPs. f. Add 1 mg of DATS@PDNs solution containing MMT suspension to the solution, stir vigorously at room temperature for 4 h, centrifuge, and filter to obtain white solid product DATS@PDNs@MMT.

[0019] Furthermore, in step S1, the feeding ratio of DATS to PDNs is 60%.

[0020] Furthermore, in step f, the mass ratio of DATS@PDNs to MMT is 1:20.

[0021] Furthermore, the particle size of DATS@PDNs@MMT is approximately 489 nm.

[0022] The beneficial effects of this invention are as follows: 1. Using DATS as an H2S donor, it was first controllably loaded into the three-dimensional network structure of lysine dendritic peptide nanogels (PDNs), and then further electrostatically adsorbed onto the surface of mucosa-derived membranes (MMTs) to construct a multifunctional drug delivery system, DATS@PDNs@MMT, which can specifically cover the damaged mucosa surface and provide long-lasting sustained release of H2S. DATS@PDNs@MMT has a particle size of approximately 489 nm, a potential of -19.2 mV, and exhibits good stability. 2. DATS@PDNs@MMT has excellent anti-inflammatory properties, which can significantly inhibit the expression of inflammatory factors inside activated macrophages and induce macrophages to polarize into anti-inflammatory macrophages; 3. DATS@PDNs@MMT has good antioxidant stress resistance, which can significantly reduce the ROS concentration inside activated macrophages and increase the expression of HO-1 protein inside cells, thereby significantly promoting the proliferation and scratch repair of colon epithelial cells; 4. DATS@PDNs@MMT has good in vivo anti-inflammatory effects and good biocompatibility. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The particle size and morphology of DPs are shown in the specific embodiments of the present invention. Figure 2 This is a schematic diagram illustrating the synthesis of DATS@PDNs according to a specific embodiment of the present invention; Figure 3 This is a schematic diagram illustrating the synthesis of DATS@PDNs@MMT according to a specific embodiment of the present invention; Figure 4 This is a diagram showing the drug loading rate and drug loading efficiency of DATS in a specific embodiment of the present invention; Figure 5 The image shows the particle size and morphology of DPs@MMT in a specific embodiment of the present invention. Figure 6 The figure shows the H2S release of DPs@MMT at different GSH concentrations under pH 7.4 environment, as a specific embodiment of the present invention. Figure 7 The figure shows the H2S release of DPs@MMT at different GSH concentrations under pH 6.5 environment, as a specific embodiment of the present invention. Figure 8 This is a stability comparison chart of DPs@MMT in a specific embodiment of the present invention; Figure 9 This is a graph showing the particle size variation of DPs@MMT under acidic conditions in a specific embodiment of the present invention. Figure 10 This is a distribution diagram of DPs@MMT on the colon surface according to a specific embodiment of the present invention; Figure 11 To evaluate the cytotoxicity of DPs@MMT on RAW264.7 cells using CCK-8 and live / dead cell staining methods in specific embodiments of the present invention; Figure 12 To evaluate the cytotoxicity of DPs and DPs@MMT on CaCO-2 cells using CCK-8 and live / dead cell staining methods in specific embodiments of the present invention; Figure 13 The distribution and quantitative fluorescence data of cy5.5-labeled DPs@MMT inside activated macrophages are shown in the figure below, representing a specific embodiment of the present invention. Figure 14 Fluorescence imaging of intracellular H2S and corresponding fluorescence intensity diagrams are shown in the specific embodiments of the present invention. Figure 15 Fluorescence imaging of intracellular ROS and corresponding fluorescence intensity in a specific embodiment of the present invention; Figure 16 The specific embodiments of the present invention show the mRNA and protein expression levels of HO-1 in activated macrophages after DPs@MMT treatment; Figure 17 The specific embodiments of the present invention show the expression levels of intracellular TNF-α, IL-6, and IL-1β. Figure 18 The specific embodiments of the present invention show the intracellular expression levels of IL-10 and IL-4. Figure 19 The image shows the protein expression of (a) p-ERK and (b) p-STAT3 in cells after DPs@MMT treatment according to a specific embodiment of the present invention, and (c) the corresponding protein imaging map. Figure 20 The expression levels of (a) iNOS and (b) CD163 proteins in activated macrophages after DPs@MMT treatment are shown in the specific embodiments of the present invention. Figure 21 This invention describes the proliferation, apoptosis, and scratch healing of CaCO-2 cells after DPs@MMT treatment in a specific embodiment. Figure 22 The following are specific embodiments of the present invention: (a) a schematic diagram of the mouse treatment process, (b) changes in mouse body weight during treatment, (c) the activity index of colitis in mice after treatment, (d) quantitative measurement of mouse colon length, and (e) macroscopic photographs. Figure 23This invention describes the retention of DPs@MMT in mouse colon tissue and the clearance of reactive oxygen species in a specific embodiment of the invention. Figure 24 For specific embodiments of the present invention, (a) imaging of H&E staining of colon tissue, (b)-(d) expression levels of inflammatory factors TNF-α, IL-6 and IL-1β in mouse colon tissue; Figure 25 Immunofluorescence imaging of iNOS and CD163 labeled macrophages in colon tissue and corresponding quantitative analysis of fluorescence intensity, as shown in the specific embodiment of the present invention. Figure 26 This diagram illustrates the expression of HO-1 and 8-OHDG in colon tissue according to a specific embodiment of the present invention. Figure 27 The following is a blood routine analysis of mice in each group on day 11 according to the specific embodiments of the present invention: (a) hemoglobin (HGB), (b) white blood cell count (WBC), (c) red blood cell count (RBC), and (d) H&E staining images of the heart, liver, spleen, lung, and kidney of the Normal group and the DPs@MMT group. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] In the description of this application, it should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. For ease of description, the dimensions of the various parts shown in the drawings are not drawn to actual scale. Techniques, methods, and devices known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques, methods, and devices should be considered part of the specification. In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values. It should be noted that similar reference numerals and letters in the following drawings denote similar items, and therefore, once an item is defined in one drawing, it does not need to be further discussed in subsequent drawings.

[0027] Example 1:

[0028] (3-Aminopropyl)triethoxysilane (60 mmol) was dissolved in 350 mL of methanol, stirred, and heated to 50 °C. 30 mL of concentrated hydrochloric acid (12 mol / L) was added dropwise, and the mixture was stirred and heated to 90 °C. After cooling and reflux for 24 hours, heating was stopped. Once the temperature had decreased to room temperature, the reaction mixture was added to 200 mL of tetrahydrofuran (THF), and stirred at room temperature for 4 hours. After washing three times by centrifugation (4000 rpm, 5 min) using THF, the resulting product was placed in a vacuum drying oven to obtain a dry white powder, POSS.

[0029] Example 2:

[0030] The POSS-based first-generation lysine peptide dendritic macromolecule G1-Lys is synthesized via a divergent method using POSS as the core and tert-butyloxycarbonyl (Boc)-protected lysine Boc-Lys(Boc)-OH as the branching unit. The specific reaction steps are as follows: Under nitrogen atmosphere, POSS (0.85 mmol), tert-butyloxycarbonyl (Boc)-protected lysine Boc-Lys(Boc)-OH (8.07 mmol), HBTU (10.29 mmol), and HOBt (10.29 mmol) were dissolved in 50 mL of DMSO. After activation in an ice bath for 20 min, 5 mL of DIEA was added, and the reaction was stirred at room temperature for 48 h. After the reaction was complete, 100 mL of chloroform was added to the reaction solution, and the mixture was stirred thoroughly. The solution was then washed with saturated brine to remove DMSO from the mixture. Anhydrous magnesium sulfate was then added to the solution for overnight drying. Magnesium sulfate was removed from the solution by filtration. The solution was then subjected to rotary evaporation under reduced pressure until it became viscous. 2 mL of acetonitrile was added dropwise to produce a small amount of precipitate. The precipitate was then placed in a -20°C refrigerator for recrystallization overnight. After recrystallization, a large amount of acetonitrile was added for washing and centrifugation (4000 rpm, 5 min) 2-3 times until the supernatant became colorless. The supernatant was dried under vacuum to obtain a white solid, POSS-Lys-G1-Boc.

[0031] The obtained POSS-Lys-G1-Boc was dissolved in chloroform, and trifluoroacetic acid (ten times the equivalent of the Boc group) was added. After vigorous stirring for 8 hours, the solution was viscous by rotary evaporation under reduced pressure. Then, it was added dropwise to 200 mL of diethyl ether. After 2-3 centrifugation and washing, it was finally dried under vacuum to obtain the white solid POSS-Lys-G1. Similarly, the synthesis methods of POSS-Lys-G2 and POSS-Lys-G3 are the same as those of POSS-Lys-G1, and will not be described in detail here.

[0032] Example 3:

[0033] The third-generation dendritic macromolecule POSS-Lys-G3 has abundant amino structures on its surface. Therefore, chemical cross-linking can be achieved by amidation reaction of the amino groups on the macromolecule surface with the cross-linking agent 3,3'-dithiodipropionate di(N-hydroxysuccinimide) ester (DSP), which contains disulfide bonds. The specific steps are as follows: POSS-Lys-G3 and DSP were weighed and dissolved separately in anhydrous DMF at a molar ratio of 1:8. DSP was added dropwise to the POSS-Lys-G3 solution at 1500 rpm and reacted at room temperature for 24 h. The reaction solution was placed in a dialysis bag (1 kD) and dialyzed against DMF for 4 h to remove unreacted material. Then, it was transferred to deionized water and dialyzed for 24 h to remove DMF from the solution. The resulting solution was freeze-dried to obtain a white flocculent solid product. A small amount of the product was weighed and dissolved in water, and the particle size and potential of the blank nanogel were measured using a Malvern particle size analyzer (DLS). The morphology of the nanoparticles was analyzed by transmission electron microscopy (TEM).

[0034] Example 4:

[0035] The previously obtained blank nanogels (PDNs) and DATS were dissolved in methanol solution. To calculate the optimal ratio of DATS to PDNs, three different DATS ratios (40%, 50%, and 60%) were designed. Specifically, methanol solutions containing 50 mg, 75 mg, and 116 mg of DATS were added dropwise to methanol solutions containing 50 mg of PDNs, respectively. After stirring and homogenization, the mixture was sonicated for 10 min. Then, the methanol solution was added dropwise to deionized water at a volume ratio of 1:20, and the mixture was vigorously stirred at 1500 rpm for 24 h at room temperature to ensure sufficient loading of DATS molecules into the nanogel network structure. The reaction solution was then placed in a dialysis bag (1 kD) and dialyzed against deionized water. Centrifugation was used to remove residual methanol and free DATS. The resulting solution was freeze-dried to obtain a slightly yellow cotton-like material, DATS@PDNs (DPs). Finally, DLS was used to determine the particle size and zeta potential of the DPs, and TEM was used to evaluate their appearance. The S ion content of the nanogel can be accurately analyzed by using an elemental analyzer (EA, Elementar VarioEL), which can accurately estimate the drug loading capacity and corresponding drug loading efficiency of the nanogel.

[0036] Hydrophobic DATS can be loaded into PDNs using physical loading methods to obtain drug-loaded gels DATS@PDNs (DPs), such as... Figure 1As shown, the particle size distribution of DPs, measured by DLS, is approximately 207 nm, with a PDI of approximately 0.205, exhibiting excellent dispersibility in aqueous solution. Furthermore, due to the hydrophobic nature of DATS, successful loading results in a more compact PDN structure due to hydrophobic interactions, leading to a smaller size of the drug-loaded gel DPs compared to the blank nanogel PDNs. DLS also revealed that the DPs potential is approximately 28.5 mV, demonstrating that DATS loading does not affect the presence of a large number of positive charges on the nanogel's periphery. Further TEM analysis of the gel's morphology reveals that the DPs are uniformly shaped, regularly spherical nanogels.

[0037] To construct a system with excellent drug loading performance, we selected three different proportions of DATS for loading experiments. The DATS accounted for 50%, 60% and 70% of the total mass of the reaction system, respectively. The concentration of S ions in the solution of the obtained DPs nanogels with the three proportions was detected by an elemental analyzer (EA, Elementar VarioEL) to calculate the drug loading amount and drug loading efficiency of the material.

[0038] result: according to Figure 4 When the feed ratio was 40%, the drug loading and drug loading efficiency reached 11.12% and 22.24%, respectively; when the feed ratio was 50%, the drug loading and drug loading efficiency reached 23.45%; and when the feed ratio was 60%, the drug loading and drug loading efficiency reached 26.65% and 38.07%, respectively. The tests showed that when the feed ratio reached 60%, the drug loading and drug loading efficiency of DATS reached their optimal levels.

[0039] Example 5:

[0040] Because the nanogel has a large number of positively charged amino groups on its periphery, while montmorillonite (MMT) is negatively charged in aqueous solution, MMT can be encapsulated around the nanogel through electrostatic adsorption. To prepare the composite material combining DPs and MMT, firstly, 3 g of MMT powder was added to 100 mL of water under gentle stirring for 48 hours to form a mixture of MMT and excipients (vanillin, glucose, and sodium saccharin). The mixture was then centrifuged at 5000 rpm for 2 minutes to remove the excipients. Next, 50 mL of NaOH solution (pH 9-10) was added to dissolve the MMT under vigorous stirring, followed by ultrasonic exfoliation to separate the MMT into a monolayer structure. The resulting solution was freeze-dried to obtain a white flocculent solid product. MMT and DPs were dissolved in deionized water to obtain MMT suspensions and pure aqueous solutions of DPs. To obtain the optimal loading rate, three different DPs feed ratios (4%, 5%, and 6%) were designed, i.e., MMT suspensions containing 15 mg, 20 mg, and 25 mg were added dropwise to 1 mg DPs solution, respectively. After vigorous stirring at room temperature for 4 h, the mixture was centrifuged and filtered to obtain a white solid product. Finally, the product was redispersed in water or lyophilized for further use. The particle size and zeta potential of CPHs were detected by DLS. The morphology of the composite material was observed by TEM. To calculate the adsorption efficiency of MMT for DPs, 2 mL of DPs (0.5 mg / mL) was mixed with 50 μL of DMSO solution containing Cy5.5NHS (1 mg / mL) and kept at room temperature for 4 h. After extensive dialyzing of the solution with ultrapure water, the prepared MMT suspension was added dropwise, stirred for 4 h, and then lyophilized to obtain Cy5.5-labeled DPs. Then, Cy5.5-labeled DPs@MMT were prepared as described above. The adsorption efficiency of MMT for DPs was calculated by measuring the change in fluorescence intensity.

[0041] The negatively charged monolayer MMT, after ultrasonic ablation, was combined with positively charged DPs to obtain the composite material DPs@MMT. To achieve better loading efficiency and targeting performance, three different mass ratios of DPs / MMT (1:10, 1:20, 1:25) were used for feeding. DLS analysis showed the results in Table 1. When the DPs to MMT ratio was 1:20, the resulting composite material DPS@MMT exhibited uniform particle size, good dispersibility, and a high negative charge, indicating that this composite material had the highest loading efficiency and could achieve a targeted effect on positively charged damaged areas of the colonic mucosa.

[0042] Table 1. Particle size and zeta potential of composites with three different DPs / MMT mass ratios 1:25 594.6nm 0.251 -32.6mv 1:20 489.2nm 0.185 -26.2mv 1:10 497.4nm 0.158 10mv like Figure 5As shown, DLS analysis revealed that the DPs@MMT particle size was approximately 490 nm, with a PDI of less than 0.2. SEM images also confirmed the successful bonding between the nanoparticles and montmorillonite. DLS analysis showed that the zeta potential of the composite material changed from positive to negative, further confirming the successful bonding between MMT and the nanoparticles.

[0043] Example 6:

[0044] To determine the H2S release efficiency of DPs@MMT under physiological conditions, hydrogen sulfide gas detection was used to assess the release behavior of DPs@MMT. First, 1 mL of DPs@MMT (20 mg / mL) suspension was added to a sample vial, followed by the addition of the hydrogen sulfide gas detector to a 1 L sealed glass container. Finally, different concentrations of GSH solution were added dropwise to the sample vial using a syringe: 1) pH 7.4 + 0 mM GSH; 2) pH 7.4 + 1 mM GSH; 3) pH 7.4 + 1.5 mM GSH; 4) pH 7.4 + 2 mM GSH. The time of addition of PBS or GSH was set to zero, and the H2S concentration (ppm) detected by the hydrogen sulfide detector was recorded every 5 minutes. The experiment was terminated after 60 minutes. Following the method reported by Urara Hasegawa et al., the H2S concentration detected by the hydrogen sulfide detector was converted into the H2S gas release rate through calculation. The specific calculation method is as follows: Assuming that the gas phase and liquid phase in the container are in equilibrium, and the pressure in the sealed system is 1 standard atmosphere, the molar amount of H2S released from DATS can be obtained using the following formula:

[0045] Where p is the partial pressure of H2S, Vg and Vl are the volumes of the gas and liquid phases, respectively, and R is the gas constant (0.08205 L·atm·mol⁻¹). -1 ·K -1 T is the temperature, c is the concentration of H2S in the liquid phase, and k is the Henry's law constant for H2S in water (9.741 L·atm·mol⁻¹ at 25°C). -1 Therefore, the measured H2S gas partial pressure (ppm) can be converted into the H2S gas concentration, thereby obtaining the amount of H2S released.

[0046] To simulate the H2S release behavior of DPs@MMT under inflammatory conditions in the colon, 1 mL of DPs@MMT (20 mg / mL) suspension was added to the sample vial. The pH was then adjusted to 6.5 using dilute hydrochloric acid. This solution, along with hydrogen sulfide gas, was then added to a 1 L sealed glass container. Finally, different concentrations of GSH solution were added to the sample vial using a syringe: 1) pH 6.5 + 0 mM GSH; 2) pH 6.5 + 1 mM GSH; 3) pH 6.5 + 1.5 mM GSH; 4) pH 6.5 + 2 mM GSH. Subsequent processing was as described above. result: The release of H2S over 60 minutes can be obtained by recording with a hydrogen sulfide gas detector. Figure 6 When DPs@MMT was mixed with different concentrations of GSH, the release of H2S was observed: at a concentration of 1 mM, the release of H2S reached 1.96 μmol; at a concentration of 1.5 mM, the release of H2S reached 2.91 μmol; and at a concentration of 2 mM, the release of H2S reached 3.69 μmol. The intracellular GSH concentration is around 1-2 mM; therefore, this experiment demonstrates that DPs@MMT can slowly release a large amount of H2S under physiological conditions.

[0047] The H2S release from DPs@MMT under acidic conditions was recorded by a hydrogen sulfide gas detector over a period of 60 minutes. Figure 7 When DPs@MMT was in an environment of pH 6.5, the release of H2S from different concentrations of GSH mixtures varied: at a concentration of 1 mM, the release of H2S reached 1.148 μmol; at a concentration of 1.5 mM, the release of H2S reached 1.558 μmol; and at a concentration of 2 mM, the release of H2S reached 2.460 μmol. The enteritis environment is acidic, and the intracellular GSH concentration in diseased tissue cells is between 1 and 1.5 mM. Therefore, this experiment demonstrates that DPs@MMT can slowly release H2S under the inflammatory environment of the colon.

[0048] Example 7:

[0049] To test the stability of DPs@MMT, a small amount of DPs@MMT composite material was dispersed in deionized water to prepare a suspension with a concentration of 1 mg / mL. The suspension was stored at room temperature, and small amounts of the solution were taken on days 1, 2, 3, 4, 5, 6, and 7 for DLS detection of particle size and zeta potential changes.

[0050] result: By testing the particle size change of DPs@MMT over 7 days, the following results were obtained. Figure 8Over a 7-day period, the particle diameter of DPs@MMT remained relatively stable with only a slight increase, and the PDI value remained below 0.2. This indicates that after 7 days, the DPs@MMT structure remained stable, with good dispersion and no aggregation or dissociation. This is because DPs carry a large amount of positive charge, while MMT carries a large amount of negative charge. In a neutral state, DPs and MMT are firmly bonded together by electrostatic adsorption, without dissociation. Simultaneously, the large amount of negative charge on the surface of the composite DPs@MMT causes mutual repulsion, leading to a stable overall system.

[0051] Example 8:

[0052] The main factors influencing the surface potential of MMT are isomorphous substitution by low-valence cations and the ionic strength in aqueous solution. Therefore, the surface zeta potential of MMT changes with the pH of the solution, leading to changes in the ionic strength. Cy5.5-labeled polymerases (DPs) were combined with MMT to form a Cy5.5-labeled composite material, DPs@MMT. DLS was used to detect the zeta potential and particle size changes of DPs@MMT under different pH conditions to confirm that DPs@MMT can maintain a negative charge under acidic colonic conditions and can dissociate the drug-loaded nanogel DPs, allowing DPs to be taken up by cells and exert a therapeutic effect. The degree of dissociation of the composite material was analyzed using a fluorescent microplate reader.

[0053] result: The dissociation of DPs@MMT was determined by detecting changes in DPs@MMT particle size and zeta potential over 3 hours under acidic conditions using DLS. Figure 9 As shown, under acidic conditions, DPs@MMT began to dissociate at 60 min, with the particle size changing from 476 nm to 621 nm and 212 nm. The 621 nm particle size represents the particle size of the remaining composite material and the dissociated MMT, while the 212 nm particle size is the same as that of DPs. This proves that the composite material DPs@MMT began to partially dissociate into MMT and DPs at 60 min. At 90 min, the particle size peak positions further separated, with the proportion at 212 nm increasing, indicating that the composite material underwent further dissociation.

[0054] Example 9:

[0055] Mice with colitis were injected rectally with 200 μL of Cy5.5-labeled DPs@MMT (30 mg / mL). One hour later, the mice were sacrificed, and colonic tissue was collected, fixed with paraformaldehyde for 24 hours, and stained with Hoechst 33342. The colonic tissue was observed using a laser confocal microscope to analyze the distribution and content of DPs@MMT within the colonic tissue. Colons from untreated mice and treated mice served as control groups.

[0056] result: The distribution of DPs@MMT on the colon surface was observed using confocal microscopy, and the results are as follows: Figure 10 As shown, DPs@MMT accumulates extensively in the colon of mice with colitis, but hardly accumulates in the colon of normal mice, demonstrating that DPs@MMT can target and cover damaged areas of the colon.

[0057] Example 11:

[0058] Mouse RAW264.7 cell lines and human colon adenocarcinoma CaCO-2 cells were selected for this study. RAW264.7 cells were cultured in a dedicated macrophage culture medium, while CaCO-2 cells were cultured in a dedicated CaCO-2 cell culture medium. Cell culture was conducted in a cell culture incubator at 37°C and 5% CO2. Untreated RAW264.7 cells were considered normal macrophages, while RAW264.7 cells co-incubated with lipopolysaccharide were considered uncontrolled activated macrophages that could potentially cause non-specific tissue damage.

[0059] Example 12:

[0060] The DPS@MMT complex was sterilized by irradiation under UV light for 2 hours, then mixed with sterile PBS solution to prepare a 10 mg / mL DPS@MMT suspension. The suspension was then mixed with pre-prepared complete culture medium and successively diluted to prepare media containing 1, 1.5, and 2 mg / mL DPS@MMT before being stored for later use. 1 × 10⁻⁶ mg / mL DPS@MMT was added to each well of a 96-well plate. 4 RAW264.7 cells were cultured overnight and then added to complete culture medium containing DPS@MMT at concentrations of 0, 1, 1.5, and 2 mg / mL. After incubation for 24 h, the cells were washed with PBS, and CCK8 was added. The cells were then incubated in a cell culture incubator in the dark for 0.5 h. The absorbance of each well at 450 nm was measured using a microplate reader. Cell viability was calculated using the following formula.

[0061]

[0062] Where A1 is the OD value of the blank control group, A2 is the OD value of the experimental group, and A3 is the OD value of the negative control group.

[0063] The cytotoxicity of two materials, Dex-P (5 μg / mL) and DPs (100 μg / mL), in activated macrophages and ordinary macrophages was also tested. Dexamethasone, a commonly used anti-inflammatory drug in clinical practice, exhibits strong cytotoxicity against macrophages and was used as a cytotoxicity control. In addition, cells were stained with AM / PI for 30 min, and cell fluorescence was observed and photographed using an inverted fluorescence microscope.

[0064] Cytotoxicity assays were also conducted using CaCO-2 cells cultured in a CaCO-2-specific medium at 37°C with 5% CO2. The culture method was the same as described above, and the subsequent AM / PI treatment after CKK8 treatment followed by the same method.

[0065] like Figure 11 As shown in (a), cytotoxicity increased slightly with increasing DPs@MMT concentration, but cell viability remained above 90% even at a material concentration of 2 mg / mL, while the cell viability of the control group, Dex-p, was below 80%. This demonstrates that the composite material does not pose a significant threat to macrophages. Using AM / PI live / dead staining technology, we were able to more accurately detect the cytotoxicity of RAW264.7 cells. Calcein (AM), present on the cell membrane, is broken down by cellular esterases, releasing green fluorescence. PI, on the other hand, penetrates into the cell interior, binding to the cell's DNA and producing red fluorescence. This binding... Figure 11 This is reflected in (b). The figure shows that cells treated with DPS and DPs@MMT exhibit predominantly green fluorescence, with very little red fluorescence, indicating a high proportion of live cells and a low proportion of dead cells. This result is similar to that of CCK8, further demonstrating that the material does not pose a significant threat to normal cells. Figure 12 As shown in (a), DPs, PDNs@MMT, and DPs@MMT exhibited minimal toxicity to CaCO-2 cells, with cell viability consistently above 90%. Live / dead cell staining further confirmed the cytotoxicity of the materials. The figure shows that cells treated with DPs, PDNs@MMT, and DPs@MMT primarily exhibited green fluorescence, with very little red fluorescence, indicating a high prevalence of live cells and a low prevalence of dead cells. This result is similar to that obtained with CCK8, further demonstrating that the materials pose little harm to normal cells and can be used as a drug in the colon. Combined with cytotoxicity experiments on RAW264.7 and CaCO-2 cells, the biocompatibility of the composite material was verified; therefore, 2 mg / mL DPs@MMT was selected for subsequent experiments.

[0066] Example 13:

[0067] To investigate the cellular uptake of DPs nanogels, we used laser confocal microscopy (CLSM) to verify that macrophages can phagocytose DPs. To this end, we designed several experimental sets to verify the phagocytic effect of activated macrophages on the nanogels.

[0068] 1) LPS: RAW264.7 cells treated with LPS were co-incubated with complete culture medium without any added materials as a negative control group; 2) LPS+DPs: RAW264.7 cells treated with LPS were co-incubated with Cy5.5-modified DPs (100 μg / mL); 3) LPS+DPs@MMT: RAW264.7 cells treated with LPS were co-incubated with Cy5.5 modified DPs@MMT (2 mg / mL).

[0069] Example 14:

[0070] After verifying that DPs@MMT can release H2S in an in vitro simulated colon environment, this invention further confirms whether DPs@MMT can successfully release H2S in cells. The method for detecting H2S is based on the method reported by Zhou et al. (see Zhou Y, Mazur F, Liang K, et al. Sensitivity and Selectivity Analysis of Fluorescent Probes for Hydrogen Sulfide Detection[J]. Chemistry, an Asian journal, 2022, 17(5): e202101399), which uses the fluorescent probe WSP-5 for detection.

[0071] Depend on Figure 13 As shown in (a), after co-incubation of activated macrophages with DPs, a large amount of red fluorescence was observed within the cells. This is because DPs have a high cellular uptake efficiency, allowing macrophages to engulf large quantities of DPs. While the fluorescence of DPs@MMT was slightly weaker than that of DPs, it still exhibited strong fluorescence, demonstrating that the composite material has a relatively high cellular uptake efficiency, capable of delivering DATS into the cells for treatment of activated macrophages. Figure 13 As shown in (b), the Cy5.5 quantification results obtained using a fluorescence microplate reader are consistent with the CLSM images, further demonstrating that the material has high cell uptake efficiency.

[0072] Example 15:

[0073] To demonstrate that intracellularly released H2S reduces intracellular ROS levels, this invention utilizes Cell ROX... TM Green assays were used to detect intracellular ROS levels in activated macrophages after treatment with different materials.

[0074] Depend on Figure 14(a) It can be seen that since untreated activated macrophages contain only endogenous H2S at a very low concentration, there is almost no fluorescence. DATS treatment of activated macrophages resulted in a small amount of fluorescence because DATS is almost insoluble in water, making it difficult for cells to take it up; only a very small amount of DATS was taken up by the cells and released hydrogen sulfide. After incubation of activated macrophages with DPs and DPs@MMT, the H2S fluorescence intensity increased significantly. This is because, compared to the DATS group, the phagocytic efficiency of DPs and DPs@MMT was significantly increased, with more DATS being phagocytosed by the cells along with the materials and decomposed inside the cells to produce H2S. The quantitative fluorescence data in 14(b) also show a significant increase in intracellular H2S concentration.

[0075] Depend on Figure 15 (a) It was found that the ROS level in activated macrophages was much higher than that in normal cells, while the ROS level was significantly reduced after treatment with DPs and DPs@MMT. Figure 15 (b) Quantitative data also show that the intracellular ROS level decreased significantly after DPs and DPs@MMT treatment.

[0076] Example 16:

[0077] To demonstrate that DPs@MMT possesses anti-inflammatory capabilities, this invention uses enzyme-linked immunosorbent assay (ELISA) to detect the expression levels of pro-inflammatory and anti-inflammatory factors within macrophages.

[0078] according to Figure 17 Data (a)-(c) show that treatment of activated macrophages with Dex-p, DPs, and DPs@MMT significantly reduced the secretion of inflammatory factors, with DPs and DPs@MMT showing a significantly greater reduction than Dex-p. This demonstrates that DPs and DPs@MMT have superior anti-inflammatory effects compared to Dex-p, and they can effectively regulate the concentration of pro-inflammatory factors within activated macrophages to normal levels, thus confirming the anti-inflammatory effect of DPs@MMT.

[0079] Figure 18 As shown in (a)-(b), when activated macrophages were treated with Dex-p, DPs, and DPs@MMT, the secretion of inflammatory factors increased to varying degrees. The increase in inflammatory factors by DPs and DPs@MMT was significantly greater than that by Dex-p, demonstrating that the anti-inflammatory effects of DPs and DPs@MMT were superior to those of Dex-p. Furthermore, we found that the levels of IL-4 and IL-10 produced by activated macrophages treated with DPs and DPs@MMT were significantly higher than those of normal macrophages. This indicates that DPs and DPs@MMT can regulate the concentration of anti-inflammatory factors within activated macrophages to a higher level, thereby promoting the transformation of macrophages to an anti-inflammatory phenotype.

[0080] Example 17:

[0081] To investigate the anti-inflammatory mechanism of H2S, this invention examines changes in the MAPK and NF-κB signaling pathways, HO-1 expression, and macrophage phenotype after DPs@MMT treatment. This invention uses Western Blot and RT-qPCR techniques to conduct a detailed study on the expression of HO-1 protein; Simultaneously, changes in two typical inflammatory signaling pathways (MAPK and NF-κB) were detected to explore whether H2S release would affect changes in intracellular signaling pathways. This invention uses Western blotting to detect the expression of several proteins. To evaluate the effect of DPs@MMT on the phenotype of activated macrophages, this invention uses RT-qPCR and Western Blot to detect the expression levels of CD163 and iNOS proteins in mRNA and the primer sequence of iNOS. At the same time, macrophages are immunolabeled with iNOS and CD163 antibodies, and intracellular fluorescence is observed and photographed using laser confocal microscopy.

[0082] like Figure 19 As shown, the expression levels of activated macrophage proteins in the LPS group were significantly increased, but the expression levels of both proteins decreased significantly when incubated with other materials. In contrast, the expression levels of p-ERK and p-STAT3 in the DPs and DPs@MMT groups were much lower, indicating that they have a stronger anti-inflammatory effect in cells. Using PDNs@MMT as a control group, we found that the secretion levels of inflammatory proteins in cells treated with the blank material PDNs@MMT were similar to those in the LPS group, demonstrating that the anti-inflammatory ability of the material is provided by DATS in the composite material.

[0083] like Figure 20 As shown in (a), LPS treatment significantly increased the intracellular iNOS expression level, while DPs and DPs@MMT treatment significantly decreased the intracellular iNOS expression level, which was similar to that of normal macrophages. This demonstrates that DPs@MMT can inhibit the activation of macrophages into M1-type macrophages. Figure 20 As shown in (b), the expression level of CD163 protein in cells was significantly increased after treatment with DPs and DPs@MMT, demonstrating that DPs@MMT can induce macrophage activation into M2-type macrophages. The results indicate that H2S has a significant effect on promoting the transformation of pro-inflammatory macrophages into anti-inflammatory macrophages.

[0084] Example 18:

[0085] To demonstrate that intracellularly released H2S can alleviate damage to colon tissue under inflammatory conditions, this invention uses a TUNEL assay kit to detect apoptosis in CaCO-2 cells co-cultured with RAW264.7 cells. This invention uses the Cell Light EdU Apollo 567 in vitro imaging kit to detect the proliferative effect of DPs@MMT on colonic mucosal cells under inflammatory conditions; This invention demonstrates the cell migration-promoting effect of DPs@MMT through a cell scratch healing experiment.

[0086] The protective effect of DPs@MMT on colon cells, such as Figure 21 As shown in (a)-(b), compared with the Normal group, the proliferation of CaCO-2 cells co-cultured with activated macrophages was significantly inhibited, and apoptosis was also significantly increased. However, after treatment with DPs@MMT, apoptosis was significantly inhibited, accompanied by a large number of proliferations. In the end, the proliferation and apoptosis of CaCO-2 cells in the material group were the same as those in normally cultured cells. This proves that DPs@MMT can effectively protect CaCO-2 cells from apoptosis induced by activated macrophages by resisting oxidative stress and alleviating inflammatory response.

[0087] The effect of DPs@MMT on promoting mucosal repair is as follows Figure 21 As shown in (c), compared with the Normal group, the migration of CaCO-2 cells in the LPS group was significantly affected by the influence of activated macrophages, resulting in a slower cell migration rate and thus affecting scratch healing. The scratch healing effect of the DPs@MMT group was comparable to that of the Normal group, indicating that DPs@MMT promotes CaCO-2 migration.

[0088] In summary, these results clearly demonstrate that DPs@MMT can effectively reduce intestinal cell apoptosis and the integrity of the gastrointestinal epithelial barrier through antioxidant and anti-inflammatory effects, which is crucial for inhibiting the further deterioration of colitis.

[0089] Example 19:

[0090] animal experiments The mouse IBD colitis model was constructed as follows: In this experiment, BALB / c female mice, 6-8 weeks old and weighing about 20g, were used. After being housed in the animal room for three days to acclimatize to the environment, the mice were fed with 3% DSS pure water solution to induce colitis.

[0091] A colitis model was established by feeding mice with DSS solution. The status of colitis in mice was evaluated by monitoring changes in mouse body weight, disease activity index (DAI), and colon length. The material's ability to scavenge reactive oxygen species and its retention in the colon were detected using an in vivo optical imaging system. The therapeutic effect of the material on colitis was detected by H&E staining and fluorescent staining. The secretion of inflammatory factors in colon tissue was detected by ELISA; the biosafety of DPs@MMT was evaluated by complete blood count and analysis of important organ sections from mice.

[0092] Five days after DSS induction, the mice were divided into four groups, each receiving a different material rectally. The specific groupings are as follows: 1) DSS group: 200 μL of normal saline was injected each time; 2) Dex-p group: 200 μL Dex-p (75 μg / mL) solution was injected each time; 3) DPs group: 200 μL LDPs (1.5 mg / mL) solution was injected each time; 4) DPs@MMT group: Each injection consisted of 200 μL of DPs@MMT (30 mg / mL) suspension; A separate group of healthy mice served as a normal control group, receiving no treatment. Mice were then injected every two days for three consecutive days. Throughout the experiment, mouse weight changes, diarrhea, and fecal bleeding were monitored daily. The Disease Activity Index (DAI) was used to quantify the severity of colitis in the mice. Treatment ended on day 11. After treatment, the mice were sacrificed, and their colons were collected and their length measured.

[0093] To evaluate the targeting effect of DPs@MMT on inflamed colonic tissue, mice with DSS-induced acute colitis were first fasted for one day, and then administered 100 μL of Cy5.5-labeled DPs@MMT (30 mg / mL) rectally. At 0, 1, 3, 6, and 9 hours after administration, the in vivo distribution of Cy5.5-DPs@MMT throughout the intestine was imaged using an in vivo imaging system. Mice were then sacrificed, and the intestines were collected for ex vivo imaging, and the fluorescence intensity of Cy5.5-DPs@MMT was quantified.

[0094] To evaluate the effect of DPs@MMT on reactive oxygen species (ROS) scavenging in inflamed colonic tissue, mice with DSS-induced acute colitis were first fasted for one day, then administered 200 μL DPs@MMT (30 mg / mL) rectally. One hour later, mice were injected abdominally with the L012 probe (20 mg / kg), and in vivo imaging was used to observe luminescent images of the intestinal region. All mice were sacrificed, and the intestines were collected for ex vivo imaging and ROS clearance was quantified.

[0095] Colon tissue samples were fixed in paraformaldehyde for 7 days, then washed overnight with PBS to remove residual paraformaldehyde. They were then dehydrated sequentially with 65%, 75%, 85%, 90%, 95%, and 100% ethanol (1 hour per concentration). After clearing with xylene, the tissues were embedded in paraffin and finally fixed onto slides using a tissue sectioner. Hematoxylin and eosin (H&E) staining and CD86 / CD163 / Hoechst fluorescence staining were then performed to observe colonic inflammation. HO-1 / 8-OHDG / Hoechst fluorescence staining was used to observe oxidative stress in the inflamed colonic tissue.

[0096] Another 100 mg of fresh colon tissue sample was taken, rinsed with physiological saline, and then ground into small tissue pieces using a homogenizer. NP-40 tissue lysis buffer containing PMSF was then added, and lysis was performed on ice for 30 min. After lysis, the sample was centrifuged (10000 rpm, 4℃, 10 min), and the supernatant was used as the protein extraction solution. The expression levels of inflammatory factors in the tissue sample were detected using a TNF-α, IL-1β, and IL-6 inflammatory factor ELISA kit.

[0097] On the last day of treatment, venous blood was collected from mice in each group using the orbital blood sampling method. Complete blood count (CBC) was performed on the venous blood to evaluate the levels of red blood cells (RBC), white blood cells (WBC), and hemoglobin (HGB) to assess the safety of the treatment. Simultaneously, vital internal organs such as the heart, liver, spleen, lungs, and kidneys of the DPs@MMT mice were removed and subjected to H&E staining to assess the biocompatibility of DPs@MMT.

[0098] The process of colitis modeling and treatment is as follows: Figure 22 As shown in (a), the changes in mouse body weight and disease activity index during treatment are as follows: Figure 22 As shown in (b)-(c). From Figure 22 (b) It can be seen that as the number of days of DSS induction increases, the body weight of colitis mice gradually decreases. However, starting from day 3 after drug injection, the rate of body weight loss slows down in the Dex-p group, DPs group, and DPs@MMT group. In particular, the rate of weight loss stops in the DPs@MMT group on day 9, and the body weight begins to increase. Figure 22 (c) It can be seen that the increase in the number of days of DSS induction is accompanied by the increase in the colonic inflammatory activity index of mice. Starting from the drug injection on the 3rd day, the rate of increase of DAI index in mice in the Dex-p group, DPs group and DPs@MMT group slowed down. In particular, the DAI upward trend in the DPs@MMT group stopped on the 9th day and the DAI index began to decline.

[0099] The colon length of mice after treatment is as follows: Figure 22As shown in (d) and 22(e), the colon length of mice in the DSS group was significantly shortened. After treatment, the colon length of mice in the Dex-p group, DPs group and DPs@MMT group increased. At the same time, we found that the colon length of mice treated with DPs@MMT was close to that of mice in the Normal uninduced group.

[0100] Combination Figure 22 (b)-(d) We were able to macroscopically evaluate the disease status of colitis in mice from three indicators: body weight, disease activity index, and colon damage. Compared with the DSS group, the colitis in the Dex-p, DPs, and DPs@MMT groups all showed different degrees of relief. In particular, the colitis in the DPs@MMT group improved significantly after four doses of treatment. The DAI index and colon length were similar to those in the Normal group, which proves that DPs@MMT has excellent therapeutic effects on colitis mice.

[0101] The retention of Cy5.5-labeled DPs@MMT in colitis mice is as follows: Figure 23 As shown in (a), the fluorescence intensity gradually weakened over time, and the distribution area also gradually decreased, reaching its lowest point after 9 hours. After 9 hours, the mice were dissected, and in vitro fluorescence imaging of the intestinal tissue revealed that drug fluorescence still remained in the intestinal tissue. Combining in vivo imaging and in vitro fluorescence results, it can be concluded that the material can remain in the intestinal tissue for more than 9 hours.

[0102] L012 probe markers indicate the distribution of reactive oxygen species in mice, as follows: Figure 23 As shown in (b), the Normal group showed almost no fluorescence, the DSS group showed high-intensity fluorescence, and the DPs@MMT group showed a significant decrease in fluorescence. After dissecting the mice, in vitro fluorescence imaging of the intestinal tissue revealed that the DSS group had strong fluorescence in the colon, while the fluorescence in the colon was significantly weakened after DPs@MMT treatment, proving that the ROS concentration in the colon decreased significantly after DPs@MMT treatment.

[0103] H&E can be used to analyze inflammatory responses and tissue damage in colonic tissue. Figure 24(a) It can be seen that H&E staining of normal tissue shows intact goblet cells, no inflammatory cell infiltration around the crypts, and no ulceration. Compared with normal tissue, DSS tissue shows a large area of ​​missing goblet cells, partial disappearance of crypts, and a large amount of inflammatory cell infiltration in the mucosa, with ulceration in the tissue. Although the Dex-p group and DPs group had already received drug treatment, they still showed missing goblet cells, disappearance of crypt tissue, and inflammatory cell infiltration. In contrast, after drug treatment, the DPs@MMT group showed basically intact goblet cells, intact crypt tissue, and almost no inflammatory cell infiltration. We also detected the secretion of inflammatory factors in colon tissue and found that the secretion of pro-inflammatory factors TNF-α, IL-1β, and IL-6 in the colon of the DSS group was 4-7 times higher than that of normal tissue. After treatment with Dex-p and CPs, the secretion level of inflammatory factors decreased slightly, but it was still significantly higher than that of normal tissue. The DPs@MMT group showed the best efficacy among the three treatment groups, with the greatest reduction in inflammatory factors in the colonic tissue after treatment, similar to the secretion level of inflammatory factors in normal tissue.

[0104] To further demonstrate the anti-inflammatory effect of DPs@MMT, M1 and M2 macrophages in colonic tissue were labeled with iNOS and CD163 antibodies, respectively. The results are as follows: Figure 25 As shown. From Figure 25 It can be seen that after DSS induction, the untreated colonic tissue exhibited abundant red fluorescence while green fluorescence was almost absent, indicating severe inflammation within the colonic tissue. Simultaneously, after DPs@MMT treatment, the red fluorescence in the colonic tissue largely disappeared while abundant green fluorescence was observed, demonstrating that the inflammation within the colonic tissue had largely subsided. This proves that DPs@MMT can transform M1 macrophages into M2 macrophages, promoting the healing of inflammation in the colon.

[0105] like Figure 26 As shown, the DSS group exhibited abundant green fluorescence, indicating significant oxidative stress damage at the site of colonic inflammation. In contrast, the DPs@MMT group showed a significant reduction in green fluorescence, while the HO-1 expression level in the colonic tissue of the DPs@MMT group was significantly increased. This demonstrates that DPs@MMT treatment can effectively alleviate oxidative stress at the site of colonic inflammation.

[0106] Figure 27 As shown in (a)-(c), the levels of HGB, RBC, and WBC in the blood of mice in each group were within the normal range, proving that neither DPs@MMT nor the H2S released by DPs reached a concentration that would cause poisoning in mice. Finally, H&E staining was performed on the important organs of mice treated with DPs@MMT, as shown in... Figure 27As shown in (d), no signs of inflammation or tissue necrosis were observed in the heart, liver, spleen, lungs, and kidneys of the DPs@MMT group mice, consistent with the H&E stained tissue images of the normal group. This indicates that DPs@MMT does not cause side effects on tissues and organs. In conclusion, DPs@MMT has good biocompatibility and broad application prospects in the treatment of colitis.

[0107] In summary, DSS-induced colonic inflammation presents with abundant ROS, severe goblet cell damage, extensive inflammatory cell infiltration, and significant secretion of inflammatory factors TNF-α, IL-1β, and IL-6. After DPs@MMT treatment, ROS levels in the colonic tissue decreased, goblet cells remained intact, inflammatory cell infiltration was significantly reduced, and the secretion levels of inflammatory factors were significantly decreased. This demonstrates that DPs@MMT has good anti-inflammatory and antioxidant effects, effectively protecting colonic tissue and promoting its healing. Furthermore, DPs@MMT exhibits good biocompatibility, not causing H2S poisoning in mice.

[0108] Based on the above embodiments and animal experiments, the multifunctional drug carrier system constructed in this system, which can target and identify damaged colonic sites and release H2S in response to GSH concentrations in a physiological environment, represents a significant innovation. This system achieves specific targeting of damaged colonic sites and on-demand release of H2S; that is, H2S release only occurs at sites of severe inflamed colonic mucosa damage. This significantly improves therapeutic efficacy and the safety of the entire treatment process. The above descriptions are merely preferred embodiments of the present invention and are not intended to limit the invention. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A dendritic polypeptide nanogel with H2S donor diallyl trisulfide (DATS) electrostatically adsorbed on montmorillonite, comprising a dendritic polypeptide nanogel carrier, wherein the dendritic polypeptide nanogel carrier is POSS-Lys-G3 with a polyhedral oligomeric silsesquioxane (POSS) core and a third-generation lysine dendritic macromolecule as the branching unit, and blank nanogel PDNs obtained by chemical crosslinking with the crosslinking agent 3,3'-dithiodipropionate di(N-hydroxysuccinimide) ester (DSP), characterized in that... H2S donor diallyl trisulfide (DATS) molecules are loaded into the network structure of a dendritic polypeptide nanogel carrier to form DATS@PDNs dendritic polypeptide nanogel. Montmorillonite (MMT) is coated onto the surface of DATS@PDNs dendritic peptide nanogel to form DATS@PDNs@MMT dendritic peptide nanogel. DATS@PDNs@MMT dendritic peptide nanogel can release H2S in the environment of colon inflammation. Montmorillonite (MMT) is encapsulated around the nanogel DATS@PDNs through electrostatic adsorption; The molar ratio of POSS-Lys-G3 and DSP crosslinking is 1:8; The steps for loading the H2S donor diallyl trisulfide (DATS) molecule into the network structure of the dendritic polypeptide nanogel carrier are as follows: S1. Add a methanol solution containing 116 mg DATS dropwise to a methanol solution containing 50 mg PDNs, stir well, and then sonicate for 10 min. S2. The methanol solution finally obtained in S1 is added dropwise to deionized water at a volume ratio of 1:20, and stirred vigorously at 1500 r / min for 24 h at room temperature. S3. Place the final reaction solution obtained in S2 into a 1kD dialysis bag and dialyze it in deionized water. Centrifuge to remove residual methanol and free DATS from the reaction solution. S4. The solution obtained in S3 was freeze-dried to obtain a slightly yellow cotton-like material DATS@PDNs; The steps for coating montmorillonite MMT onto the surface of DATS@PDNs dendritic peptide nanogel are as follows: a. First, while gently stirring for 48 hours, add 3g of MMT powder to 100mL of water to form a mixture of MMT and excipients; the excipients are vanillin, glucose, and sodium saccharin. b. Centrifuge the mixture at 5000 rpm for 2 minutes to remove excipients; c. Add 50 mL of pH 9-10 NaOH solution to dissolve MMT under vigorous stirring, and then use ultrasound to exfoliate MMT into a monolayer structure. d. The solution obtained in step c is freeze-dried to obtain a white flocculent solid product; e. Dissolve the MMT and DATS@PDNs obtained in step d in deionized water to obtain a suspension of MMT and a solution of DATS@PDNs. f. Add the suspension containing MMT dropwise to the DATS@PDNs solution, stir vigorously at room temperature for 4 hours, centrifuge, and filter to obtain the white solid product DATS@PDNs@MMT dendritic peptide nanogel. In step f, the mass ratio of DATS@PDNs to MMT is 1:

20.

2. The dendritic polypeptide nanogel with H2S donor diallyl trisulfide (DATS) electrostatically adsorbed on montmorillonite according to claim 1, characterized in that, The particle size of DATS@PDNs@MMT is 489 nm.

Images