Osalazine-loaded hollow mesoporous prussian blue / polydopamine nano-enzyme as well as preparation method and application thereof

Abstract

The invention relates to an oxsalazine-loaded hollow mesoporous prussian blue / polydopamine nano-enzyme as well as a preparation method and application thereof, and belongs to the technical field of material synthesis and molecular biology. Osalazine is loaded in hollow mesoporous Prussian blue (HMPB), and the surface of the hollow mesoporous Prussian blue is coated with a polydopamine layer. The composition is used for promoting IBD treatment. The drug loading system has good peroxidase activity, can quickly release drugs in an inflammatory environment for a long time, plays effective anti-inflammatory and anti-oxidation roles in IBD treatment, and has good biological safety.

Description

Technical Field

[0001] The present invention belongs to the technical fields of material synthesis and molecular biology, and particularly relates to a hollow mesoporous Prussian blue / polydopamine nanozyme loaded with olsalazine, a preparation method thereof, and an application thereof. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the overall understanding of the present invention, and does not necessarily constitute an admission or imply in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Inflammatory bowel disease (IBD) is a chronic gastrointestinal inflammatory disease with a high recurrence rate and complex pathogenic causes, and is clinically divided into Crohn's disease and ulcerative colitis. Patients with IBD are troubled by symptoms such as abdominal pain, diarrhea, and bloody stools in their daily lives, and are also accompanied by a further risk of canceration. However, due to its complex pathogenic mechanism, there is still a lack of reliable and effective treatment means. The core of the pathological mechanism of IBD is the formation of reactive oxygen species (ROS) and reactive nitrogen species, which can stimulate the overproduction of inflammatory cytokines, thereby exacerbating oxidative stress / nitrosative stress. Recent studies have explored the use of antioxidant natural small molecules / proteins and synthetic inorganic / polymeric nanomaterials to regulate immune balance, providing a new antioxidant treatment strategy for the treatment of IBD.

[0004] Prussian blue (PB) has been approved by the Food and Drug Administration as an antidote for the treatment of radioactive element thallium and cesium poisoning. PB has superoxide dismutase (SOD), catalase, and peroxidase mimetic enzyme activities, and has extremely high biosafety. It has been reported in the literature that in a dextran sulfate sodium (DSS)-induced colitis mouse model, PB nanoparticles (NPs) can scavenge ROS in the intestine and alleviate intestinal lesions.

[0005] Polydopamine (PDA) is a natural polymer inspired by mussels. Multiple functional groups on PDA, such as catechol and quinone groups, endow it with excellent redox ability. Summary of the Invention

[0006] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a hollow mesoporous Prussian blue / polydopamine nanozyme loaded with olsalazine, its preparation method and application, which is formed by coating the clinical anti-colitis drug olsalazine (Olsa) with hollow mesoporous Prussian blue (HMPB) / PDA, and is used to promote the treatment of IBD. This drug delivery system has good peroxidase activity, can rapidly and long-term release drugs in the inflammatory environment, plays an effective anti-inflammatory and antioxidant role in the treatment of IBD, and has good biosafety.

[0007] In order to achieve the above object, the technical solution of the present invention is as follows:

[0008] In the first aspect, a hollow mesoporous Prussian blue / polydopamine nanozyme loaded with olsalazine (Olsa@HMPB / PDANPs) includes: hollow mesoporous Prussian blue particles (HMPBNPs), wherein the hollow mesoporous Prussian blue particles (HMPBNPs) are loaded with olsalazine (Olsa), and the surface of the hollow mesoporous Prussian blue particles (HMPB NPs) is coated with a polydopamine (PDA) layer.

[0009] Optionally, the particle size of the hollow mesoporous Prussian blue / polydopamine nanozyme loaded with olsalazine is 180 - 300 nm.

[0010] Optionally, the drug loading amount of olsalazine in the hollow mesoporous Prussian blue / polydopamine nanozyme loaded with olsalazine is 25 - 35%.

[0011] Optionally, the thickness of the polydopamine layer is 5 - 30 nm.

[0012] In the second aspect, a preparation method of the above-mentioned hollow mesoporous Prussian blue / polydopamine nanozyme loaded with olsalazine includes the following steps:

[0013] S1. Dispersing the hollow mesoporous Prussian blue particles (HMPBNPs) and olsalazine (Olsa) in water at a mass ratio of (1 - 5):1, centrifuging and collecting the precipitate to obtain a hollow mesoporous Prussian blue nanozyme loaded with olsalazine (Olsa@HMPB NPs);

[0014] S2. Adding the hollow mesoporous Prussian blue nanozyme loaded with olsalazine (Olsa@HMPB NPs) and dopamine hydrochloride to the buffer solution at a mass ratio of (4.5 - 5):1, stirring and reacting for 1 - 2 h to obtain a hollow mesoporous Prussian blue / polydopamine nanozyme loaded with olsalazine (Olsa@HMPB / PDANPs).

[0015] Optionally, in S1, the ratio of hollow mesoporous Prussian blue particles (HMPBNPs) to water is (1 - 1.5) mg / mL.

[0016] Optionally, in S1, the dispersion method includes: adding hollow mesoporous Prussian blue particles (HMPBNPs) to deionized water, ultrasonicating for 2 - 3 min to obtain a first dispersion system; then adding olsalazine (Olsa) to the first dispersion system, ultrasonicating for 5 - 6 min, and stirring at a speed of 300 - 400 rpm for 20 - 28 h.

[0017] Optionally, in S1, the method for collecting the precipitate after centrifugation includes: centrifuging at a speed of 16000 - 18000 rpm for 15 - 20 min, dispersing the obtained precipitate in deionized water at a ratio of (5 - 6) mg / mL, centrifuging at a speed of 16000 - 18000 rpm for 15 - 20 min, and collecting the precipitate.

[0018] Optionally, in S2, the buffer solution is Tris-HCl buffer solution, and the ratio of hollow mesoporous Prussian blue nanozyme (Olsa@HMPBNPs) to the buffer solution is (0.5 - 1) mg / mL.

[0019] Optionally, in S2, adding the olsalazine-loaded hollow mesoporous Prussian blue nanozyme (Olsa@HMPB NPs) to the buffer solution, ultrasonicating for 2 - 3 min to obtain a second dispersion system; then adding dopamine hydrochloride to the second dispersion system, stirring at a speed of 600 - 700 rpm for 1 - 2 h, and centrifuging and washing to obtain olsalazine-loaded hollow mesoporous Prussian blue / polydopamine nanozyme (Olsa@HMPB / PDA NPs).

[0020] Optionally, the preparation method of the hollow mesoporous Prussian blue particles includes: adding potassium ferricyanide polyvinylpyrrolidone to an HCl solution, stirring and reacting, centrifuging and washing to obtain Prussian blue particles, adding the Prussian blue particles and PVP to an HCl solution, reacting at 140 - 160 °C for 4 - 6 h, centrifuging to remove the supernatant, and obtaining the hollow mesoporous Prussian blue particles.

[0021] In the third aspect, the above-mentioned olsalazine-loaded hollow mesoporous Prussian blue / polydopamine nanozyme (Olsa@HMPB / PDANPs) is used in at least one of the following a1) - a4):

[0022] a1) Preparing a product for reducing the expression of pro-inflammatory factors and / or increasing the expression of anti-inflammatory factors in colon tissues;

[0023] a2) Preparing a product for increasing the protein level of TOM20 in colon epithelium and / or inhibiting the decrease of SOD2 level;

[0024] a3) Preparing a product for reducing oxidative stress response and / or mitochondrial damage in colon cells;

[0025] a4) Preparing a product for treating inflammatory bowel disease.

[0026] The product is a drug or a common experimental reagent for non-medical use, and the common experimental reagent can be used for basic research.

[0027] According to the present invention, when the product is a drug, the drug further comprises at least one pharmaceutically inactive ingredient.

[0028] The pharmaceutically inactive ingredient may be a carrier, excipient, diluent, etc. commonly used in pharmacy. Moreover, according to the usual method, it can be made into dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc. for oral administration, external use, suppositories and sterile injection solutions for use.

[0029] The non-drug active ingredients such as the carrier, excipient and diluent that can be included are well-known in the art, and those of ordinary skill in the art can determine that they meet the clinical standards.

[0030] Preferably, the drug of the present invention can be administered into the body by known means. For example, it can be delivered systemically via intravenous injection or locally injected into the tissue of interest. Optionally, it can be administered via intravenous, percutaneous, intranasal, mucosal or other delivery methods. Such administration can be carried out via a single dose or multiple doses. Those skilled in the art understand that the actual dose to be administered in the present invention can vary to a large extent depending on various factors such as the target cells, biological type or its tissue, the general condition of the subject to be treated, the route of administration, the mode of administration, and so on.

[0031] Preferably, the drug administration object can be humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, chimpanzees, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0033] Figure 1 It is a test result diagram of the preparation and characterization of hollow mesoporous Prussian blue / polydopamine nanozyme loaded with olsalazine in the specific embodiment. Among them, (A) TEM images of PB, HMPB and HMPB / PDANPs;

[0034] (B) Elemental mapping of HMPB / PDANPs, C (blue), N (cyan), O (red), S (green), and Fe (yellow); (C) Hydrated particle size distribution of HMPB / PDA and Olsa@HMPB / PDA; (D) FTIR spectra of PDA, HMPB, and HMPB / PDA NPs; (E) Characterization of the crystal structure of NPs by XRD; (F) Zeta potential of HMPB / PDA and Olsa@HMPB / PDA NPs. (G-H) UV-visible absorption spectra of Olsa; (I)

[0035] Olsa cumulative release rate of Olsa@HMPB / PDA NPs in normal intestinal environment (pH 7.4) and inflamed intestinal environment (pH 5.5); (J) Absorbance at 650 nm after NPs were treated with H2O2 and TMB; Results are expressed as mean ± SD.

[0036] Figure 2 Are the characterization results of cell experiments and animal experiments in the specific implementation manners Figure 1 , (A) Experimental flow chart. (B) Photos of mouse colon tissues. (C) Mouse colon length (n = 5). (D) Photos of mouse rectal bleeding. (E) Changes in mouse body weight (n = 5). (F) Changes in DAI scores (n = 5). (G) Photos of mouse spleen tissues. (H) Mouse spleen index (n = 5). (I) H&E staining of mouse colon tissues. (J) Colon tissue injury score (n = 5). Results are expressed as mean ± SD. Figures C and H were detected by one-way ANOVA with Bonferroni correction, and Figures E and F were detected by two-way repeated measures ANOVA with Bonferroni correction. *p < 0.05, **p < 0.01, ***p < 0.001.

[0037] Figure 3 Are the characterization results of cell experiments and animal experiments in the specific implementation manners Figure 2 , (A) Immunofluorescence staining of CD68 and F4 / 80 in colon tissues. (B) Number of CD68+ / F4 / 80+ cells in colon tissues (n = 6). (C) Immunofluorescence staining of CD206 and F4 / 80 in colon tissues. (D) Number of CD206+ / F4 / 80+ cells in colon tissues (n = 6). (E-F) mRNA expression levels of TNF-α, iNOS, TGF-β, and IL-10 in colon tissues (n = 4). Results are expressed as mean ± SD. Figures B, D, E, and F were detected by one-way ANOVA with Bonferroni correction. *p < 0.05, **p < 0.01, ***p < 0.001.

[0038] Figure 4It is the characterization results of cell experiments and animal experiments in the specific implementation mode Figure 3 , (A) Quantitative analysis results of ROS. (B) Quantitative analysis results of mitoSOX. (C) Analysis results of JC-1 fluorescence intensity. (D) Quantitative analysis results of ATP content (n = 5). (E) Immunohistochemistry was used to detect the expression of TOM20 protein in the colon of each group. (F) Statistical analysis of the TOM20+ area in the colon tissues of each group (n = 6). (G) Western blot was used to detect the expression levels of TOM20 and SOD2 proteins in the colon. (H) Statistical analysis of the relative expression levels of TOM20 and SOD2 (n = 4). The results are expressed as mean ± SD. Figures A, B, C, F, and H were detected by one-way ANOVA with Bonferroni correction. Figure D was tested by Kruskal-Wallis test. p < 0.05. *p < 0.05, **p < 0.01, ***p < 0.001..

[0039] Figure 5 It is the characterization results of cell experiments and animal experiments in the specific implementation mode Figure 5 , (A) The cell viability was measured after incubating CCD841 cells with HMPB / PDA NPs and Olsa@HMPB / PDA NPs for 24 h (n = 6). (B) The cell viability was measured after incubating BV2 cells with HMPB / PDA NPs and Olsa@HMPB / PDA NPs for 24 h (n = 6). (C) Immunofluorescence images of CCD841 cells after incubating with HMPB / PDA NPs and Olsa@HMPB / PDA NPs for 24 h and 48 h. Live cells (green) and dead cells (red). (D) Statistical analysis of the live / dead cell ratio (n = 12). (E) Cell migration photos of CCD841 cells incubated with HMPB / PDA NPs and Olsa@HMPB / PDA NPs for 24 h and 48 h. (F-G) Statistical analysis of the migration rates of CCD841 cells incubated with HMPB / PDA NPs and Olsa@HMPB / PDA NPs for 24 h and 48 h (n = 12). (H) Hemolysis test results and hemolysis rates (n = 4). The results are expressed as mean ± SD.

[0040] Figure 6 It is a schematic diagram of the technical route in the specific implementation mode. Specific implementation mode

[0041] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0042] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular forms are also intended to include the plural forms. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they specify the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0043] For the experimental methods without specific conditions noted in the following examples, they are generally in accordance with conventional conditions. Each of the raw materials and reagents used in the following examples, unless otherwise specified, can be obtained commercially.

[0044] The preparations of the present invention can conveniently exist in unit dosage forms and can be prepared by any method well known in the pharmaceutical field. The amount of the active ingredient combined with the carrier substance to prepare a single dosage form is generally the amount of the compound that produces a therapeutic effect.

[0045] Pharmaceutically acceptable carriers, which are well recognized in the art, include pharmaceutically acceptable materials, compositions or carriers suitable for administering the compounds of the present invention to mammals. The carriers include liquid or solid fillers, diluents, excipients, solvents or encapsulating materials that participate in carrying the main substance or transferring it from one organ or part of the body to another organ or another part of the body. Each carrier must be "acceptable" in the sense of being compatible with other components in the preparation and harmless to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: saccharides such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate, powdered tragacanth, malt, gelatin, talc, excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; diols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; and other non-toxic and compatible substances used in pharmaceutical preparations.

[0046] Wetting agents, emulsifying agents and lubricants such as sodium dodecyl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening agents, flavoring agents and fragrances, preservatives and antioxidants can also be present in the composition.

[0047] The overall technical route is as Figure 6 shown and specifically includes the following steps.

[0048] (I) Preparation and characterization of hollow mesoporous Prussian blue / polydopamine nanozyme loaded with olsalazine

[0049] 1. The preparation method of olsalazine-loaded hollow mesoporous Prussian blue / polydopamine nanozyme (Olsa@HMPB / PDA NPs) includes:

[0050] S0. Weigh 131.7 mg of potassium ferricyanide (K3FeC6N6) and 3 g of polyvinylpyrrolidone (PVP) respectively, add them into 40 mL of HCl solution (0.01 M), stir at room temperature for 0.5 h, and then heat at 80 °C for 20 h. After centrifugation, discard the supernatant, wash three times, and vacuum dry to obtain blue powdery PB NPs; add 20 mg of PB NPs and 100 mg of PVP into 20 mL of HCl solution (1 M), seal the mixture in an autoclave and react at 140 °C for 4 h. This step is used to generate a hollow mesoporous structure in PB NPs; after centrifuging the reactant, discard the supernatant, wash five times, and vacuum dry to obtain HMPB NPs.

[0051] S1. Weigh 10 mg of HMPB NPs and add them into 10 mL of deionized water, sonicate (45 kHz, 70 W) for 2 min to mix well; then, according to the mass ratio of HMPB NPs to Olsa of 2:1, add 5 mg of Olsa into the above system, sonicate for 5 min, and stir magnetically at 300 rpm for 24 h; centrifuge (16,000 rpm, 15 min) to collect the supernatant, disperse the precipitate in 2 mL of deionized water, and centrifuge again (16,000 rpm, 15 min) to collect the supernatant; the obtained precipitate is Olsa@HMPB NPs; analyze the absorbance of the supernatant at a wavelength of 360 nm with a UV-visible spectrophotometer, calculate the content of unloaded drug according to the Olsa standard curve, and then obtain the drug loading of Olsa.

[0052] S2. Add 20 mg of Olsa@HMPB NPs into 40 mL of Tris-HCl (10 mM, pH 8.5), sonicate to mix well; then add 4 mg of dopamine hydrochloride into this system, so that the mass ratio of Olsa@HMPB NPs to dopamine hydrochloride is 5:1, stir at room temperature for 1 h (600 rpm), and keep the indoor air flowing during this period; after centrifugation and washing, obtain Olsa@HMPB / PDA NPs.

[0053] 2. The characterization methods included: using a Transmission electron microscope (TEM) and an X-ray diffraction (XRD) instrument to characterize the microstructure and morphology of the prepared nanoparticles; using a Dynamic light scattering (DLS) instrument to measure the hydrodynamic diameter and potential of the nanoparticles; using a Fourier transform infrared spectrometer (FTIR) to characterize the crystal structure, elemental valence state, and chemical groups of the NPs.

[0054] The characterization results included: as Figure 1 shown in A, the sizes of PB, HMPB, and HMPB / PDA NPs detected by TEM were 250 ± 30 nm, 200 ± 20 nm, and 200 ± 20 nm, respectively; HMPB NPs presented a hollow nanostructure with uniform pore size, and a PDA layer was uniformly formed on the surface of HMPB after PDA wrapping, and the thickness of the PDA layer was about 20 nm; as Figure 1 shown in B, the elemental mapping results further indicated that Fe, C, N, O, and S elements were uniformly distributed within HMPB / PDA NPs.

[0055] The hydrodynamic particle size of the NPs was measured by DLS, as Figure 1 shown in C, and the results showed that the average hydrodynamic particle sizes of HMPB / PDA and Olsa@HMPB / PDA NPs were 272.4 nm (PDI: 0.252) and 301.1 nm (PDI: 0.266), respectively.

[0056] The chemical groups of the obtained NPs were characterized using FTIR spectra, as Figure 1 shown in D. In HMPB and HMPB / PDA NPs, due to the presence of C≡N bonds, Fe-CN bonds, and C=O bonds, strong absorption peaks appeared at 2080 cm -1 , 590 cm -1 , and 1656 cm -1 ; due to the presence of O-H, N-H, and NH2, absorption peaks in the range of 3500 - 3200 cm -1 existed in both PDANPs and HMPB / PDA NPs.

[0057] The crystal structure of the prepared NPs was characterized by XRD, as Figure 1 shown in E. Diffraction peaks of HMPB NPs appeared at 2θ angles of 17.5°, 24.8°, 35.4°, and 39.8°, indicating that the PDA wrapping did not cause obvious changes; meanwhile, the Zeta potential measurement results were as Figure 1As shown in F, the Zeta potential of HMPB@PDA was -21 mV, and there was no obvious change after loading Olsa; the ultraviolet-visible absorption spectrum of Olsa is as shown in G in Figure 1 . The results showed that Olsa had a strong absorption peak at 360 nm, and as shown in H in Figure 1 , the concentration of Olsa was linearly correlated with its absorbance at 360 nm. Therefore, in this experiment, the content of unloaded drug was calculated according to the Olsa standard curve, further proving the successful loading of Olsa; in short, these results indicated that Olsa@HMPB / PDANPs were successfully prepared in this experiment.

[0058] 3. Testing of drug release rate, the method includes: adding Olsa@HMPB / PDA (Olsa = 3.47 mg, HMPB / PDA = 10 mg) into simulated intestinal fluids in normal intestinal environment (37 °C, 0 M H2O2, 10 mL) and inflammatory intestinal environment (37 °C, 10×10-3 M H2O2, 10 mL) respectively, and placing them in a shaker (120 rpm, 37 °C); taking out 1 mL of the above solution from the buffer at different time points (0.5, 1, 4, 8, 12 and 24 h), centrifuging and calculating the cumulative release amount of Olsa by measuring the absorbance of the supernatant at 360 nm. The release curve is as shown in Figure 1 I. In the normal intestinal environment represented by the black broken line, the release rates of Olsa at 1 h and 24 h were 5.3% and 31.7% respectively; while in the inflammatory intestinal environment represented by the red broken line, the release rates of Olsa at 1 h and 24 h were 12.2% and 87.8% respectively. This result indicated that the drug release rate of the Olsa@HMPB / PDA system was increased in the inflammatory environment, which was helpful to increase the drug concentration in the gastrointestinal tract.

[0059] 4. Measurement of peroxidase-like activity, the method includes: preparing solutions of TMB (4 mg / mL, DMSO), H2O2 (30%, ddH2O), HMPB (20 μg / mL, PBS), PDA (20 μg / mL, PBS) and HMPB / PDA (20 μg / mL (quantitatively equal amount of HMPB), PBS) respectively; then mixing equal amounts of solutions in the order of HMPB + H2O2 + TMB, PDA + H2O2 + TMB, HMPB / PDA + H2O2 + TMB, TMB group; detecting the absorbance value at 650 nm after 15 min. The results are as shown in Figure 1 J. Compared with TMB + H2O2 (yellow dotted line), the absorbance at 650 nm of HMPB + H2O2 (red dotted line) and HMPB@PDA + H2O2 (green dotted line) increased significantly, indicating that HMPB and HMPB@PDA NPs had good peroxidase-like activity.

[0060] (2) Cell experiments

[0061] 1. Cell viability detection, the method includes: using the Cell Counting Kit CCK-8 to evaluate cell viability. CCD841 cells and BV2 cells were respectively inoculated into 96-well plates, and then the cells were treated with one of HMPB / PDA and Olsa@HMPB / PDA (0, 5, 20, 40, 80, 100 μg / mL) for 24 h. The CCK-8 solution was added to the 96-well plates and incubated at 37 °C for 2 h. The cell viability was measured at 450 nm using a microplate reader. The results of CCD841 cells are as shown in Figure 5 Figure A, and the viability of BV2 cells is as shown in Figure 5 Figure B. Compared with the control group, HMPB / PDA NPs and Olsa@HMPB / PDA NPs had no obvious cytotoxic effect on CCD841 cells and BV2 cells at a concentration of 0-100 μg / mL for 24 h.

[0062] 2. Live / dead cell analysis, the method includes: evaluating the cell survival rate of cells treated with HMPB / PDA (100 μg / mL) and Olsa@HMPB / PDA (100 μg / mL) according to the instructions of the live / dead staining kit, and observing and counting under a fluorescence microscope after culturing for 24 h. The staining results are as shown in Figure 5 Figure C, and the statistical results are as shown in Figure 5 Figure D. After incubating CCD841 cells with Olsa@HMPB / PDA NPs and HMPB / PDA NPs at a concentration of 100 μg / mL for 24 h and 48 h, there was no significant difference in the ratio of live / dead cells, indicating that Olsa@HMPB / PDA NPs have good biocompatibility.

[0063] 3. Cell scratch test, the method includes: inoculating 4×10 5 cells / well into 6-well plates and culturing for 24 hours; scratching the cells with a pipette tip and washing with PBS to remove floating cells and debris; adding serum-free fresh medium with or without HMPB / PDA and Olsa@HMPB / PDA (100 μg / mL) to the 6-well plates and culturing for 24 h and 48 h. The scratch distance of the cells was photographed and recorded at 0 h, 24 h, and 48 h, and the migration rate was calculated. The pictures of the scratch test results are as shown in Figure 5 Figure E, the statistical results of the migration rate at 24 h are as shown in Figure 5 Figure F, and the statistical results of the migration rate at 48 h are as shown in Figure 5 Figure G: Compared with the control group, incubating CCD841 cells with Olsa@HMPB / PDA NPs and HMPB / PDA NPs at a concentration of 100 μg / mL for 24 h and 48 h had no obvious effect on the cell migration and proliferation ability.

[0064] 4. Hemolysis test, the method includes: placing fresh mouse blood in a centrifuge tube and centrifuging at 3000 rpm for 10 min, then discarding the supernatant, diluting and washing with PBS more than 3 times to obtain red blood cells; resuspending the obtained red blood cells with 5 mL of PBS; mixing 50 μL of the red blood cell suspension with 200 μL of PBS (negative control), 1% Triton-100 (positive control), or PBS solutions of HMPB / PDA (1 mg / mL) and Olsa@HMPB / PDA (1 mg / mL); after incubating at 37 °C for 1 h, all samples are centrifuged at

[0065] 10000 rpm for 5 min; finally, take pictures of the samples and measure the absorbance of the supernatant at 540 nm; Hemolysis rate (%) = (A 1 / A + ) × 100%; where A 1 is the absorbance value of the sample at 540 nm, and A + is the absorbance value of the positive control at 540 nm. The results of the hemolysis experiment are as shown in Figure 5 Figure H, and there is no obvious hemolysis reaction for 1 mg / mL of HMPB / PDA NPs and Olsa@HMPB / PDA NPs.

[0066] (III) Animal experiments

[0067] 1. Induction of colitis and drug treatment, including: C57BL / 6J male mice (21 - 23 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and raised under SPF conditions in the Experimental Animal Center of Shandong University; all mouse experiments were approved by the Animal Ethics and Welfare Committee of Shandong University (Approval No.: ECSBMSSDU2024 - 02 - 310); after 7 days of mice adapting to the environment, they were randomly divided into 4 groups, namely: control group (Control), model group (DSS), DSS + HMPB / PDA group, DSS + Olsa@HMPB / PDA group; mice in the control group were fed with mouse food + water, and mice in the other 3 groups were fed with mouse food + 2.5% DSS drinking water to induce acute colitis in mice for 7 consecutive days; meanwhile, on the 1st, 3rd, 5th, and 7th days, mice were intragastrically administered with HMPB / PDA (20 mg / kg) or Olsa@HMPB / PDA NPs (20 mg / kg). Observe and record body weight, stool characteristics, and bloody stool conditions every day. On the 8th day, mice were anesthetized and sacrificed, record the colon length and spleen weight, and collect colon tissues for subsequent molecular detection. The experimental flow chart is as shown in Figure 2 Figure A. As shown in Figure 2 Figures B - H, Olsa@HMPB / PDA NPs treatment significantly alleviated DSS-induced colitis, manifested as an increase in colon length ( Figure 2 B and Figure 2 C), an increase in body weight ( Figure 2E), In summary, Olsa@HMPB / PDANPs treatment can alleviate DSS-induced colitis in mice.

[0068] 2. Disease activity index score, the disease severity of mice was evaluated using DAI: Calculate the scores for each category (from 0 to 4), and then take the sum. Briefly, weight loss: 0 points, <1%; 1 point, 1-5%; 2 points, 5-10%; 3 points, 10-15%; 4 points, >15%. Fecal traits: 0 points, normal stool; 1-2 points, soft and formed; 3 points, soft stool; 4 points, diarrhea. Bloody stool: 0 points, none; 2 points, occult blood; 4 points, gross bloody stool. The DAI evaluation results are as Figure 2 shown in F. Compared with the DSS group, HMPB / PDA NPs treatment can alleviate the increase in DSS-induced DAI score; the spleen is as Figure 2 shown in G, and the spleen index is as Figure 2 shown in H. HMPB / PDANPs treatment can alleviate the increase in the numerical value of the spleen index induced by DSS.

[0069] 3. Detection of reactive oxygen species level, oxidative damage is a basic pathological mechanism of most inflammatory diseases; ROS is an important factor inducing the inflammatory environment, which is mainly produced in the cytoplasm and mitochondria; Take the same mass of fresh colon tissue from mice, prepare a single-cell suspension, and after centrifugation, take the cell pellet and co-incubate it with a fluorescent probe (one of DCFH-DA or MitoSOX) for 30 min (37 °C), and perform fluorescence quantitative detection using an enzyme-linked immunosorbent assay (ELISA) reader. The ROS detection results obtained by DCFH-DA are as Figure 4 shown in A, and the ROS detection results obtained by MitoSOX are as Figure 4 shown in B: Olsa@HMPB / PDANPs treatment not only inhibits the cellular ROS level but also inhibits mtROS. At the same time, HMPB / PDANPs treatment only inhibits the cellular ROS level but has no obvious improvement effect on mtROS.

[0070] 4. Detection of mitochondrial membrane potential, take the same mass of fresh colon tissue from mice, prepare a single-cell suspension, and after centrifugation, take the cell pellet and co-incubate it with the fluorescent probe JC-1 for 30 min (37 °C), and perform fluorescence quantitative detection using an ELISA reader to calculate the Red / Green ratio. The JC-1 staining results are as Figure 4 shown in C, indicating that Olsa@HMPB / PDANPs treatment inhibits the decrease in mitochondrial membrane potential in DSS-induced colitis mice.

[0071] 5. Detection of mitochondrial ATP content, take the same mass of fresh colon tissue from mice, prepare samples using an ATP detection kit, prepare a standard curve, and perform detection using an ELISA reader. The results are as Figure 4As shown in D, treatment of colitis mice with Olsa@HMPB / PDANPs can reverse the decrease in colonic mitochondrial ATP levels.

[0072] 6. For H&E staining, paraffin-embedded colon tissues were made into 4-μm sections. After dewaxing and dehydration, the operation was carried out according to the instructions of the H&E staining kit. Finally, the sections were scanned with a microscope. The results of H&E staining are shown in Figure 2 I. In the DSS group, the colon tissues showed inflammatory cell infiltration, goblet cell reduction, and destruction of the colonic crypt structure. Figure 2 J represents the score of colonic tissue damage. Figure 2 I and Figure 2 J indicate that treatment with Olsa@HMPB / PDA NPs can alleviate the colonic structural damage caused by DSS.

[0073] 7. For immunofluorescence staining, paraffin-embedded colon tissues were cut into 4-μm sections. Next, the colon sections were incubated with the primary antibodies overnight at 4°C. The primary antibodies were: anti-CD206 (1:200, YM3050, Immunoway), anti-CD68 (1:200, YM4678, Immunoway), anti-F4 / 80 (1:100, 29414-1-AP, Proteintech). Image acquisition was performed using a fluorescence microscope. Inflammatory factors are closely related to the pathogenesis of IBD, and macrophages are the main immune cells secreting inflammatory factors. Macrophages have two phenotypes: inflammatory-related M1 macrophages and anti-inflammatory M2 macrophages. In this study, the changes in M1 and M2 macrophages were detected by immunofluorescence staining of CD68, CD206, and F4 / 80. The results showed that treatment with HMPB / PDA or Olsa@HMPB / PDA NPs significantly reversed the increase in M1 macrophages in DSS-induced colitis mice (as shown in Figure 3 A and Figure 3 B), while treatment with Olsa@HMPB / PDANPs significantly increased the reduction of M2 macrophages in DSS-induced colitis mice (as shown in Figure 3 C and Figure 3 D), suggesting that Olsa@HMPB / PDA NPs can promote the transformation of macrophages from the M1 type to the M2 type.

[0074] 8. For immunohistochemical staining, paraffin-embedded colon tissues were made into 4-μm sections. After dewaxing and dehydration, the colon sections were incubated with the primary antibody overnight at 4°C. The primary antibody was: anti-TOM20 (1:200, 66777-1-Ig, Proteintech); Image acquisition was performed using a microscope. We passed through as shown in Figure 4Immunohistochemical staining as shown in E observed a decrease in the level of TOM20 in the colon of DSS mice, and Olsa@HMPB / PDANPs treatment significantly increased the protein level of TOM20 in the colon epithelium of DSS mice. Figure 4 F). Taken together, these results indicate that Olsa@HMPB / PDA NPs can scavenge ROS and alleviate mitochondrial dysfunction.

[0075] 9. qRT-PCR was used to detect mRNA expression. RNA was extracted using an Ultra-pure RNA kit (CoWin Biosciences, China). RNA was reverse-transcribed into cDNA using a reverse transcription kit (TOYOBO, Osaka, Japan). qRT-PCR was performed on a Bio-rad IQ5 RealTime PCR system (Bio-rad, CA, USA). Using β-actin as an internal reference, the relative expression levels of TNF-α, iNOS, TGF-β, and IL-10 mRNA were calculated by the 2 -ΔΔCT -method. The primers are shown in Table 1.

[0076] The results of qRT-PCR showed as Figure 3 shown in E and Figure 3 F, after DSS treatment, the levels of TNF-α and iNOS mRNA were significantly increased, and the levels of TGF-β and IL-10 mRNA were significantly decreased; Olsa@HMPB / PDANPs treatment significantly decreased the expression of TNF-α and iNOS mRNA and increased the expression of TGF-β and IL-10 mRNA. The results suggest that Olsa@HMPB / PDANPs can inhibit the pathological development of IBD and prevent the exacerbation of IBD by reducing the expression of pro-inflammatory factors and increasing the expression of anti-inflammatory factors in the colon tissue of mice.

[0077] 10. Western blot was used to detect protein expression

[0078] Collect colon tissues and freeze them at -80°C. Lyse the tissues with RIPA lysis buffer containing cholinesterase inhibitor and phosphatase inhibitor, centrifuge at 4°C and 12,000 rpm for 15 min, and then add 5× or 2× loading buffer to the protein supernatant. Western blots were detected using the following primary antibodies: anti-TOM20 (1:1000, 66777-1-Ig, Proteintech), anti-SOD2 (1:1000, 66474-1-Ig, Proteintech), anti-β-actin (1:1000, Zhongshan GoldenBridge Biotechnology). Incubate the primary antibody overnight at 4°C and the secondary antibody for 1 h at room temperature. Image using a Tanon imaging system. The primers used are shown in Table 1.

[0079] Table 1

[0080]

[0081] We observed a decrease in TOM20 levels in the colon of DSS mice by Western blot as shown in Figure 4 G, and the Western blot results of the antioxidant enzyme SOD2 shown in Figure 4 H showed that Olsa@HMPB / PDA NPs treatment significantly reversed the decrease in SOD2 levels caused by DSS. Taken together, these results indicate that Olsa@HMPB / PDA NPs can scavenge ROS and alleviate mitochondrial dysfunction.

[0082] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A hollow mesoporous Prussian blue / polydopamine nanozyme loaded with olsalazine, characterized in that, Comprising: Hollow mesoporous Prussian blue particles, wherein olsalazine is loaded in the hollow mesoporous Prussian blue particles, and a polydopamine layer is coated on the surface of the hollow mesoporous Prussian blue particles.

2. The hollow mesoporous Prussian blue / polydopamine nanozyme loaded with olsalazine according to claim 1, characterized in that, The particle size of the olsalazine-loaded hollow mesoporous Prussian blue / polydopamine nanozyme is 180 - 300 nm; Optionally, the drug loading amount of olsalazine in the olsalazine-loaded hollow mesoporous Prussian blue / polydopamine nanozyme is 25 - 35%; Optionally, the thickness of the polydopamine layer is 5 - 30 nm.

3. A method for preparing hollow mesoporous Prussian blue / polydopamine nanozyme loaded with olsalazine as described in any one of claims 1-2, characterized in that, Including the following steps: S1. Dispersing hollow mesoporous Prussian blue particles and olsalazine in water at a mass ratio of (1 - 5):1, centrifuging and collecting the precipitate to obtain olsalazine-loaded hollow mesoporous Prussian blue nanozyme; S2. Adding the olsalazine-loaded hollow mesoporous Prussian blue nanozyme and dopamine hydrochloride to a buffer solution at a mass ratio of (4 - 4.5):1, stirring and reacting for 1 - 2 h to obtain olsalazine-loaded hollow mesoporous Prussian blue / polydopamine nanozyme.

4. The preparation method of the hollow mesoporous Prussian blue / polydopamine nanozyme loaded with olsalazine according to claim 3, characterized in that, In S1, the ratio of the hollow mesoporous Prussian blue particles to water is (1 - 1.5) mg / mL.

5. The preparation method of the hollow mesoporous Prussian blue / polydopamine nanozyme loaded with olsalazine according to claim 4, characterized in that, In S1, the dispersion method includes: adding the hollow mesoporous Prussian blue particles into deionized water, ultrasonically treating for 2 - 3 min to obtain a first dispersion system; then adding olsalazine into the first dispersion system, ultrasonically treating for 5 - 6 min, and stirring at a rotation speed of 300 - 400 rpm for 20 - 28 h.

6. The preparation method of the hollow mesoporous Prussian blue / polydopamine nanozyme loaded with olsalazine according to claim 3, characterized in that, In S1, the method for centrifuging and collecting the precipitate includes: centrifuging at a rotation speed of 16000 - 18000 rpm for 15 - 20 min, dispersing the obtained precipitate in deionized water at a ratio of (5 - 6) mg / mL, centrifuging at a rotation speed of 16000 - 18000 rpm for 15 - 20 min, and collecting the precipitate.

7. The preparation method of the hollow mesoporous Prussian blue / polydopamine nanozyme loaded with olsalazine according to claim 3, characterized in that, In S2, the buffer solution is Tris-HCl buffer solution, and the ratio of the hollow mesoporous Prussian blue nanozyme to the buffer solution is (0.5 - 1) mg / mL.

8. The preparation method of the hollow mesoporous Prussian blue / polydopamine nanozyme loaded with olsalazine according to claim 3, characterized in that, In S2, adding the olsalazine-loaded hollow mesoporous Prussian blue nanozyme into the buffer solution, ultrasonically treating for 2 - 3 min to obtain a second dispersion system; then adding dopamine hydrochloride into the second dispersion system, stirring at a rotation speed of 600 - 700 rpm for 1 - 2 h, centrifuging and washing to obtain olsalazine-loaded hollow mesoporous Prussian blue / polydopamine nanozyme.

9. The preparation method of the hollow mesoporous Prussian blue / polydopamine nanozyme loaded with olsalazine according to claim 3, characterized in that, The preparation method of the hollow mesoporous Prussian blue includes: adding potassium ferricyanide polyvinylpyrrolidone to an HCl solution, stirring and reacting, centrifuging and washing to obtain Prussian blue powder, adding the Prussian blue powder PVP to an HCl solution, reacting at 140 - 160 °C for 4 - 6 h, centrifuging to remove the supernatant to obtain hollow mesoporous Prussian blue.

10. Use of an olsalazine-loaded hollow mesoporous Prussian blue / polydopamine nanozyme as described in any one of claims 1 - 2 in at least one of the following a1) - a4): a1) Preparing a product for reducing the expression of pro-inflammatory factors and / or increasing the expression of anti-inflammatory factors in colon tissues; a2) Preparing a product for increasing the protein level of TOM20 in colon epithelium and / or inhibiting the decrease of SOD2 level; a3) To prepare a product for reducing oxidative stress response and / or mitochondrial damage in colon cells; a4) To prepare a product for treating inflammatory bowel disease.

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