Preparation and application of nanoparticles for effectively treating ulcerative colitis

Abstract

The invention relates to the technical field of food medicines and health care products, in particular to preparation and application of nanoparticles for effectively treating ulcerative colitis, which comprises the following steps: A, taking a diselenide compound as a raw material, and carrying out amidation reaction on the diselenide compound and N-alpha-fluorenylmethoxycarbonyl-N-epsilone-t-butyloxycarboryl-D-lysine to obtain Se-NHBoc; b, the Se-NHBoc is subjected to a deprotection reaction, Boc groups are removed, and Se-NH2 is obtained; and C, carrying out condensation reaction on the Se-NH2 and a carrier, and purifying to obtain the nanoparticles. The ROS-responsive nano-carrier has the beneficial effects that the constructed ROS-responsive nano-carrier is safe in component, has good drug controlled release ability, and can effectively resist gastrointestinal digestion, pH change and thermal degradation.

Description

Technical Field

[0001] The invention relates to the technical field of food, medicine and health care products, and in particular to the preparation and application of nanoparticles for effectively treating ulcerative colitis. Background Art

[0002] The pathogenesis of ulcerative colitis (UC) is complex and has not yet been fully elucidated. Traditional treatments such as oral medications and injections are poorly absorbed in UC patients, making it difficult for drug concentrations to reach the level required for treatment, affecting the treatment effect. Long-term use of drugs may lead to systemic side effects, such as nephrotoxicity, osteoporosis, susceptibility to infection, gastrointestinal bleeding, etc. In addition, some patients develop drug resistance and require higher doses or more frequent medication, which increases the complexity of drug treatment and patient discomfort. Therefore, the development of a drug with good therapeutic effects, low toxicity and side effects, and wide applicability is a current research hotspot. The ROS level in the UC disease site is abnormally elevated. In response to this situation, the development of a targeted delivery system for ROS to efficiently deliver active substances to the inflammatory site is the key to improving drug treatment and reducing toxic side effects.

[0003] Curcumin (CUR) has a wide range of pharmacological activities such as anti-inflammatory, antioxidant, hypolipidemic, antiviral, anti-infective, and anti-tumor, and has low toxicity and few adverse reactions. In recent years, it has become a potential candidate drug for the treatment of various diseases. In addition, many studies have found that CUR can inhibit inflammatory mediators such as TNF-α, IL-6, and interferon-γ (IFN-γ), signal pathway conduction, and affect intestinal flora, and has the potential to treat UC.

[0004] Studies have been conducted on the use of natural polymers to prepare various active compound delivery systems to resist the degradation of active substances during gastrointestinal digestion and improve their bioavailability. However, these delivery systems lack targeting and cannot specifically identify the site of inflammation, thus limiting their therapeutic effects. In addition, there are currently few reports on the synthesis of ROS-responsive delivery carriers based on structural modification of natural polymers. Most of the precursor polymer materials currently used are synthetic high-molecular polymers, which have limited application in the food field. The preparation process of these delivery systems is complicated and requires the addition of multiple organic reagents or toxic reagents, which to a certain extent limits their feasibility in food and clinical applications.

[0005] CN113577299B proposes a ROS-responsive monoclonal antibody oral nanoparticle and a preparation method thereof. This method is an example of using hyaluronic acid to embed infliximab for the treatment of colitis. However, infliximab has side effects such as nausea, vomiting and discomfort. And there is no study on the stability of the nanoparticles, and whether it can be successfully released in vitro.

[0006] CN117919434A proposes a self-frame nano drug delivery system and its preparation method and application. This method is to mix natural polyphenols, functional molecules containing diselenide bonds and hexachlorocyclotriphosphazene monomers, and obtain a nanosystem through polymerization. However, hexachlorocyclotriphosphazene itself is corrosive and has certain preparation risks. And there is no research on the elimination performance of the nanoparticles on ROS, and whether it can be successfully released in vitro.

[0007] In summary, the delivery systems used by existing active substances for the treatment of UC have poor tissue selectivity, the synthetic raw materials have potential safety hazards, cannot be used in the food field, and the preparation process is complicated, which greatly limits the targeted therapeutic application of active substances. Summary of the invention

[0008] The purpose of the present invention is to disclose the preparation and application of nanoparticles for the effective treatment of ulcerative colitis. The present invention mainly uses carboxymethyl cellulose, a raw material permitted by food regulations, introduces selenium, a trace metal element essential to the human body, constructs ROS-responsive nanoparticles (CUR@CMC-Se) containing diselenide, and improves the bioavailability and targeted controlled release performance of curcumin. The CUR@CMC-Se prepared in this study is safe in composition and has good drug controlled release, resistance to gastrointestinal digestion, resistance to pH degradation and thermal degradation. In vivo experimental results show that oral CUR@CMC-Se can effectively reduce the degree of colon inflammation in mice and restore intestinal health. Through exploration, it was found that CUR@CMC-Se can not only promote intestinal health recovery by regulating the expression of inflammatory factors. More importantly, CUR@CMC-Se can regulate the intestinal flora in a disordered state by adjusting the structure of the intestinal flora, enhance the intestinal barrier function, and thus promote the recovery of intestinal health.

[0009] Methods include:

[0010] 1. Construction and characterization of ROS-responsive nanocarrier CMC-Se: ① Construction of CMC-Se ② FTIR analysis ③ DSC analysis ④ Selenium content and element distribution ⑤ Particle size distribution and Zeta potential determination; The results showed that the selenide fragments were successfully connected to carboxymethyl cellulose, and the constructed carrier could form a stable system.

[0011] 2. Investigation of the structural changes of CMC-Se during oxidation: ① FTIR analysis ② Electron energy spectrum analysis ③ DSC analysis ④ Particle size distribution and Zeta potential measurement ⑤ TEM morphology observation; CMC-Se can respond in a H2O2 environment and cause structural dissociation.

[0012] 3. Preparation, characterization and functional investigation of CUR@CMC-Se: ① Preparation and characterization ② In vitro oxidation response functional investigation ③ In vitro simulated digestion stability investigation ④ pH stability investigation ⑤ Thermal stability investigation; CUR@CMC-Se has the potential to precisely deliver drugs to high ROS sites in UC and can help CUR resist digestive degradation in the gastrointestinal tract and stability in thermal treatment.

[0013] 4. Investigation of the effect and mechanism of CUR@CMC-Se in treating UC mice: ① Biosafety experiment ② Distribution of ROS-responsive nanocarriers in mice ③ Establishment of UC mouse model ④ Evaluation of UC mouse model ⑤ Evaluation of the effect of CUR@CMC-Se in treating UC mice ⑥ Investigation of the mechanism of CUR@CMC-Se in treating UC; CUR@CMC-Se effectively treats UC through anti-inflammatory effects and regulation of intestinal flora, providing new ideas and methods for the treatment of related diseases.

[0014] This study designed a new treatment strategy for UC. By constructing a ROS-responsive nanocarrier CMC-Se and loading CUR into it to form CUR@CMC-Se, the precise controlled release of the drug was successfully achieved. In vivo experimental results have shown that oral CUR@CMC-Se can effectively reduce the degree of colon inflammation in mice and restore intestinal health. It was also explored that the possible mechanism is that CUR@CMC-Se promotes intestinal health recovery by regulating the expression of inflammatory factors and the composition of intestinal flora. This study provides new ideas and methods for the treatment of UC and has important clinical application prospects.

[0015] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0016] A method for preparing nanoparticles for effectively treating ulcerative colitis comprises the following steps:

[0017] A. Using diselenide as raw material, it reacts with N-α-fluorenylmethoxycarbonyl-N-epsilon-tert-butyloxycarbonyl-D-lysine to obtain Se-NHBoc;

[0018] B. subjecting the Se-NHBoc to a deprotection reaction to remove the Boc group to obtain Se-NH2;

[0019] C. Carry out condensation reaction between the Se-NH2 and the carrier, and purify the mixture to obtain nanoparticles.

[0020] Preferably, the method comprises the following steps:

[0021] A. In an ice bath and under the protection of an inert gas, the diselenide compound is used as a raw material and dissolved in an amide reaction solvent, then an auxiliary solvent is added, stirred, and then the N-α-fluorenylmethoxycarbonyl-N-epsilon-tert-butyloxycarbonyl-D-lysine is added, stirred and dissolved, and finally a catalyst and a condensing agent are added, stirred and dissolved, and the mixture is transferred to room temperature and stirred to perform the amide reaction, and after purification, the Se-NHBoc is obtained;

[0022] B. dissolving the Se-NHBoc in a deprotection solvent, then adding a deprotection agent to carry out the deprotection reaction to remove the Boc group, and obtaining the Se-NH2 after purification;

[0023] C. Dissolve the carrier in a buffer solvent, then add a condensation solvent to fully dissolve it, add an activation reagent and a condensation reagent, stir, and obtain a carrier solution; dissolve the Se-NH2 in the buffer solvent, then add the condensation solvent to fully dissolve it, and obtain a Se-NH2 solution; slowly add the Se-NH2 solution dropwise to the carrier solution, carry out the condensation reaction, and purify to obtain the nanoparticles.

[0024] Preferably, in step A, the diselenide compound includes selenocystamine dihydrochloride, the inert gas includes nitrogen, the amide reaction solvent includes DMF, the auxiliary solvent, the catalyst includes 1-hydroxybenzotriazole, and the condensation agent includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; in step B, the deprotection solvent includes dichloromethane, and the deprotection agent includes trifluoroacetic acid; in step C, the carrier includes CMC, the buffer solvent includes MES, the condensation solvent includes DMSO, the activation reagent includes NHS, and the condensation reagent includes EDCI.

[0025] In step A, DMF and N-methylmorpholine are used as solvents at the same time. In the presence of both, Fmoc can be effectively inhibited from participating in the reaction, the generation of diketopiperazine can be reduced, and the occurrence of side reactions can be reduced.

[0026] Preferably, in step A, the mass volume ratio of the diselenide compound, the amide reaction solvent, the auxiliary solvent, the N-α-fluorenylmethoxycarbonyl-N-epsilon-tert-butyloxycarbonyl-D-lysine, the catalyst, and the condensation agent includes 5-10g: 100-200mL: 5-10mL: 15-20g: 2-4g: 5-15g; in step B, the volume ratio of the deprotection solvent to the deprotection agent includes 1:4; in step C, the volume ratio of the buffer solvent to the condensation solvent includes 5:1, the concentration of the buffer solvent includes 50-100mM, the concentration of the carrier in the carrier solution includes 1wt%, the concentration of the activation reagent includes 50-100mM, the concentration of the condensation reagent includes 100-200mM, and the concentration of the Se-NH2 solution includes 1wt%.

[0027] Preferably, the method comprises the following steps:

[0028] A. In an ice bath at 0°C and under the protection of the inert gas, the diselenide compound is used as a raw material, dissolved in an amide reaction solvent, and stirred for 10 minutes. Then, the auxiliary solvent is added, stirred for 5 minutes, and then the N-α-fluorenylmethoxycarbonyl-N-epsilon-tert-butyloxycarbonyl-D-lysine is added, stirred and dissolved for 10 minutes. Finally, the catalyst and the condensing agent are added, stirred and dissolved for 20 minutes, and the mixture is transferred to room temperature and stirred overnight to perform the amide reaction. After purification, the Se-NHBoc is obtained;

[0029] B. Dissolving the Se-NHBoc in the deprotection solvent, then adding the deprotection agent, and performing the deprotection reaction for 6 hours to remove the Boc group. After purification, the Se-NH2 is obtained and stored at 4°C for future use;

[0030] C. Dissolve the carrier in the buffer solvent, adjust the pH to 5.5, then add the condensation solvent to fully dissolve for 12 hours, add the activation reagent and the condensation reagent, stir for 2 hours, and obtain the carrier solution; dissolve the Se-NH2 in the buffer solvent, adjust the pH to 5.5, then add the condensation solvent to fully dissolve for 12 hours, and obtain the Se-NH2 solution; slowly add the Se-NH2 solution dropwise to the carrier solution to carry out the condensation reaction, the reaction temperature includes room temperature, the reaction time includes 24 to 28 hours, and purify to obtain the nanoparticles.

[0031] Preferably, in step A, the purification operation includes one or more of extraction, drying, washing, and precipitation; in step B, the purification operation includes one or more of neutralization, filtration, and drying; in step C, the purification operation includes one or more of dialysis and freeze-drying.

[0032] Preferably, in step A, the purification operation includes: adding ethyl acetate and water for extraction, collecting the upper organic phase, and then continuing to add ethyl acetate to the lower aqueous phase for extraction, repeating 3 times, discarding the aqueous phase, collecting the upper organic phase together, transferring it to a rotary evaporator and rotary evaporating it until it is almost dry, then rinsing it twice with a saturated sodium chloride solution to remove impurities, then adding anhydrous sodium sulfate to remove residual water, then adding dichloromethane until it is just dissolved, then adding methanol until the solution just becomes turbid, standing for 24 hours to precipitate and filtering to obtain the Se-NHBoc;

[0033] In step B, the purification operation includes: adding saturated sodium bicarbonate solution for neutralization until no bubbles emerge, that is, the reaction is completed, filtering and drying to obtain the Se-NH2;

[0034] In step C, the purification operation includes: using a dialysis bag with a molecular weight cutoff of 10,000 Da, dialyzing in ultrapure water for 3 days, changing the water every 4 hours to remove unreacted small molecule impurities, and freeze-drying for 3 days to obtain the nanoparticles.

[0035] A nanoparticle effective in treating ulcerative colitis obtained by the preparation method of the nanoparticle effective in treating ulcerative colitis.

[0036] An application of the above nanoparticles that are effective in treating ulcerative colitis is used to prepare a preparation for targeted delivery of ROS.

[0037] Preferably, it is used for preparing a preparation for treating ulcerative colitis.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] The beneficial effects of the present invention are that the constructed ROS-responsive nanocarrier has safe ingredients, good drug controlled release ability, and can effectively resist gastrointestinal digestion, pH changes and thermal degradation. In addition, it can significantly reduce the degree of colon inflammation in mice, restore intestinal health, and promote the healthy recovery of the intestine by regulating the expression of inflammatory factors. More importantly, the study found that the ROS-responsive nanoparticles also showed significant effects in regulating intestinal flora, promoting the recovery of intestinal inflammatory sites from the perspective of flora composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is the reaction equation of the present invention.

[0041] Figure 1-1 Structural characterization of CMC-Se. (A) FTIR image; (B) 1 H NMR image; (C) DSC image; (D) Selenium element distribution spectrum; (E) Particle size distribution and Zeta potential.

[0042] Figure 1-2 The structural changes of CMC-Se after oxidation. (A) FTIR image; (B) XPS image; (C) DSC image; (D) particle size distribution and Zeta potential; (E) TEM image.

[0043] Figure 2-1 Preparation and characterization of CUR@CMC-Se. (A) FTIR image; (B) DSC image; (C) particle size distribution and Zeta potential.

[0044] Figure 2-2 is the CUR standard curve.

[0045] Figure 2-3 In vitro oxidation response function and in vitro simulated gastrointestinal digestibility of CUR@CMC-Se (A) Release curve of CUR under different H2O2 concentrations. ****P<0.0001; (B) Particle size distribution and Zeta potential after treatment with 10 mM H2O2 for different time periods; (C) SEM and TEM images; (D) CUR retention rate after in vitro simulated digestion; (E) Particle size distribution and Zeta potential before and after in vitro simulated digestion.

[0046] Figure 2-4 Stability of CUR@CMC-Se (A) CUR retention rate after incubation at different pH environments for 6 hours. ****P<0.0001; (B) CUR retention rate after incubation at different temperatures for 6 hours; (C) TEM images of CUR@CMC-Se morphology after treatment at different pH for 6 hours; (D) Changes in particle size and Zeta potential at different temperatures.

[0047] Figure 3-1 Biosafety experiment (A) Organ coefficient. (B) Number of immune cells; (C) Serum biochemical indexes of liver and kidney function; (D) H&E staining results of various organs of mice, ns means no statistical significance.

[0048] Figure 3-2 The distribution of CMC-Se-Cy5.5 in mice in the Control group and DSS group. (a) Distribution of colon fluorescence signals at different time periods; (b) Distribution of mouse organ fluorescence signals at different time periods; (c) Average fluorescence signal intensity of colon tissue at different time periods. *P<0.05, **P<0.01, ns indicates no statistical significance.

[0049] Figure 3-3The recovery of UC symptoms in each group of mice. (a) Percentage of body weight change; (b) Spleen coefficient; (c) DAI score; (d) Colon tissue pathology score; (e) ROS content in colon tissue. *P<0.05, **P<0.01, ****P<0.001, ns indicates no statistical significance.

[0050] Figure 3-4 To investigate the effect and mechanism of CUR@CMC-Se in treating UC mice (A) Recovery of colon length in each group of mice (a) Colon length statistics; (b) Colon photos. *P<0.05, **P<0.01; (B) H&E staining of colon tissue in each group; (C) ZO-1 immunohistochemical staining analysis of colon tissue in each group; (D) Relative expression of inflammatory-related factors in colon tissue in each group *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0051] Figure 3-5 (A) Alpha diversity analysis (a) Observed OTUs; (b) Simpson index; (c) Shannon index; (d) Chao index; (B) Beta diversity analysis. (a) Principal component analysis (PCA); (b) Principal coordinate analysis (PCoA); (c) Non-metric multidimensional scaling (NMDS). *P<0.05, **P<0.01, ***P<0.001.

[0052] Figure 3-6 (A) Species level analysis a) Based on species level Venn diagram; (b) Based on phylum level community composition diagram; (B) Species difference analysis. (a) Circos diagram; (b) Species difference analysis between groups.

[0053] Figure 4 These are comparative experimental diagrams of Example 2. (A) Structural comparison diagram; (B) ROS responsiveness sensitivity comparison diagram; (C) Digestion resistance comparison diagram. DETAILED DESCRIPTION

[0054] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0055] Example 1

[0056] 1Construction and characterization of ROS-responsive nanodelivery system (CMC-Se)

[0057] 1.1 Construction of CMC-Se

[0058] like Figure 1As shown, selenocystamine dihydrochloride is used as a raw material to undergo an amide reaction with N-α-fluorenylmethoxycarbonyl-N-epsilon-tert-butyloxycarbonyl-D-lysine to obtain Se-NHBoc, and then trifluoroacetic acid is used to remove the Boc groups at both ends as protection to obtain the final product Se-NH2; finally, EDCI is used as a condensation reagent and MES is used as a buffer solvent. After Se-NH2 and CMC react at room temperature for 24 hours, the target product (CMC-Se) is prepared by dialysis, and a light yellow fibrous solid CMC-Se is obtained after freeze-drying.

[0059] The specific operations are as follows:

[0060] Synthesis of Se-NHBoc: Except for the instructions, all reactions in this step were carried out in an ice bath at 0℃ and nitrogen. Take 5g of selenocystamine dihydrochloride and dissolve it in 100mL of DMF and stir for 10min. After fully dissolved, add 5.7mL of N-methylmorpholine and stir for 5min. Then add 17.82g of N-α-fluorenylmethoxycarbonyl-N-epsilon-tert-butyloxycarbonyl-D-lysine and stir and dissolve for 10min. Finally, add 2.332g of 1-hydroxybenzotriazole and 9.933g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, stir for 20min, and then transfer to room temperature and stir overnight.

[0061] After the reaction is completed, add appropriate amounts of ethyl acetate and water for extraction, collect the upper organic phase, then continue to add ethyl acetate to the lower aqueous phase for extraction, collect the upper organic phase together, repeat this step 3 times, discard the aqueous phase, and further process the collected organic phase.

[0062] The collected organic phase was transferred to a rotary evaporator and evaporated until almost dry, then rinsed twice with a saturated sodium chloride solution to remove impurities, and then anhydrous sodium sulfate was added to remove residual moisture. Then, an appropriate amount of dichloromethane was added to the reactant until it was just dissolved, and then an appropriate amount of methanol was added until the solution just became turbid, and then filtered after standing for 24 hours. The product was a light yellow solid, and 10.287 g of the product was obtained, with a yield of 52.69%.

[0063] Verification was performed by silica gel chromatography, using dichloromethane:methanol = 20:1 as the elution system, and climbing the plate to verify whether the obtained Se-NH-Boc was the target product.

[0064] Dissolve the purified Se-NHBoc in dichloromethane until it is completely dissolved, then add trifluoroacetic acid 4 times the volume of dichloromethane, and react for 6 hours. Finally, add saturated sodium bicarbonate solution to neutralize the remaining trifluoroacetic acid in the solution until no bubbles emerge, indicating that the reaction is complete. Filter, dry, and store at 4°C for later use.

[0065] First, 2g CMC was dissolved in 50mM MES buffer (pH 5.5) at a ratio of 1wt%, and 1 / 5 volume of DMSO was added to fully dissolve for 12h. Subsequently, 50mM NHS and 100mM EDCI were added and stirred for 2h. Finally, 500mg of selenide fragments were dissolved in a solution of the same system as CMC, and then slowly added dropwise to the CMC solution, and reacted at room temperature for 24h. In a dialysis bag with a molecular weight cutoff of 10000Da, dialyzed in ultrapure water for 3d, and the water was changed every 4h to remove unreacted small molecule impurities. After freeze drying for 3 days, a light yellow fibrous solid CMC-Se was obtained.

[0066] 1.2 Structural characterization of CMC-Se

[0067] Fourier Transform Infrared Spectrometer (FTIR) NICOLET IS50 (Thermo Fisher Scientific Co., Waltham, MA, USA) was used to analyze CMC powder and freeze-dried product CMC-Se. ATR mode was selected, the number of scans was set to 32, and the resolution was 4 cm -1 , the wave number scanning range is 4000~400cm -1 .

[0068] The CMC powder and the freeze-dried product CMC-Se were dissolved in D2O and placed in a nuclear magnetic resonance tube. The samples were tested using an AVANCE nuclear magnetic resonance spectrometer (NMR) (Brucker Biospin, Germany) at 400 MHz, 25°C, and 32 scans. 1 H and 13 C.

[0069] The thermal properties of CMC and CMC-Se were analyzed using a TA-50H integrated thermal analyzer (Shimadzu Corporation, Japan). The samples were sealed in an aluminum pan, and the temperature program was set from 30°C to 600°C, the heating rate was 10°C / min, and the nitrogen flow rate was 20mL / min. A control empty pan under the same heating conditions was used as a reference.

[0070] Take an appropriate amount of CMC-Se solid for acid dissolution pretreatment. After the treatment, use inductively coupled plasma-optical emission spectrometry (ICP-OES) to determine the selenium content in the sample.

[0071] Transmission electron microscope (TEM) and energy dispersive X-ray spectrometry (EDX) were used to analyze the elemental distribution of selenium.TEM sample preparation: CMC-Se aqueous solution was dropped on the copper mesh, and after the nanoparticles were adsorbed and deposited for 30 minutes, the excess nano solution was absorbed with filter paper, and dried in an oven at 45°C. The morphology of CMC-Se in aqueous solution was observed by transmission electron microscopy.

[0072] Based on the dynamic light scattering (DLS) technology, the particle size and Zeta potential of CMC-Se were measured using a nanoparticle size potentiometer. The specific experimental conditions and parameters were set as follows: CMC and CMC-Se aqueous solutions were diluted to appropriate concentrations with ultrapure water before measurement. The operating temperature of the measurement was set at 25°C, and 6 parallels were used.

[0073] The results are shown in Table 1-1 and Figure 1-1 shown.

[0074] Table 1-1 Selenium (Se) content

[0075]

[0076] like Figure 1-1 As shown in A, three new characteristic peaks appeared in CMC-Se modified by Se-NH2: 1690.88 cm -1 The characteristic peak of the amide bond C=O at the Fmoc group is at 1644.59 cm -1 , NH bond and CN bond appear at 1539cm -1 and 1254cm -1 At, and 552.95cm -1 Se-Se, 1233.9cm -1 and 951.8cm -1 These results indicate that Se-NH2 is successfully branched onto CMC.

[0077] from Figure 1-1As can be seen in B, characteristic peaks belonging to Se-NH2 (3.25ppm, 1.5ppm, 1ppm) appear in the spectrum of CMC-Se, indicating that Se-NH2 is successfully branched onto CMC.

[0078] like Figure 1-1 C, CMC shows two characteristic peaks during the heating process, of which the endothermic peak at 76°C is the loss of CMC moisture content, and the exothermic peak at 287°C is the splitting of various functional groups and the depolymerization and degradation of the polymer main chain. The CMC-Se curve retains the characteristics of CMC and has three new endothermic peaks, indicating that the appearance of new functional groups leads to changes in the thermal properties of the structure, and CMC is successfully modified by Se-NH2.

[0079] The selenium content of CMC-Se was tested. The ICP-OES test results are shown in Table 1-1. The selenium content of CMC-Se modified with selenide fragments is 46705.1 mg / kg, accounting for 4.67% by mass. EDX was used to explore the distribution of selenium, such as Figure 1-1 D, it can be seen that selenium is mainly concentrated inside the small sphere formed by CMC-Se. The above results prove that selenium is successfully grafted onto CMC, and the carrier can form small spheres in water by self-assembly. The energy spectrum results show that in the process of forming small spheres, the elements of selenide fragments concave toward the center of the small sphere are mainly concentrated inside, which is in line with the carrier design principle.

[0080] like Figure 1-1 E, the average particle size of CMC-Se in the solution is 310.7nm, and the PDI value is 0.332, indicating that CMC-Se has good dispersion and uniform distribution in the solution. According to previous research reports, when the absolute value of the Zeta potential is greater than 25mV, it can be determined that the solution system has good stability. CMC-Se modified with Se-NH2 exhibits a higher negative charge (-35.67mV), indicating that it can form a stable system in the solution.

[0081] 1.3 Study on the structural changes of CMC-Se oxidation

[0082] CMC-Se was dissolved in a PBS solution with a pH of 6.8 at a concentration of 10 mg / mL. H2O2 was used as the active oxygen representative. H2O2 was added to the CMC-Se solution to a concentration of 10 mM. After reacting for 36 hours, the oxidized product (CMC-Se-COOH) was freeze-dried. CMC-Se-COOH was then characterized by FTIR, X-ray photoelectron spectroscopy (XPS), DSC analysis, particle size distribution and Zeta potential determination, and TEM morphology observation. Figure 1-2 shown.

[0083] like Figure 1-2 A, CMC-Se at 552.95cm -1 The disappearance of the characteristic peak at 1690.88cm indicates that the Se-Se bond is completely oxidized. In addition, the oxidation of the Se-Se bond causes the spatial structure of the carrier to change. -1 The C=O at 1714.15 cm -1 The results showed that the structure of CMC-Se changed after being treated with 10 mM H2O2 for 36 h.

[0084] Figure 1-2 B The results show that the exothermic peak of CMC-Se at 287°C decreased significantly after being treated with 10 mM H2O2 for 36 h. The reason may be that the cross-linked cellulose main chain disintegrated after the Se-Se bond was oxidized and broken, resulting in a decrease in the heat released by the main chain dissociation.

[0085] like Figure 1-2 As shown in C, the valence state of Se changes. Based on spin-orbit coupling, the main peak of Se at 55.9 eV is attributed to the lower energy sublevel 3d 5 , while the binding energy of Se-Se in the H2O2 environment jumps to 59.5eV, indicating that Se changes to a higher energy state, which means that Se is oxidized to form selenious acid or oxidation products. XPS results show that Se-Se in CMC-Se can respond to fracture in the H2O2 environment, providing assistance for the dissociation of the carrier structure.

[0086] Figure 1-2 As can be seen in D, the particle size of CMC-Se increases from 310.7nm to 420-4702nm unevenly, and the potential decreases from -35.67mV to -22.32mV. The above results show that after CMC-Se is oxidized by H2O2, the cross-linked structure of CMC connected by Se-NH2 is destroyed and the overall structure becomes loose. The hydrophobic fragments are transformed into hydrophilic fragments, causing CMC-Se to be unable to maintain a spherical structure and thus become irregular clusters. The results of the Zeta potential change show that the amount of charge carried by the polymer decreases, indicating that the system has changed to an unstable state, further proving that CMC-Se has the potential to respond to H2O2 and release.

[0087] from Figure 1-2 As can be seen in E, the carrier without H2O2 treatment forms a spherical structure in the solution, while after H2O2 treatment, it can be observed that the morphology of the carrier changes significantly from a complete sphere to an irregular loose agglomerate.

[0088] Preparation and characterization of ROS-responsive curcumin nanoparticles (CUR@CMC-Se)

[0089] 2.1 Preparation and characterization of CUR@CMC-Se

[0090] 2.1.1 Preparation and characterization

[0091] Weigh a certain amount of CMC-Se and dissolve it in deionized water at a concentration of 10 mg / ml. Weigh a certain amount of curcumin and dissolve it in acetone at room temperature at a concentration of 10 mg / ml. Add an appropriate amount of curcumin acetone solution to 10 ml of CMC-Se solution and treat it ultrasonically. Remove the residual acetone in the system by rotary evaporation (40°C, 100 rpm). The sample was further centrifuged at 3000 rpm for 5 min to take the supernatant, and after high-pressure homogenization, a uniform and stable ROS-responsive nanoparticle CUR@CMC-Se loaded with curcumin was obtained. CUR@CMC-Se was then characterized by FTIR, analyzed by DSC, and its particle size distribution and Zeta potential were determined. Figure 2-1 shown.

[0092] like Figure 2-1 A, CUR at 3503cm -1 、1626cm -1 and 1605cm -1 There are three typical peaks at 951.8cm, which correspond to the stretching vibration of OH, the stretching vibration of the inter-ring chain C=C and C=O groups, and the stretching vibration of the aromatic ring C=C. Analysis of the infrared spectrum of CUR@CMC-Se loaded with CUR shows that the characteristic peak signal of CUR disappears, and the peak at 951.8cm -1 The C-Se signal at the site is weakened, which may be due to the π-π stacking effect. The aromatic ring of CUR interacts with the Fmoc group on CMC-Se, resulting in the weakening of the C-Se signal.

[0093] like Figure 2-1 The CUR molecule shown in B has a unique endothermic peak at 185°C, which corresponds to the melting point of the CUR crystal structure. Compared with the single component, CUR@CMC-Se shows different endothermic peaks, retaining the characteristic peaks of CMC-Se at 76°C and 287°C, and the endothermic peak of CUR at 187°C disappears due to the transformation of the crystalline state of CUR into an amorphous state, indicating that CUR is successfully loaded into CMC-Se. The reason may be that the Fmoc group in CMC-Se has a strong π-π stacking effect with the aromatic ring molecules, resulting in a decrease in the crystallinity of CUR and its transformation into an amorphous state, forming a eutectic mixture with CMC-Se.

[0094] like Figure 2-1As shown in Fig. a and Fig. b, the PDI of CUR@CMC-Se is 0.212 and the particle size is 387.12 nm. The potential of CUR@CMC-Se loaded with CUR is -44.35 mV, indicating that a stable composite system is formed after loading CUR. The absolute value of the Zeta potential of CUR@CMC-Se increases to a large extent, which may be due to the fact that CUR mainly binds to CMC-Se through hydrophobic force, and the aggregation tendency between micelles is reduced.

[0095] 2.1.2 Determination of curcumin standard curve

[0096] Using ethanol as solvent, curcumin solutions with gradient concentrations of 1.5 μg / ml, 3.0 μg / ml, 4.5 μg / ml, 6.0 μg / ml, and 7.5 μg / ml were prepared. The absorbance of the six groups of solutions at 430 nm was tested using an ultraviolet-visible spectrophotometer (UV-7600). A curcumin standard curve was plotted with the curcumin solution concentration as the abscissa and the absorbance as the ordinate.

[0097] The standard curve of CUR concentration and its absorbance at 430 nm is as follows Figure 2-2 As shown in the figure, the two showed an obvious linear relationship. It can be seen from the results that the CUR standard curve has good linearity in the range of 1.5-7.5 μg / mL. After linear fitting, the standard curve equation is: y=0.1548x+0.0045(R 2 =0.9999), where x is the CUR concentration (μg / mL), y is the absorbance at 430 nm, and R 2 is the linear regression coefficient. This equation can be used to calculate the CUR content in subsequent studies.

[0098] 2.1.3 Determination of loading rate and encapsulation rate

[0099] Take 0.5 ml of sample CUR@CMC-Se, add 8 ml of ethyl acetate, and ultrasonically crush until curcumin is completely extracted from the nanopolymer. Collect the ethyl acetate layer, dilute it with ethanol to a suitable concentration, measure its absorbance at 430 nm, and substitute it into the standard curve to calculate the total amount of curcumin. Use formula 2-1 and formula 2-2 to calculate its encapsulation efficiency and loading rate.

[0100] Encapsulation efficiency (%, W / W) = total amount of encapsulated curcumin / total amount of added curcumin*100 (2-1)

[0101] Loading rate (%, W / W) = total amount of embedded curcumin / total amount of CMC-Se*100 (2-2)

[0102] Table 2-1 CUR@CMC-Se embedding rate and loading rate

[0103]

[0104] As shown in Table 2-1, in the previous study of this laboratory, it was found that the loading rate of CUR on unmodified CMC was only 0.16%. The embedding rate of CMC-Se modified with Se-NH2 fragment was 97.36%, and the loading rate was 4.87%, which was 30 times higher than that of unmodified CMC. The results showed that the introduction of Se-NH2 fragment can effectively increase the drug loading capacity of CMC and provide help for the oral use of CUR.

[0105] 2.2 Study on the in vitro oxidation response function of CUR@CMC-Se and in vitro simulated gastrointestinal digestibility

[0106] Taking H2O2 as the representative of active oxygen, the centrifugation method was used to study the in vitro release characteristics of CUR@CMC-Se under different concentrations of H2O2. 50mL of sample was placed in PBS buffer with a pH of 1.2 and incubated for 2h. Then the pH was adjusted to 6.8, and different concentrations of H2O2 were added to make the content in the solution reach 0mM / 50μM / 100μM / 200μM, respectively, and placed in a 37℃ shaker with a shaker speed of 100rpm. 2ml of sample was taken out at each time point, centrifuged at 3000rpm for 5min, and the supernatant was taken to detect the CUR content. 2mL of the corresponding PBS solution was added, and the absorbance was measured at 430nm, and the release rate was calculated using formula 2-3. The oxidized sample was named CUR@CMC-Se-H2O2. Then the particle size distribution and Zeta potential of CUR@CMC-Se-H2O2 were measured and the TEM morphology was observed.

[0107] Release rate (%) = curcumin content in release solution / curcumin content in sample * 100 (2-3)

[0108] Simulated gastric fluid (SGF) was prepared by dissolving 0.16 g NaCl, 0.35 g pepsin (enzyme activity = 3000 U / g) in 50 ml deionized water, and the pH was adjusted to 1.2 with HCl. Simulated intestinal fluid (SIF) was prepared by dissolving 0.44 g NaCl, 0.1 g trypsin (enzyme activity = 250000 U / g), 0.6 g bile salt in 50 ml deionized water, and the pH was adjusted to 6.8 with HCl. The sample was mixed with SGF in a ratio of 1:1 and reacted for 120 min (37°C, 100 rpm). After gastric digestion, SIF was added to the mixture in a ratio of 1:1 and reacted for 180 min (37°C, 100 rpm). After the sample was digested, the total digestion mixture including the digestion fluid and nanocomplex was extracted with ethyl acetate and centrifuged (3000 rpm, 5 min, 4°C). The ethyl acetate phase was collected and diluted with ethanol. The concentration of curcumin was measured at 425 nm and the retention rate was calculated using formula 2-4. Then, the particle size and Zeta potential of the digested sample were measured using a nanoparticle size potentiometer. Figure 2-3 shown.

[0109] Retention rate (%) = total amount of curcumin after digestion / total amount of curcumin in sample * 100 (2-4)

[0110] like Figure 2-3 As shown in A, only a small amount of CUR was released from CUR@CMC-Se in a 2-hour simulated gastric acid environment, while it showed a rapid release trend under two concentrations of H2O2. In an environment of pH 6.8 and 200μM H2O2, about 36% of CUR was released into the solution outside the micelles after 3 hours of oxidation treatment, while about 29% of CUR was released into the solution under a 100μM H2O2 environment. In a lower concentration of H2O2 environment, the burst release of curcumin was delayed until 9 hours after treatment. The groups treated with H2O2 showed significant differences compared with those not treated with H2O2. After 36 hours of oxidation treatment, the CUR release rate of the 200μM group reached 80%, the release rate of the 100μM group reached 56%, and the release rate of the 50μM group also reached 48%, while the CUR release of the PBS group after 36 hours was only 26%, which was much lower than the three experimental groups. The results show that CUR@CMC-Se has different sensitive responses to different concentrations of H2O2. The higher the H2O2 concentration, the faster the rate of drug release from CUR@CMC-Se. In the absence of H2O2, CUR@CMC-Se releases drugs only because CMC swells in an environment of pH 6.8. The results show that CUR@CMC-Se helps release drugs more accurately in the inflammatory area, reduces nonspecific drug release, and reduces the risk of drug side effects.

[0111] from Figure 2-3 As can be seen from B a, the particle size of CUR@CMC-Se increases with time. At 24h of oxidation, the particle size of CUR@CMC-Se begins to be unevenly distributed, which may be due to the complete dissociation of the carrier structure to form blocks of different sizes. At the same time, the present invention also performs potential characterization analysis on the potential of CUR@CMC-Se before and after oxidation. After H2O2 stimulation oxidation, the PDI is 0.839, and the absolute value of the Zeta potential decreases and is lower than 25mV, indicating that the system is converted from a stable state to an unstable state, and the aggregation trend between micelles increases, proving that the selenide fragments in the system respond to H2O2, and the selenium fragments are degraded to destroy the cross-linked structure of carboxymethyl cellulose, resulting in the redispersion of the originally aggregated charges, so the potential is on a downward trend.

[0112] from Figure 2-3 As can be seen in C a and c, CUR@CMC-Se presents a regular nearly round spherical structure. After being oxidized by H2O2, Figure 2-3 In C b and d, needle-shaped CUR crystals can be seen precipitating from the carrier, and the structure of the carrier becomes loose and irregular. The above results show that CUR loaded inside CMC-Se can be quickly released in different concentrations of H2O2 environments, and can be stably loaded in an environment without H2O2, indicating that CMC-Se has the function of sensitive response to H2O2.

[0113] like Figure 2-3 As shown in Figure D, the retention curve of CUR in simulated digestion in vitro showed a slow downward trend. After 2 hours of simulated gastric juice digestion, 90% of CUR was retained, and after further 3 hours of simulated intestinal juice digestion, the retention rate of CUR still reached 60%. In previous studies in this laboratory, it was found that free CUR was almost completely degraded during the in vitro simulated digestion process, and only 0.34% was detectable. In this experiment, the CUR@CMC-Se prepared by the present invention effectively protected CUR from degradation during the simulated digestion process, and the retention rate after digestion was increased by 176 times compared with the free CUR.

[0114] like Figure 2-3 E a, b, the particle size of CUR@CMC-Se after digestion increases to about 688.5nm, and although the Zeta potential decreases, it is still -34.15mV and remains in a stable state. The results show that CUR@CMC-Se can maintain the stability of the system during the simulated digestion process.

[0115] 2.3 Study on the stability of CUR@CMC-Se

[0116] The sample was immersed in a PBS solution with pH = 1.2 / 6.8 / 7.4, and 3 mL of the sample was taken every 1 h, and 3 mL of the corresponding PBS solution was added. Centrifugation (3000 rpm, 5 min, 4 ° C), the supernatant was collected, diluted with ethanol, and the CUR content was measured at 430 nm using a UV-visible spectrophotometer, and the CUR retention rate was calculated using formula 2-5. The CUR@CMC-Se digested sample was centrifuged (3000 rpm, 5 min, 4 ° C), and the morphology was observed using TEM after drying.

[0117] Place the CUR@CMC-Se solution of appropriate concentration in a 5 mL centrifuge tube and heat it in a water bath at 25°C, 37°C, 60°C, 80°C and 100°C for 6 hours. Take 2 mL of sample every hour and dilute it with ethanol. Use a UV-visible spectrophotometer to measure the CUR content at 430 nm and use formula 2-6 to calculate the CUR retention rate. Take 1 mL of each sample every hour and use a nanoparticle size potentiometer to measure the particle size and Zeta potential changes of the samples at different times and temperatures. Figure 2-4 shown.

[0118] Retention rate (%) = (total amount of CUR after 6 hours of incubation) / total amount of CUR in the original sample × 100 (2-5)

[0119] Retention rate (%) = (total amount of CUR after heating at each temperature for x hours) / total amount of CUR in the original sample × 100 (2-6)

[0120] from Figure 2-4 It can be found in Figure A that CUR@CMC-Se exhibits the best protection performance in an environment with a pH of 1.2. After 6 hours of treatment, the retention rate of CUR can still reach 95%, while in a more neutral environment, the retention rate of CUR decreases, but still reaches more than 85%. The retention rate in a neutral environment is slightly weaker than that in an acidic environment. This may be because the carboxyl groups of CMC-Se macromolecules dissociate in the water medium and are converted into negatively charged carboxyl ions, resulting in higher electrostatic repulsion, causing the structure of CUR@CMC-Se to swell, and more water molecules enter the interior, resulting in leakage and degradation of CUR.

[0121] like Figure 2-4 C. Through TEM observation, it was found that under the environment of pH 1.2 and 6.8, CUR@CMC-Se swelled and the particle size became larger, but the spherical structure was not destroyed and dissociated, and it was still able to form a stable spherical structure to protect CUR inside. The above results show that CUR@CMC-Se has good stability in the gastrointestinal environment and can reduce the proportion of CUR being degraded during the simulated digestion process. This may be because after modification, CMC still retains its original gastrointestinal digestion resistance, protecting CUR from damage to the gastrointestinal environment.

[0122] The retention rate of curcumin after CUR@CMC-Se was treated at different temperatures for 6 h is shown in Figure 2. Figure 2-4 B. After incubation at 100℃ for 6h, the retention rate of curcumin was only 10%, but the retention rate of CUR could still reach 75% and 48% after incubation for 1h and 2h. After treatment at 60℃ and 80℃ for 6h, it showed good protection performance, and the retention rate of CUR could still reach 83% and 65%. The results showed that CMC-Se significantly improved the thermal stability of CUR, which provided help for the processing and application of CUR@CMC-Se.

[0123] Figure 2-4 D can be observed that the results are consistent with the CUR retention rate. In the 80℃ and 100℃ environments ( Figure 2-4 D a, b), the particle size of CUR@CMC-Se gradually increases from 1h, indicating that the CUR@CMC-Se structure expands under this temperature, and as time goes by, the carrier gradually becomes a loose structure and cannot effectively form an effective protective barrier outside the CUR molecules, resulting in the leakage and degradation of CUR. In an environment below 60℃ ( Figure 2-4 D c), the particle size of CUR@CMC-Se hardly changes, indicating that CUR@CMC-Se has good thermal resistance. The Zeta potential results also further reveal the thermal stability of CUR@CMC-Se, such as Figure 2-4 D f, the absolute value of the potential of CUR@CMC-Se at 60℃, 80℃ and 100℃ decreased, although it was still greater than 25mV and in a stable state, but the results showed that the structure gradually changed from a stable state to an unstable state. However, there was no significant difference between CUR@CMC-Se at 25℃ and 37℃ and it could remain consistent with the initial state ( Figure 2-4 D d, e).

[0124] 3. Study on the effect and mechanism of CUR@CMC-Se in treating UC mice

[0125] 3.1 Biosafety Experiment

[0126] Before using it for treatment, the in vivo safety of CUR@CMC-Se was evaluated. BALB / c mice aged 6-8 weeks were divided into the Control group and the DSS group, with 6 mice in each group, half male and half female. The drug group was gavaged with 25 mg / kg of CUR@CMC-Se every day, and the Control group was gavaged with an equal amount of PBS. Body weight was monitored every day for 10 consecutive days. On the 11th day, the mice were anesthetized and serum was obtained by eye bleeding to test biochemical indicators, and whole blood was collected for routine blood tests. After the mice were euthanized, they were dissected and the heart, liver, spleen, lung, and kidney organs were taken. After washing with PBS, they were fixed with paraformaldehyde, gradient dehydrated, and embedded in paraffin. After making pathological sections, H&E staining was performed, and the histological differences of the organs between the two groups were observed under a microscope. Figure 3-1 shown.

[0127] Serum biochemical tests, routine blood tests and H&E staining of pathological sections were commissioned to Wuhan Saiweier Biotechnology Co., Ltd.

[0128] The organ coefficient is the ratio of the weight of an organ of an experimental animal to its body weight. Under normal circumstances, the ratio of each organ to body weight is relatively constant. When a disease occurs in an animal, the weight of the damaged organ will change accordingly. An increase in the organ coefficient indicates congestion, edema, or hyperplasia of the organ; a decrease in the organ coefficient indicates organ atrophy and other degenerative changes. Therefore, the organ coefficient is one of the indicators for judging the biosafety of drugs. Figure 3-1 A, There was no significant difference in the organ coefficients between the Control group and the CUR@CMC-Se group, indicating that feeding mice with CUR@CMC-Se for 10 consecutive days had no significant effect on the weight of various organs of mice, indicating that CUR@CMC-Se did not cause toxic and side effects on various organs of mice.

[0129] Serum biochemical indicators of liver and kidney function of mice were tested to explore whether CUR@CMC-Se would cause metabolic organ abnormalities. Figure 3-1 As shown in B, there was no significant difference in the liver function indicators alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and albumin (ALB) and the renal function indicators creatinine (CREA) and uric acid (UA) between the two groups of mice. The results showed that the liver and kidney functions of mice orally taking CUR@CMC-Se could remain normal, without causing excessive burden on the liver and kidneys.

[0130] The results are as follows Figure 3-1 C, The number of four immune cells in the blood of the two groups of mice was within the normal range and there was no significant difference. The results showed that the administration of CUR@CMC-Se did not cause abnormal immune response in mice.

[0131] like Figure 3-1As shown in D, no obvious damage and necrosis were observed in the organs of mice in the Control group and the CUR@CMC-Se group, indicating that CUR@CMC-Se has no side effects on organ tissues.

[0132] 3.2 Distribution of ROS response system in mice

[0133] 3.2.1 Mouse husbandry

[0134] Twenty-four 6-8 week old BALB / c male mice (ethics review number: SUMC2023-482, Shantou University Medical College Medical Experiment Animal Ethics Committee) were purchased and fed adaptively in an SPF animal breeding room for one week. The mice were raised normally, and mouse food and water were available for free access. The temperature in the animal room was kept at 25°C, and the relative humidity was controlled at 40%.

[0135] 3.2.2 Study on the distribution of CMC-Se in DSS-induced UC mice

[0136] The mice were divided into two groups, the Control group and the DSS group, with 12 mice in each group, and kept in 6 cages. The DSS group was given drinking water containing 3.5% DSS every day, with free drinking water, 7-10 mL / mouse / day, and new DSS drinking water was replaced every two days for 7 days. After the last day of modeling, the mice were fasted for 24 hours and had free drinking water. The mice were gavaged with Cy5.5-labeled CMC-Se, and killed at 0h, 2h, 6h, 12h, and 24h. The colon tissues and organs (heart, liver, spleen, lungs, and kidneys) were dissected, and the fluorescent signals in the colon were detected using the IVIS in vivo imaging system. The distribution of CMC-Se in the body was observed. Figure 3-2 shown.

[0137] To evaluate the selective accumulation of CMC-Se in the inflamed colon, in vivo and ex vivo imaging was performed using Cy5.5 as a fluorescent probe. Figure 3-2 As shown in a, 2 hours after oral administration, CMC-Se can be seen to be concentrated in the cecum, and the distribution trends in the colon of the two groups are similar. When the oral administration is 6 hours, the fluorescent signal begins to accumulate in the inflamed colon, while the fluorescent signal of the mice in the Control group is basically concentrated in the cecum, and the fluorescent signal only appears in the colon with feces. With the increase in administration time, it can be observed that the distribution area of ​​the fluorescent signal in the colon of the DSS group gradually increases, while the colon of the control group mice still has no observed adhesion distribution of the fluorescent signal. Calculate the fluorescence signal intensity in the colon at 2, 6, 12, and 24 hours. Figure 3-2In c, it can be seen that the fluorescence signal intensity of the DSS group was higher than that of the control group at each time period. After 12 hours, the fluorescence intensity of the colon of the DSS group mice was significantly higher than that of the control group, and the fluorescence signal intensity was similar to that at 2 hours. The results show that CMC-Se-Cy5.5 has the ability to target and adhere to the colon. Further, the present invention studies the distribution of nanoparticles in specific organs, and takes the heart, liver, spleen, lung, and kidney of mice at various time points for in vitro imaging. Figure 3-2 As shown in b, the fluorescence signal of CMC-Se-Cy5.5 was not detected in other organs. The results showed that CMC-Se had good selective accumulation in the UC colon and had no distribution in the healthy colon and other organs, which effectively increased the concentration and retention time of the drug reaching the inflammatory area.

[0138] 3.3.3 Establishment of UC mouse model

[0139] BALB / c mice were selected as experimental mice, and mice were cultured in an SPF-level laboratory. Mice with similar weights were randomly divided into 6 groups: control group, modeling (DSS) group, positive drug (5-ASA) group, pure curcumin (CUR) group, pure carrier (CMC-Se) group, and curcumin nanoparticle (CUR@CMC-Se) group. There were 6 mice in each group. The DSS group was given drinking water containing 3.5% DSS, free drinking water, 7-10mL / mouse / day, and new DSS drinking water was replaced every two days for 7 days. The Control group was given the same volume of distilled water. The DSS group was replaced with distilled water after the seventh day and continued to be raised until the 10th day. The mice in the 5-ASA group were given 200 mg / kg of mesalazine per day, the mice in the CUR group were given 25 mg / kg of CUR per day, the mice in the CMC-Se group were given 250 mg / kg of CMC-Se per day, and the mice in the CUR@CMC-Se group were given 25 mg / kg of CUR equivalent per day for 10 days. After the last administration, the mice in each group were fasted but not watered for 12 hours, and serum, spleen and colon tissue were obtained under anesthesia. After measuring the length, the colon tissue was transected 1-2 cm above the anus, washed with PBS, embedded and fixed for pathological sections for subsequent histopathological analysis, and other colon tissues were frozen at -80℃ for later use.

[0140] 3.4 Establishment and evaluation of UC mouse model

[0141] 3.4.1 Mouse weight

[0142] The experiment was conducted after one week of adaptive feeding. The mice in each group were weighed and marked before the experiment. The weight of the mice was then weighed and recorded at a fixed time every day. The formula for calculating the weight change rate of mice is shown in formula (3-1):

[0143] Weight change rate (%) = (weight on day n (g) / (weight on day 1 (g)) - 1) × 100 (3-1)

[0144] 3.4.2 Stool characteristics

[0145] Fresh feed, bedding and DSS drinking water were replaced every 48 h, and feces of mice in each group were collected every 24 h for observation and fecal occult blood test and fecal viscosity scoring.

[0146] 3.4.3 Disease Activity Index (DAI) score

[0147] The symptoms of mice were observed in vivo and the disease activity index was evaluated, as shown in Table 3-1. The DAI score was the sum of the scores of the three indicators (3-2). After scoring, statistical analysis was performed. The success standard of modeling depended on whether there were statistical differences between the groups.

[0148] result Figure 3-3 shown.

[0149] Table 3-1 DAI scoring criteria

[0150]

[0151] DAI score = weight loss (%) + stool status + fecal occult blood (3-2)

[0152] On the 11th day, the mice were killed and colon tissues were obtained. The length of colon tissues of mice in each group was measured using a vernier caliper. The spleens of mice were obtained after dissection, and the spleen coefficient was calculated according to the following formula (3-3).

[0153] Spleen coefficient = spleen weight (g) / mouse weight (g) * 100 (3-3)

[0154] The ROS content in the colon of the Control group, DSS group and CUR@CMC-Se group was determined according to the instructions of the mouse reactive oxygen species (ROS) enzyme-linked immunosorbent assay kit. Three mouse colons were selected in each group for the experiment.

[0155] This experiment used a treatment model of modeling and intervention to study UC mice. Figure 3-3 a, The mice in the DSS group lost weight and had blood in their stools during the first 7 days of modeling. By day 10, the UC symptoms had not improved significantly. To quantify the severity of UC, the present invention scored the changes in feces and occult blood, and calculated the DAI score for each mouse. Figure 3-3b, c, d, e, the results showed that the DAI score, spleen coefficient, and ROS of the mice in the DSS group were significantly higher than those in the control group, further confirming that DSS successfully induced a mouse ulcerative colitis model. In addition, except for the control group and the CUR@CMC-Se group, the other groups began to have blood in the stool on the 4th day of the experiment, while the blood in the stool of the DSS group continued until the 10th day and did not improve. The results show that the present invention successfully established a UC mouse model.

[0156] 3.5 Effect and mechanism of CUR@CMC-Se in treating UC mice

[0157] The fixed colon tissues were stained with H&E and the histopathological scores were performed as shown in Table 3-2.

[0158] Table 3-2 Histopathological scoring criteria

[0159]

[0160] Immunohistochemistry (IHC) is an antibody-based technique used to detect protein expression in tissues whose structure and organization have been preserved. After embedding and fixing, the pathological sections are immunostained, prepared and observed under a microscope.

[0161] RNA was extracted according to the instructions of the UE total RNA miniprep kit, and after reverse transcription, RT-qPCR reaction was performed to detect the expression levels of β-actin, TNF-α, IL-1β, MUC3, IL-10, and TGF-β genes. Figure 3-4 shown.

[0162] Next, the present invention evaluated the recovery of mice. Figure 3-3 As can be seen from a and c, administration of 5-ASA, CUR, CMC-Se, and CUR@CMC-Se all effectively maintained the weight of mice and reduced the DAI score. Among them, the DAI of the CUR@CMC-Se group was the lowest, indicating that it can effectively improve the UC condition of mice. The measurements after dissecting the colon showed ( Figure 3-4 Aa, b), the colon length of mice in the CUR@CMC-Se group recovered well, and there was a significant difference compared with the colon of the DSS group. Here, the present invention further detected indicators related to inflammation. The spleen, as the largest immune and lymphoid organ of mice, plays a vital role in the immune response involving lymphocytes and macrophages. The spleen coefficient is a manifestation of inflammation in mice. When inflammation occurs, the spleen of mice will increase in weight and hypertrophy. Figure 3-3 b It can be seen that the spleen coefficient of mice treated with CUR@CMC-Se was significantly reduced, indicating that the inflammation in the mice was alleviated. Figure 3-3e The results showed that CUR@CMC-Se could effectively eliminate the abnormally elevated ROS in UC mice and restore it to a level similar to that of healthy mice.

[0163] Figure 3-4 The results of B a and b showed that the number of epithelial cells and goblet cells in the colon tissue of mice in the DSS group was significantly reduced, severe tissue destruction and inflammatory infiltration occurred on the mucosal surface and the submucosa of the colon, and the crypt upward structure was atrophied and distorted. The pathological scores of mice in the DSS group ( Figure 3-3 c, d) were significantly higher than those in the control group, indicating that the colon of mice in the DSS group was severely inflammatory. The colon crypt atrophy of mice in the oral CUR@CMC-Se group was restored, the number of goblet cells increased, the area of ​​inflammatory infiltration decreased, the submucosal layer became denser, and the inflammatory condition was alleviated ( Figure 3-4 B f). In contrast, although the other groups showed some improvement, a large number of inflammatory infiltration areas were still observed, the crypt surface was irregularly broken, the abnormal crypt structure was not repaired, and the epithelial cells were abnormal ( Figure 3-4 B c, d, e). These results indicate that CUR@CMC-Se loaded with CUR can effectively reverse the colon damage in mice. After significant difference analysis of H&E staining scores, it was found that there was no significant difference between the CUR@CMC-Se group and the control group, indicating that CUR@CMC-Se can significantly alleviate the symptoms of UC in mice and help promote intestinal damage repair.

[0164] Intestinal barrier damage is one of the main pathological features of UC. ZO-1 is an essential tight junction protein for the epithelial barrier and mainly appears on the cell membrane. It plays an important role in protecting intestinal health. Therefore, restoring the content of ZO-1 protein in the colon helps to restore the intestinal barrier function and prevent the penetration of harmful microorganisms and substances in the intestine. Figure 3-4 As can be observed in C a and b, the expression of ZO-1 on the goblet cell membrane in the colon tissue of the DSS group was significantly reduced and there was no continuous and complete distribution, which revealed that the connection between cells in the colon of the DSS group mice was destroyed, resulting in functional abnormalities. Except for the CMC-Se group, the expression of ZO-1 in the other groups increased significantly. The expression of ZO-1 in the 5-ASA group and the CUR group basically recovered to a level similar to that of the Control group, but there were still gaps in the distribution of ZO-1 on the cell membrane and it had not yet fully recovered to the state of tight connection ( Figure 3-4 C c, d, e). From the slice results of CUR@CMC-Se group, we can see that ( Figure 3-4 C f), ZO-1 is evenly, continuously and completely distributed on the goblet cell membrane in the colon. The results of immunohistochemical staining showed that CUR@CMC-Se can increase the expression of ZO-1 and the degree of tight junction, which helps to repair the intestinal barrier and restore intestinal health.

[0165] Here, the present invention uses real-time fluorescence quantitative PCR analysis to explore the expression of pro-inflammatory factors (TNF-α, IL-1β), anti-inflammatory factors (IL-10, TGF-β) and mucin 3 (MUC3). Figure 3-4 Da and b show that the expression of TNF-α and IL-1β in the DSS group increased significantly, while the other treatment groups, except 5-ASA, effectively reduced the expression of these two in the intestine of mice. The reason why the level of pro-inflammatory factors in the 5-ASA group did not decrease significantly may be the side effects caused by long-term use. IL-10 and TGF-β are markers of anti-inflammatory factors, such as Figure 3-4 As shown in D d, e, CMC-Se and CUR@CMC-Se can significantly promote the expression of anti-inflammatory factors, and have a synergistic effect on improving the therapeutic effect of CUR. MUC3 is an important mucosal adhesion protein and an important component of the mucosal surface. Its main function is to provide a protective mucus barrier to maintain the moisture and defense of the mucosal surface. Figure 3-4 As can be seen in D c, CUR@CMC-Se increased the expression of MUC3, which helped the intestine to secrete mucin 3 to repair the intestine. Real-time fluorescence quantitative PCR results showed that CUR@CMC-Se could inhibit the expression of pro-inflammatory factors, promote the expression of anti-inflammatory factors and mucosal proteins that help intestinal recovery, and regulate and treat UC mice at the molecular level.

[0166] 3.6 16S rRNA sequencing analysis of intestinal microorganisms

[0167] Before dissecting the mice, collect the mouse feces first, collect 3 pieces of feces from each mouse and place them in a sterile EP tube. The sequencing part was entrusted to Shanghai Meiji Biotechnology Co., Ltd. Specifically, after the genomic DNA is extracted, the purity is detected by 1% agarose gel electrophoresis. According to the specified sequencing area, specific primers with barcodes are synthesized, and PCR amplification is performed using TransStart FastpfuDNA Polymerase. All samples were processed according to the formal experimental conditions, and each sample was repeated 3 times. The PCR products of the same sample were mixed and detected by 2% agarose gel electrophoresis. The PCR products were cut and recovered using the AxyPrepDNA gel recovery kit (AXYGEN), and eluted with Tris_HCl and detected using 2% agarose electrophoresis. Referring to the preliminary quantitative results of electrophoresis, the PCR products were quantified using QuantiFluor TM-ST blue fluorescence quantitative system (Promega) was used for detection and quantification, and then the samples were mixed in the corresponding proportions according to the sequencing requirements of each sample. The library was constructed using the TruSeqTM DNA Sample PrepKit. After the constructed library was qualified, it was sequenced on the machine and then analyzed by bioinformatics.

[0168] Bioinformatics analysis:

[0169] The OUT clustering steps are as follows: extract non-repetitive sequences from the optimized sequences to reduce the amount of redundant calculations in the intermediate analysis process, remove single sequences without repetition, perform OTU clustering on non-repetitive sequences (excluding single sequences) at 97% similarity, remove chimeras during the clustering process, and obtain representative sequences of OTUs. Map all optimized sequences to OTU representative sequences, select sequences with a similarity of more than 97% with OTU representative sequences, and generate an OTU table. In order to obtain the species classification information corresponding to each OTU, the RDP classifier Bayesian algorithm was used to perform taxonomic analysis on the OTU representative sequences at a similarity level of 97%, and the community species composition of each sample was statistically analyzed at each taxonomic level: Domain, Kingdom, Phylum, Class, Order, Family, Genus, and Species. The comparative data of 16s bacteria are as follows: Silva (Release 138 http: / / www.arb-silva.de); RDP (Release 11.5 http: / / rdp.cme.msu.edu / ); Greengene (Release 13.5 http: / / greengenes.secondgenome.com / ); functional gene: FGR, RDP is compiled from the functional gene database of GeneBank (Release 7.3 http: / / fungene.cme.msu.edu / ).

[0170] Data analysis software Mothur (version 1.30.2) was used to calculate Chao1, Shannon, Simpson, and obs of the control group, DSS group, and CUR@CMC-Se group at the OUT level to evaluate alpha diversity. Qiime (version 1.9.1) weighted UniFrac was used to perform principal component analysis (PCA) and principal coordinate analysis (PCoA). PICRUSt2 (version 2.2.0) was used for KEGG and COG functional analysis. R language (Version 3.3.1) was used to classify the species composition of different groups (or samples) at each taxonomic level (such as domain, kingdom, phylum, class, order, family, genus, species, OTU, etc.) based on the results of taxonomic analysis. The dominant species and corresponding relative abundance of each sample at a certain taxonomic level were analyzed. Figure 3-5 and Figure 3-6 shown.

[0171] In recent years, the imbalance of intestinal flora is considered to be crucially related to the onset of UC. DSS-induced UC can cause changes in the abundance and composition of intestinal microorganisms in UC mice. Next, the present invention performed 16S rRNA sequencing on the intestinal flora of mice to explore whether CUR@CMC-Se can restore intestinal health by affecting the intestinal flora of UC mice. Alpha diversity was used to evaluate the diversity and changes in the abundance of microorganisms in the intestine of each group. Figure 3-5 A a, b, c, d Simpson and Shannon indexes represent community diversity. The Chao index results show the abundance of communities in the intestine. As can be seen from the figure, the observed OTUs, Chao1 and Simpson index values ​​of the DSS group were significantly lower than those of the Control group. The results show that the diversity of intestinal flora in DSS-induced colitis mice was destroyed. From the results of the CUR@CMC-Se group, although the community abundance has not yet recovered to the same level as the Control group, the community diversity has recovered and is significantly different from DSS.

[0172] Subsequently, the present invention conducted Beta diversity analysis on the differences between the groups, and the results were as follows: Figure 3-5 As shown in B a, the distances between the three groups are far apart, indicating that there are significant differences in the distribution of microbial communities among the three groups of samples, which may be due to the fact that CUR can reshape the intestinal flora to treat UC. From the PCA and PCoA results ( Figure 3-5B b, c), the CUR@CMC-Se treatment group was able to form a more stable microbial community in the intestine, which was beneficial to maintain the stability of the intestinal flora. The results of Alpha diversity and Beta diversity indicated that CUR@CMC-Se may have changed the composition structure of the intestinal flora of UC mice to restore the diversity of the mice's flora and maintain the stability of the intestinal flora ecology.

[0173] Further analysis of the species composition of each group was performed, such as Figure 3-6 As shown in Aa, at the species level, 78 species were common to the three treatment groups, while 21, 12, and 9 species were unique to the control group, DSS group, and CUR@CMC-Se group, respectively. The community structure components were further analyzed by statistical analysis methods to observe the community structure of different samples at different classification levels, such as Figure 3-6 As shown in A b, at the phylum level, the main flora of the healthy group mice were Bacteroidota and Firmicutes. In the DSS-induced UC mouse model, the abundance of Verrucomicrobiota and Firmicutes increased, and the abundance of Bacteroidota decreased, resulting in an increase in the ratio of Firmicutes / Bacteroidota, indicating that the intestinal flora was in a dysregulated state. On the other hand, the main intestinal flora of the CUR@CMC-Se group changed from Bacteroidota to Verrucomicrobiota, and the abundance of species that rely on external energy for survival decreased. This may be related to the fact that the amount of food intake of UC is reduced, and species that rely on external energy for growth find it difficult to obtain sufficient energy, resulting in a decrease in abundance. Verrucomicrobiota is a type of species that mainly uses mucin in the intestinal mucosa as an energy source and does not rely on external energy. Therefore, the abundance of Verrucomicrobiota is increased in the intestine of UC mice.

[0174] Further analysis of the differences in species composition between the groups revealed Figure 3-6 As can be seen in B a and b, at the genus level, norank_f__Muribaculaceae was the dominant flora in the intestine of the Control group mice, while Akkermansia of Verrucomicrobiota became the dominant flora in the DSS and CUR@CMC-Se groups. Akkermansia is a type of oval Gram-negative bacteria that belongs to intestinal commensal bacteria. The abundance of this bacterium is usually positively correlated with the thickness of the mucus layer. A high abundance of Akkermansia helps the intestine increase mucus secretion, thereby enhancing the intestinal barrier function.

[0175] Akkermansia feeds on mucin in the mucosal layer and does not rely on external food for nutrition. Mucin is the main structural and functional component of intestinal mucus, with a concentration of about 1%-5%. Under normal circumstances, due to the low content of mucin in the intestine, Akkermansia is not a dominant flora in the intestine, while Bacteroidetes plays a role in utilizing and metabolizing external carbohydrates in a healthy intestine.

[0176] However, when UC occurs, the intestinal barrier of mice is damaged, the food intake of mice decreases, and the content of exogenous carbohydrates decreases, resulting in a significant decrease in the number of non-mucus-consuming species. This phenomenon is also reflected in the Beta diversity analysis. Akkermansia can specifically degrade mucin to produce acetate, propionate and ethanol to regulate the host's immune response and lipid metabolism, and provide energy for the host to promote the colonization of other bacteria. The degradation of mucin prompts the host to compensate and produce more mucin to maintain the dynamic balance of these proteins, which is also verified by qPCR data. Recent studies have found that Akkermansia supplementation increases the thickness of the colonic mucus layer by about 3 times, which is significantly higher than the increase in thickness caused by the beneficial bacteria Lactobacillus plantarum. In addition, Akkermansia can also activate the TLR2 pathway through the AMPK signaling pathway, change the expression of tight junction proteins occludin and ZO-1, thereby regulating intestinal permeability and helping mice restore damaged intestinal barriers. In addition, the increase in the abundance of Akkermansia may be due to its ability to secrete a large amount of sulfatase to help mice metabolize and degrade ingested DSS, thereby helping mice resist DSS damage to the intestine.

[0177] At the same time, the abundance of Erysipelatoclostridium, which is usually identified as a pathogen in the intestine, was significantly increased in the DSS group, while the abundance of Erysipelatoclostridium in the intestine of mice treated with CUR@CMC-Se was effectively inhibited. Rombutsia, which can produce SCFAs, was specifically increased in CUR@CMC-Se, which may help the host restore intestinal health.

[0178] Example 2

[0179] CN118340908A proposes a method for preparing a highly sensitive ROS-responsive carboxymethyl cellulose and its application in the delivery of active substances. The method selects one or more of sodium carboxymethyl cellulose, sodium alginate, hyaluronic acid and pectin, and grafts selenide on a carrier as a nanocarrier to embed drugs.

[0180] According to the method of CN118340908A, the corresponding ROS response material was prepared and compared with the nanoparticles of the present invention. The results are as follows Figure 4 shown.

[0181] Compare Figure 4 It can be seen that compared with CN118340908A, the structure proposed in this study is more stable (Figure A), and the ROS responsiveness is more sensitive (Figure B), and it performs better in resisting digestion (Figure C). First of all, in terms of preparation method and nanoparticle structure, the nanoparticles of this study are very different from the nanoparticles proposed by CN118340908A (Figure A). The active group of this study is a diselenide structure. The nanoparticles of this study can release curcumin by 40% after 6 hours under 100μM hydrogen peroxide conditions; while the nanoparticles proposed by CN118340908A need to react for the same time under 250μM hydrogen peroxide to achieve similar effects, indicating that the nanoparticles of this study are more sensitive to ROS responsiveness. In addition, from the perspective of resisting gastrointestinal digestion, the retention rates of curcumin in the nanoparticles of CN118340908A were 57.42% and 41.39% respectively after 2 hours of simulated gastric and intestinal digestion. After 2 hours of simulated gastric digestion, the retention rate of curcumin in the nanoparticles of this study was as high as 90%, and after 3 hours of simulated intestinal digestion, the retention rate was still 60%. This shows that the nanoparticles of this study have significantly improved their ability to resist digestion.

[0182] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A method for preparing nanoparticles effective in treating ulcerative colitis, characterized in that: The following steps are involved: A. Using diselenide as raw material, it reacts with N-α-fluorenylmethoxycarbonyl-N-epsilon-tert-butyloxycarbonyl-D-lysine to obtain Se-NHBoc; B. subjecting the Se-NHBoc to a deprotection reaction to remove the Boc group to obtain Se-NH2; C. Carry out condensation reaction between the Se-NH2 and the carrier, and purify the mixture to obtain nanoparticles.

2. The method for preparing nanoparticles effective for treating ulcerative colitis according to claim 1, characterized in that: The following steps are involved: A. In an ice bath and under the protection of an inert gas, the diselenide compound is used as a raw material and dissolved in an amide reaction solvent, then an auxiliary solvent is added, stirred, and then the N-α-fluorenylmethoxycarbonyl-N-epsilon-tert-butyloxycarbonyl-D-lysine is added, stirred and dissolved, and finally a catalyst and a condensing agent are added, stirred and dissolved, and the mixture is transferred to room temperature and stirred to perform the amide reaction, and after purification, the Se-NHBoc is obtained; B. dissolving the Se-NHBoc in a deprotection solvent, then adding a deprotection agent to carry out the deprotection reaction to remove the Boc group, and obtaining the Se-NH2 after purification; C. Dissolve the carrier in a buffer solvent, then add a condensation solvent to fully dissolve it, add an activation reagent and a condensation reagent, stir, and obtain a carrier solution; dissolve the Se-NH2 in the buffer solvent, then add the condensation solvent to fully dissolve it, and obtain a Se-NH2 solution; slowly add the Se-NH2 solution dropwise to the carrier solution, carry out the condensation reaction, and purify to obtain the nanoparticles.

3. The method for preparing nanoparticles effective for treating ulcerative colitis according to claim 2, characterized in that: In step A, the diselenide compound includes selenocystamine dihydrochloride, the inert gas includes nitrogen, the amide reaction solvent includes DMF, the auxiliary solvent includes N-methylmorpholine, the catalyst includes 1-hydroxybenzotriazole, and the condensation agent includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; in step B, the deprotection solvent includes dichloromethane, and the deprotection agent includes trifluoroacetic acid; in step C, the carrier includes CMC, the buffer solvent includes MES, the condensation solvent includes DMSO, the activation reagent includes NHS, and the condensation reagent includes EDCI.

4. The method for preparing nanoparticles effective for treating ulcerative colitis according to claim 1, characterized in that: In step A, the mass volume ratio of the diselenide compound, the amide reaction solvent, the auxiliary solvent, the N-α-fluorenylmethoxycarbonyl-N-epsilon-tert-butyloxycarbonyl-D-lysine, the catalyst, and the condensation agent includes 5-10g:100-200mL:5-10mL:15-20g:2-4g:5-15g; in step B, the volume ratio of the deprotection solvent to the deprotection agent includes 1:4; in step C, the volume ratio of the buffer solvent to the condensation solvent includes 5:1, the concentration of the buffer solvent includes 50-100mM, the concentration of the carrier in the carrier solution includes 1wt%, the concentration of the activation reagent includes 50-100mM, the concentration of the condensation reagent includes 100-200mM, and the concentration of the Se-NH2 solution includes 1wt%.

5. The method for preparing nanoparticles effective for treating ulcerative colitis according to claim 1, characterized in that: The following steps are involved: A. In an ice bath at 0°C and under the protection of the inert gas, the diselenide compound is used as a raw material, dissolved in an amide reaction solvent, and stirred for 10 minutes. Then, the auxiliary solvent is added, stirred for 5 minutes, and then the N-α-fluorenylmethoxycarbonyl-N-epsilon-tert-butyloxycarbonyl-D-lysine is added, stirred and dissolved for 10 minutes. Finally, the catalyst and the condensing agent are added, stirred and dissolved for 20 minutes, and the mixture is transferred to room temperature and stirred overnight to perform the amide reaction. After purification, the Se-NHBoc is obtained; B. Dissolving the Se-NHBoc in the deprotection solvent, then adding the deprotection agent, and performing the deprotection reaction for 6 hours to remove the Boc group. After purification, the Se-NH2 is obtained and stored at 4°C for future use; C. dissolving the carrier in the buffer solvent, adjusting the pH to 5.5, adding the condensation solvent to fully dissolve for 12 hours, adding the activation reagent and the condensation reagent, stirring for 2 hours, and obtaining the carrier solution; The Se-NH2 is dissolved in the buffer solvent, the pH is adjusted to 5.5, and then the condensation solvent is added to fully dissolve for 12 hours to obtain the Se-NH2 solution; the Se-NH2 solution is slowly added dropwise to the carrier solution to carry out the condensation reaction, the reaction temperature includes room temperature, the reaction time includes 24 to 28 hours, and purification is carried out to obtain the nanoparticles.

6. The method for preparing nanoparticles effective for treating ulcerative colitis according to claim 2, characterized in that: In step A, the purification operation includes one or more of extraction, drying, washing, and precipitation; in step B, the purification operation includes one or more of neutralization, filtration, and drying; in step C, the purification operation includes one or more of dialysis and freeze-drying.

7. The method for preparing nanoparticles effective for treating ulcerative colitis according to claim 2, characterized in that: In step A, the purification operation includes: adding ethyl acetate and water for extraction, collecting the upper organic phase, and then continuing to add ethyl acetate to the lower aqueous phase for extraction, repeating 3 times, discarding the aqueous phase, collecting the upper organic phase together, transferring it to a rotary evaporator and rotary evaporating it until it is almost dry, then rinsing it twice with a saturated sodium chloride solution to remove impurities, then adding anhydrous sodium sulfate to remove residual water, then adding dichloromethane until it is just dissolved, then adding methanol until the solution just becomes turbid, standing for 24 hours to precipitate and filtering to obtain the Se-NHBoc; In step B, the purification operation includes: adding saturated sodium bicarbonate solution for neutralization until no bubbles emerge, that is, the reaction is completed, filtering and drying to obtain the Se-NH2; In step C, the purification operation includes: using a dialysis bag with a molecular weight cutoff of 10,000 Da, dialyzing in ultrapure water for 3 days, changing the water every 4 hours to remove unreacted small molecule impurities, and freeze-drying for 3 days to obtain the nanoparticles.

8. Nanoparticles effective in treating ulcerative colitis obtained according to the method for preparing nanoparticles effective in treating ulcerative colitis according to claim 1.

9. A use of the nanoparticles for effectively treating ulcerative colitis according to claim 8, characterized in that: Formulations for the preparation of targeted delivery of ROS.

10. The use of nanoparticles for effectively treating ulcerative colitis according to claim 9, characterized in that: Used for preparing preparations for treating ulcerative colitis.

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