Preparation and application of humic acid-bismuth nanomaterials

Abstract

The application provides preparation and application of humic acid-bismuth nanomaterials, and belongs to the technical field of medicinal chemistry.The humic acid-bismuth nanomaterials can successfully reach the small intestine from the stomach, and are enriched at the inflammation site of the colon, so that the humic acid-bismuth nanomaterials can be selectively visualized in IBD lesions after being covalently coupled with a fluorescent dye Cy5.5.In addition, the humic acid-bismuth nanomaterials have high oxygen free radical scavenging capacity, and have the function of accelerating the healing of the ulcer surface.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry technology, and in particular relates to the preparation and application of a humic acid-bismuth nanomaterial. Background Technology

[0002] Inflammatory bowel disease (IBD), primarily including ulcerative colitis (UC) and Crohn's disease (CD), is the most common chronic inflammatory disease of the gastrointestinal tract and a refractory autoimmune disease. Depending on the severity and location of the inflammation, IBD symptoms can range from diarrhea, rectal bleeding, or unexpected weight loss to complications including intestinal perforation, intestinal obstruction, and colorectal cancer. Traditional diagnostic methods rely on invasive procedures such as colonoscopy, causing significant patient discomfort and, more importantly, hindering early diagnosis. Furthermore, clinical treatments for IBD mainly include non-targeted therapies (such as aminosalicylate, glucocorticoids, and immunomodulators) and targeted biological therapies (such as anti-TNF agents), making precise dosage control difficult, leading to insufficient or excessive drug intake and failing to cure the disease. Therefore, developing new technologies and methods for the diagnosis and treatment of IBD is crucial for providing effective evidence for its comprehensive prevention and treatment.

[0003] Humic acid (HAs) is a large organic molecule rich in hydroxyl, carboxyl, and other groups, formed from plant and microbial residues through humification. In Traditional Chinese Medicine (TCM), HAs have been known as Wujin San since 1786. Numerous medical classics and pharmacologists have documented and discussed its use in detail. Li Shizhen's *Compendium of Materia Medica* records the medicinal use of humic acid, primarily for treating nosebleeds, women's blood stasis pain, and abdominal pain, among other ailments. In recent years, the applications of humic acid in tumor ablation, bacterial infection, and cerebral ischemia have been widely reported, indicating its significant potential as a tissue repair agent, particularly in wound healing from bacterial infections. However, its application in inflammatory bowel disease is rarely reported. Furthermore, the solubility of humic acid is pH-dependent, limiting its application and efficacy. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes a method for preparing and applying humic acid-bismuth (BiHA) nanomaterials. The humic acid-bismuth nanomaterials of this invention can successfully pass through the stomach and small intestine, thereby accumulating at sites of colonic inflammation. After covalently coupling the BiHA nanomaterials with the fluorescent dye Cy5.5, IBD lesions can be selectively visualized. Furthermore, the BiHA nanomaterials possess high ROS scavenging capacity, accelerating ulcer healing.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] One of the technical solutions of the present invention:

[0007] A method for preparing humic acid-bismuth nanomaterials includes the following steps:

[0008] Add a bismuth-soluble acidic solution dropwise to a humic acid solution;

[0009] Add ferric iron, purify, centrifuge, freeze and vacuum dry the resulting precipitate to obtain humic acid-bismuth nanomaterials.

[0010] Furthermore, the weight-average molecular weight of humic acid is 3,000-50,000.

[0011] Furthermore, the weight-average molecular weight of humic acid is 20,000-50,000.

[0012] Furthermore, the mass ratio of humic acid solution to bismuth-soluble acidic solution is (0.5:1) to (5:1).

[0013] Furthermore, the concentration of the humic acid solution is 2-5% (w / v).

[0014] Furthermore, the method for preparing a bismuth-soluble acidic solution is as follows: add 50 mg Bi(NO3)3·5H2O to 1.0-2.0 mL of HNO3 solution (2M) and allow it to dissolve completely to obtain a bismuth-soluble acidic solution.

[0015] Furthermore, the trivalent iron is FeCl3·6H2O.

[0016] Furthermore, the preparation method of humic acid-bismuth nanomaterials specifically includes the following steps:

[0017] 1. Take 200mg of humic acid (M W =3000-50000) Dissolved in 8.0 mL of water to obtain humic acid solution. Stirred at room temperature, 50 mg Bi(NO3)3·5H2O was added to 1.0-2.0 mL of HNO3 solution (2M). After it was fully dissolved, it was added dropwise to the above humic acid solution. Stirred at room temperature for 30-60 min.

[0018] 2. Add 30 mg of FeCl3·6H2O and stir for 12-18 h. Then, further purify the obtained Bi-HA by dd H2O dialysis for 24 h to remove excess reactants.

[0019] 3. After 24 hours, the sample was removed and centrifuged at 14000g for 5 minutes. The supernatant was discarded, and the precipitate was frozen at -80℃ for 6 hours and then freeze-dried under vacuum overnight to finally obtain BiHA nanomaterials, which were uniform BiHA nanoparticles.

[0020] This invention presents a humic acid-bismuth nanomaterial prepared via a hydrothermal method. This nanomaterial can successfully pass through the stomach and small intestine, thereby accumulating at inflamed sites in the colon. After covalently coupling the humic acid-bismuth nanomaterial with the fluorescent dye Cy5.5, IBD lesions can be selectively visualized. Furthermore, the humic acid-bismuth nanomaterial exhibits high ROS scavenging capacity, accelerating ulcer healing.

[0021] The second technical solution of the present invention:

[0022] A humic acid-bismuth nanomaterial prepared by the above method is a uniform particle, which is square, quasi-spherical, spherical or triangular in shape, with a particle size of 120-600 nm.

[0023] The humic acid-bismuth nanomaterials of this invention exhibit variations in particle size and uniformity due to differences in the molecular weight of humic acid. For example, when the molecular weight of humic acid M... W When the M=20000-50000, the particle size of the prepared nanoparticles is mostly distributed between 120-150 nm; when the M of humic acid is... W When the M=3000-10000, the particle size of the prepared nanoparticles is mostly distributed between 400-600 nm; when the M of humic acid is... W When the ratio is 10000-30000, the particle size of the prepared nanoparticles is mostly distributed between 300-600 nm.

[0024] The third technical solution of the present invention:

[0025] The application of the humic acid-bismuth nanomaterials in the preparation of drugs for treating diseases related to oxygen free radical damage.

[0026] The fourth technical solution of the present invention:

[0027] The application of the humic acid-bismuth nanomaterial in the preparation of drugs for treating inflammatory bowel disease involves inhibiting the expression of inflammatory factors such as IL-6, IL-1β, and TNF-α to treat inflammatory bowel disease.

[0028] The fifth technical solution of the present invention:

[0029] The application of the humic acid-bismuth nanomaterials in the preparation of small molecule carrier drugs or nucleic acid carrier drugs.

[0030] This invention provides a humic acid-bismuth nanomaterial that can be used to treat mitochondrial damage, inflammatory bowel disease, and as a carrier for delivering small molecules or nucleic acids, especially for integrated diagnosis and treatment of ulcerative colitis. This invention is the first to propose that humic acid-bismuth nanomaterials can be used for integrated diagnosis and treatment of inflammatory colitis, and the first to apply this nanomaterial to the diagnosis and treatment of inflammatory colitis. This nanomaterial formulation has excellent diagnostic and therapeutic effects on DSS-induced colitis in mice.

[0031] Compared with the prior art, the present invention has the following advantages and technical effects:

[0032] 1. Humic acid-bismuth nanoparticles were synthesized via a one-step hydrothermal method. Transmission electron microscopy and mapping analysis revealed that the prepared nanoparticles had a spherical structure of 120-150 nm. Cytotoxicity and hemolysis experiments demonstrated the good safety profile of the nanomaterials. After chemically conjugating the humic acid-bismuth nanoparticles with Cy5.5 fluorescent molecules, oral administration of the nanomaterials to small animals revealed that the humic acid-bismuth nanoparticles primarily accumulated in the inflamed areas of the colon in mice, as observed by in vivo and in vitro imaging. These results suggest that this nanomaterial could serve as a targeted diagnostic reagent for colitis.

[0033] 2. After co-incubating humic acid-bismuth nanomaterials with Rhodamine B molecules, the nanomaterials were purified by centrifugation and then co-incubated with RAW264.7 cells. Confocal microscopy revealed that the humic acid-bismuth nanomaterials could effectively load Rhodamine B into the cells, indicating that humic acid-bismuth has the potential to deliver small molecule dyes or nucleic acids. Furthermore, after co-incubating humic acid-bismuth nanomaterials with siRNA labeled with the fluorescent molecule Cy3 for 6 hours, and then co-incubating them with A549 cells, confocal microscopy showed that BiHA could carry siRNA into A549 cells, indicating that BiHA nanoparticles have the potential to act as nucleic acid carriers.

[0034] 3. An in vitro inflammation model was constructed using lipopolysaccharide (LPS). The expression levels of IL-6, IL-1β, and TNF-α in RAW264.7 cells from the normal control group, LPS-stimulated group, and LPS-stimulated + humic acid-bismuth group were detected by real-time quantitative PCR. The results showed that the relative mRNA expression levels of inflammatory factors IL-6, IL-1β, and TNF-α in RAW264.7 cells of the BiHA nanoparticle + LPS group were significantly lower than those in the LPS-stimulated group (P < 0.05). This indicates that BiHA nanoparticles can inhibit cellular inflammation.

[0035] 4. The ability of humic acid-bismuth nanomaterials to scavenge reactive oxygen species (ROS) was evaluated by in vitro ROS measurement and cellular ROS measurement. The results showed that humic acid-bismuth nanomaterials have a good ability to scavenge intracellular ROS. The effect of humic acid-bismuth nanomaterials on mitochondria was evaluated by mitochondrial membrane potential detection technology. The results showed that humic acid-bismuth nanomaterials can repair mitochondrial damage caused by inflammation.

[0036] 5. Chronic colitis was induced in mice via DSS, and humic acid-bismuth nanomaterials were administered to these mice via gavage. Disease activity score (DAI) was calculated, colon length was measured, MPO in colonic tissue was detected, and the expression levels of inflammatory factors IL-6, IL-1β, and TNF-α in mouse colonic tissue were detected by real-time quantitative PCR. Colonic tissue damage was assessed by HE staining. The results showed that humic acid-bismuth nanomaterials could alleviate DSS-induced ulcerative colitis in mice. Attached Figure Description

[0037] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments and descriptions of this application are used to explain this application and do not constitute an undue limitation of this application. In the drawings:

[0038] Figure 1 The following is a characterization of the BiHA nanoparticles in Example 1, where A: image of BiHA nanoparticles after freeze-drying; B: TEM image; C: elemental analysis of BiHA nanoparticle mapping; D: particle size analysis of BiHA nanoparticles; E: Zeta potential; F: UV absorption spectra of HA and BiHA.

[0039] Figure 2 The images shown are transmission electron microscope (TEM) images of BiHA nanomaterials prepared in Examples 2-4 and Comparative Examples 1-3, where A is Example 2, B is Example 3, C is Comparative Example 1, D is Example 4, E is Comparative Example 2, and F is Comparative Example 3.

[0040] Figure 3 The images show the cytotoxicity, cellular uptake, and anti-inflammatory experiments of BiHA nanoparticles in Example 1. A and B represent the toxicity experiments of different concentrations of BiHA nanoparticles on L-929 and RAW264.7 cells; C represents the hemolysis experiment of BiHA nanoparticles; D shows a confocal image of RAW264.7 cells uptake of Rhodamine B-loaded BiHA nanoparticles after 24 hours; E, F, and G represent the anti-inflammatory experiment of BiHA nanoparticles on RAW264.7 cells. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0041] Figure 4In Example 1, BiHA nanoparticles carrying siRNA were introduced into A549 cells. Specifically: the BiHA+NC group consisted of BiHA nanoparticles carrying si-NC chains co-incubated with cells for 24 hours; the BiHA group consisted of BiHA nanoparticles carrying cells for 24 hours; and the BiHA+siRNA group consisted of BiHA nanoparticles carrying si-lnc070974 co-incubated with cells for 24 hours.

[0042] Figure 5 This is an experiment on the antioxidant and mitochondrial damage repair effects of BiHA nanoparticles in Example 1. A: H2O2 scavenging experiment of different concentrations of BiHA nanoparticles; B: Quantitative analysis of ROS scavenging by BiHA using laser confocal fluorescence; C: Statistical analysis of mitochondrial membrane potential, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001; D: Confocal image of the experiment on LPS-induced ROS scavenging in RAW264.7 cells by BiHA; E: Experiment on the repair of LPS-induced mitochondrial damage in RAW264.7 cells by BiHA nanoparticles.

[0043] Figure 6 In Example 1, BiHA nanoparticles labeled with Cy5.5 were used for in vivo imaging of colonic inflammation sites in mice. In A: DSS-induced ulcerative colitis mice were administered Cy5.5-labeled BiHA by gavage, and in vivo imaging was performed at 6h, 12h, 24h, 48h, 72h, and 96h, respectively. In B: DSS-induced ulcerative colitis mice were administered Cy5.5-labeled BiHA by gavage, and anatomical tissue imaging was performed at 6h, 12h, 24h, 48h, 72h, and 96h, respectively, mainly including serum, heart, lung, liver, spleen, kidney, stomach, colon, and small intestine.

[0044] Figure 7 The BiHA nanoparticles used in Example 1 to alleviate DSS-induced ulcerative colitis in mice are shown in the figures. A: mouse body weight change; B: mouse DAI change; C: mouse spleen weight; D: mouse colon length; E: MPO activity in mouse liver tissue; F: HE staining results of colon tissue in each group of mice; G, H, I: detection results of inflammatory factors in mouse colon tissue. * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001. Detailed Implementation

[0045] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0046] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0047] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0048] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0049] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0050] In the embodiments of the present invention, room temperature refers to 25±2℃.

[0051] All raw materials used in the embodiments of the present invention were obtained through commercial purchase.

[0052] The technical solution of the present invention will be further illustrated by the following embodiments.

[0053] Example 1: Preparation of humic acid-bismuth (BiHA) nanomaterials

[0054] BiHA nanoparticles were prepared by hydrothermal method

[0055] 1. Add 200mg of humic acid (Humic Acid, Sigma, M) W =20000-50000) was dissolved in 8.0 mL ddH2O and stirred at room temperature to obtain a humic acid solution. 50 mg Bi(NO3)3·5H2O (Adamas, catalog number: 01000537) was added to 1.0 mL HNO3 solution (2M) and allowed to dissolve completely. Then, it was added dropwise to the above humic acid solution and stirred at room temperature for 30 min.

[0056] 3. Add 30 mg of FeCl3·6H2O and stir for 12 h. Then, further purify the obtained Bi-HA by dd H2O dialysis for 24 h to remove excess reactants.

[0057] 4. After 24 hours, the sample was removed, centrifuged at 14000g for 5 minutes, the supernatant was discarded, and the precipitate was frozen at -80℃ for 6 hours and then freeze-dried under vacuum overnight to finally obtain BiHA nanomaterials, which were uniform BiHA nanoparticles. Figure 1 (A)

[0058] Characterization of BiHA nanoparticles in Example 1:

[0059] 1. Observation of Bi-HA morphology by transmission electron microscopy (TEM)

[0060] (1) Weigh 100 mg of BiHA nanoparticles, add 10 mL of pure water, then add NaOH to dissolve, and then add HCl to adjust the pH to neutral to obtain a BiHA (10 mg / mL) solution.

[0061] (2) Take 1 mL of the above solution, dilute with pure water until the solution is almost colorless, and drop 20 μL onto a copper grid. Let it stand overnight at room temperature, then observe and photograph under an electron microscope. Figure 1 In the middle B, it can be observed that the BiHA nanoparticles are spherical with a particle size of 120-150 nm.

[0062] 2. Elemental mapping analysis of humic acid-bismuth nanoparticles

[0063] Elemental mapping detection and analysis of humic acid-bismuth nanoparticles, from Figure 1 The presence of a high Bi element in the BiHA nanoparticles was observed in the C-cell analysis, indicating that the BiHA nanoparticles were successfully synthesized.

[0064] 3. Nanoparticle tracking and analysis (NTA) instrument for analyzing BiHA particle size and quantity.

[0065] (1) Take 30 μL of BiHA solution (10 mg / mL) and add 15 mL of pure water to dilute it 500 times.

[0066] (2) Samples are fed into a nanoparticle tracking analyzer, and the average velocity of the particles is measured using the Stokes-Einstein equation to estimate the particle size and quantity. The nanoparticle size is mostly distributed between 120-150 nm. Figure 1 (D).

[0067] 4. Zeta potential

[0068] (1) Take 200 μL of BiHA solution (10 mg / mL) and add 1 mL of pure water to dilute it 6 times.

[0069] (2) The Zeta potential of the sample was detected on the instrument. The BiHA nanoparticles are negatively charged, and the potential is around -30mV. Figure 1 (E).

[0070] 5. Ultraviolet absorption spectrum

[0071] (1) Take 50 μL of HA / BiHA solution (10 mg / mL), add 950 μL of pure water to dilute 20 times, and take 200 μL to add to a 96-well plate.

[0072] (2) The absorbance at 230 nm-1000 nm was detected using a SpectraMax iD3 multi-microplate reader. Compared with HA, BiHA showed a significant absorption peak at 500-600 nm. Figure 1 (F) proves that BiHA was successfully synthesized.

[0073] Example 2

[0074] Same as Example 1, except for the M of humic acid. W =3000-10000. The BiHA nanoparticles synthesized in this embodiment are square or spherical in shape, with a particle size of 400-600 nm. Figure 2 (A)

[0075] Example 3

[0076] Same as Example 1, except for the M of humic acid. W =10000-30000. The BiHA nanoparticles synthesized in this embodiment are spherical or triangular in shape, with a particle size of 300-600 nm. Figure 2 (B)

[0077] Example 4

[0078] 1. Add 200mg of humic acid (Humic Acid, Sigma, M) W =20000-50000) was dissolved in 8.0 mL ddH2O and stirred at room temperature to obtain a humic acid solution. 100 mg Bi(NO3)3·5H2O (Adamas, catalog number: 01000537) was added to 2.0 mL HNO3 solution (2M) and allowed to dissolve completely. Then, it was added dropwise to the above humic acid solution and stirred at room temperature for 60 min.

[0079] 3. Add 30 mg of FeCl3·6H2O and stir for 18 h. Then, further purify the obtained Bi-HA by dd H2O dialysis for 24 h to remove excess reactants.

[0080] 4. After 24 hours, the sample was removed, centrifuged at 14000g for 5 minutes, the supernatant was discarded, and the precipitate was frozen at -80℃ for 6 hours and then freeze-dried under vacuum overnight. This example yielded uniform BiHA nanoparticles with a particle size of 500-600 nm. Figure 2 (D).

[0081] As can be seen from Examples 1-4, the molecular weight of humic acid can lead to differences in the particle size and uniformity of the prepared particles.

[0082] Comparative Example 1

[0083] Same as Example 1, except that step 2 is as follows: Take 8.0 mL of humic acid solution, stir at room temperature, add 0 mg Bi(NO3)3·5H2O (Adamas, catalog number: 01000537) to 1.0 mL of HNO3 solution (2M) (i.e., Bi(NO3)3·5H2O is not added in this comparative example), and wait for it to dissolve completely. Then add it dropwise to the purified humic acid solution and stir at room temperature for 30 min.

[0084] The BiHA nanoparticles obtained in this comparative example have irregular morphologies, mostly in the form of sheets or long chains. Figure 2 (C)

[0085] Comparative Example 2

[0086] Same as Example 1, except that step 2 is as follows: Take 8.0 mL of the purified humic acid solution, stir at room temperature, add 200 mg Bi(NO3)3·5H2O (Adamas, catalog number: 01000537) to 1.0 mL of HNO3 solution (2M) (i.e., Bi(NO3)3·5H2O is in excess in this comparative example), and after it is fully dissolved, add it dropwise to the purified humic acid solution and stir at room temperature for 30 min.

[0087] The BiHA nanoparticles obtained in this comparative example have a particle size of 500-800 nm and an uneven particle size distribution. Figure 2 (E).

[0088] Comparative Example 3

[0089] Same as Example 1, except that step 2 is as follows: Take 8.0 mL of the purified humic acid solution, stir at room temperature, add 400 mg Bi(NO3)3·5H2O (Adamas, catalog number: 01000537) to 1.0 mL of HNO3 solution (2M) (i.e., Bi(NO3)3·5H2O is in excess in this comparative example), and after it is fully dissolved, add it dropwise to the purified humic acid solution and stir at room temperature for 30 min.

[0090] The BiHA nanoparticles obtained in this comparative example had a particle size of 1-2 μm and an uneven particle size distribution. Figure 2 (F).

[0091] All subsequent tests were performed using the BiHA nanoparticles prepared in Example 1 as an example.

[0092] I. Cytotoxicity (MTT) Detection

[0093] 1. Take two 96-well plates and seed 10,000 L-929 or RAW264.7 cells into each well. For L-929 cells, add 100 μL of complete medium containing MEM, and for RAW264.7 cells, add complete medium containing DMEM (purchased from Gibico). Put them back into the incubator for culture.

[0094] 2. After culturing for 12 hours, remove the 96-well plate, discard the original culture medium, and add 100 μL of complete culture medium with BiHA concentrations of 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, and 6.25 μg / mL, respectively. For L-929 cells, add 100 μL of complete culture medium containing MEM, and for RAW264.7 cells, add complete culture medium containing DMEM. Continue culturing for 24 hours.

[0095] 3. After treating with different concentrations of BiHA for 24 hours, remove the MTT reagent, dilute it 10 times with MEM or DMEM complete medium, remove the 96-well plate, discard the original medium, add 100 μL of diluted MTT reagent to each well, and incubate in the incubator for 4 hours.

[0096] 4. After incubation for 4 hours, remove the 96-well plate, add 100 μL of Formazan solution to each well, and return it to the incubator for 4 hours.

[0097] 5. After incubation, remove the 96-well plate and use a SpectraMax iD3 multi-mode microplate reader to measure the OD value of each well at 570 nm. Figure 3 As shown in A and B, BiHA nanoparticles at concentrations of 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, and 6.25 μg / mL had no significant toxicity to L-929 and RAW264.7 cells.

[0098] II. Hemolytic Reaction Experiment of BiHA Nanoparticles

[0099] 1. Whole blood was obtained from healthy C57BL / 6J mice using ocular blood collection. The blood was collected in anticoagulant tubes containing EDTA or sodium citrate, shaken well, and centrifuged at 3000 rpm for 15 min to obtain blood cells for evaluation of the hemolysis rate of nanoparticles.

[0100] 2. Prepare nanoparticles of different concentrations. Take 1 mL of nanoparticle solution (25-125 μg / mL), 1 mL of pure water (ddH2O), and 1 mL of 1×PBS solution, and mix them with 20 μL of blood cells respectively.

[0101] 3. Incubate at 37℃ for 4 hours, then centrifuge at 3000 rpm for 15 minutes. Place the samples on a horizontal line and take a picture of the hemolysis phenomenon with a mobile phone. Figure 3 (C)

[0102] 4. Use a pipette to aspirate the sample supernatant (a 96-well plate or a quartz dish can be used, the latter being more sensitive), and measure the absorbance of the sample at 542 nm using a microplate reader. [Three parallel samples per group, calculate SD] Hemolysis rate calculation: Hemolysis rate (%) = [OD(sample) - OD(PBS group)] / [OD(ddH2O) - OD(PBS group)]. The results show that even at a concentration as high as 125 μg / mL, BiHA nanoparticles only have a hemolysis rate of 5.6% ( Figure 3 The presence of C indicates that BiHA has good biocompatibility.

[0103] III. BiHA nanoparticles as small molecule or nucleic acid carriers

[0104] 1. Co-incubation of Rhodamine B fluorescent dye with BiHA nanoparticles

[0105] (1) Take 500 μL of BiHA (10 mg / mL) and 600 μL of Rhodamine B (0.3 mg / mL), mix them evenly, and incubate them together at 37°C for 24 h in the dark.

[0106] (2) Under light-protected conditions, the above reaction solution was transferred into a 1.5 mL ultrafiltration tube (3 kd), and the reaction was carried out at 14000 rpm for 20 min. The upper filter residue was resuspended in 1 mL of 1×PBS.

[0107] (3) The above-mentioned BiHA loaded with Rhodamine B was filtered through a 0.22 μm filter membrane and stored at 4 °C in the dark.

[0108] 2. Treatment of RAW264.7 cells with BiHA nanoparticles loaded with Rhodamine B fluorescent dye, preparation of cell slides, and laser confocal imaging.

[0109] (1) Take a 24-well plate, place a round cell spreader in each well, and seed 5 × 10⁶ cells. 4 One RAW24.7 cell was placed back into the incubator for further culture.

[0110] (2) After culturing the cells for 12 hours, 2.5 μL of Rhodamine B-labeled BiHA nanoparticles were added to each well, and the cells were cultured for another 24 hours.

[0111] (3) Then collect the samples, add 500 μL of 1×PBS buffer to the corresponding wells, shake gently a few times, discard the PBS, and repeat the washing three times.

[0112] (4) Add 200 μL of 4% paraformaldehyde and fix at room temperature for 1 h.

[0113] (5) Take out the fixed smear, discard the 4% paraformaldehyde, add 500μL 1×PBS buffer, shake gently a few times, discard the PBS, and repeat the washing three times.

[0114] (6) Add 300 μL of DAPI staining solution and stain at room temperature in the dark for 15 min.

[0115] (7) Add 500 μL of 1×PBS buffer, shake gently a few times, discard the PBS, and repeat the washing process three times, leaving a small amount of PBS in the well plate.

[0116] (8) Take a clean glass slide, add a drop of anti-fluorescence quenching agent, pick up the slide with tweezers, carefully use the edge to contact the filter paper to absorb the moisture, invert it onto the anti-fluorescence quenching agent, drip neutral resin around the edge, let it stand at room temperature in the dark to solidify, and store it in the dark after it has stabilized.

[0117] (9) Laser confocal observation (see...) Figure 3 The results showed that RAW264.7 cells could take up BiHA nanoparticles after being co-incubated with BiHA nanoparticles for 24 h.

[0118] 3. Co-incubation of BiHA and siRNA

[0119] The siRNA used in this experiment was si-lnc070974, modified with 5'cy3, synthesized by Gemma Genetics, and its sequence is as follows:

[0120] Table 14 Interference sequences of lncRNA070974

[0121]

[0122] The BiHA nanoparticles prepared in Example 1 were dissolved in 1×PBS at a concentration of 1 μg / μL, and the siRNA was dissolved in 62.5 μL of DEPC water at a concentration of 20 μM. 10 μL of BiHA solution was taken and co-incubated with 5 μL of si-NC and 5 μL of si-lnc070974 at 4 °C in the dark for 6 h.

[0123] 4. BiHA-loaded siRNA treatment of A549 cells, slide preparation, and laser confocal imaging

[0124] (1) Take a 24-well plate, place a round cell spreader in each well, and seed 4 × 10⁶ cells. 4 One A549 cell was placed back into the incubator for further culture.

[0125] (2) After culturing the cells for 12 hours, add 5 μL of si-lnc070974 and 5 μL of BiHA+siRNA to each well and continue culturing for 24 hours.

[0126] (3) Then collect the samples, add 500 μL of 1×PBS buffer to the corresponding wells, shake gently a few times, discard the PBS, and repeat the washing three times.

[0127] (4) Add 200 μL of 4% paraformaldehyde and fix at room temperature for 1 h.

[0128] (5) Take out the fixed smear, discard the 4% paraformaldehyde, add 500μL 1×PBS buffer, shake gently a few times, discard the PBS, and repeat the washing three times.

[0129] (6) Add 300 μL of DAPI staining solution and stain at room temperature in the dark for 15 min.

[0130] (7) Add 500 μL of 1×PBS buffer, shake gently a few times, discard the PBS, and repeat the washing process three times, leaving a small amount of PBS in the well plate.

[0131] (8) Take a clean glass slide, add a drop of anti-fluorescence quenching agent, pick up the slide with tweezers, carefully use the edge to contact the filter paper to absorb the moisture, invert it onto the anti-fluorescence quenching agent, drip neutral resin around the edge, let it stand at room temperature in the dark to solidify, and store it in the dark after it has stabilized.

[0132] (9) Laser confocal observation (see...) Figure 4 The results showed that BiHA could carry siRNA into A549 cells.

[0133] The above results demonstrate that BiHA nanoparticles can serve as nucleic acid carriers.

[0134] IV. In vitro anti-inflammatory activity (RAW264.7)

[0135] 1. Take a 24-well plate and inoculate each well with 8 × 10⁸ g of the solution. 4 Add 500 μL of DMEM (purchased from Gibico) complete medium to each RAW264.7 cell and return them to the incubator for further culture.

[0136] 2. After culturing for 12 hours, remove the 24-well plate, discard the original culture medium, and add 500 μL of DMEM complete culture medium with BiHA concentrations of 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, and 6.25 μg / mL, respectively. Continue culturing for 20 hours, then add 500 μL of DMEM complete culture medium containing LPS, with a final LPS concentration of 10 μg / mL, and continue culturing for 4 hours.

[0137] 3. Extraction of total RNA from cells

[0138] RNA was extracted using the Invitrogen TRIzol method (purchased from TakaRa) according to the kit instructions; 1000 ng of total RNA was used for reverse transcription according to the instructions of the Nanjing Novizan kit (NO:R223-01).

[0139] (1) Discard the original culture medium in the 24-well plate, add an appropriate amount of 1×PBS buffer, gently shake the plate to wash the cells, and aspirate the PBS.

[0140] (2) Repeat step (1) to wash the cells again and remove the PBS.

[0141] (3) Add an appropriate amount of Trizol to each well (500 μL per well for 6-well plates, 300 μL per well for 12 / 24-well plates), gently shake the plate to completely dissolve the cells at the bottom of the plate with Trizol, transfer to an enzyme-free 1.5 mL centrifuge tube, place on ice, and let stand for 10 min.

[0142] (4) Add 0.2 times the volume of chloroform to each tube, immediately vortex vigorously for 10 seconds to mix thoroughly, insert into ice, and let stand for 10 minutes.

[0143] (5) Transfer the centrifuge tube to a centrifuge and centrifuge at 4°C and 14,000 rpm for 15 minutes.

[0144] (6) Take a new 1.5 mL deenzyme-free centrifuge tube and add 0.35 times the total volume of the liquid in step (5) to each tube (if the total volume in step 5 is 600 μL, add 210 μL of isopropanol here; if the total volume in step 5 is 360 μL, add 130 μL of isopropanol here), insert it on ice, and pre-cool until the centrifugation is finished.

[0145] (7) After centrifugation, carefully aspirate an equal amount of supernatant to isopropanol and add it to pre-cooled isopropanol. Mix by pipetting three times and then place on ice for RNA precipitation for 10 minutes.

[0146] (8) While waiting for precipitation, prepare 75% ethanol. Take a 50mL centrifuge tube without enzyme, add 10mL DEPC water and 30mL anhydrous ethanol in sequence, mix by inversion, and then insert it into ice for pre-cooling.

[0147] (9) After sedimentation, transfer the centrifuge tube to a centrifuge and centrifuge at 4°C and 14,000 rpm for 10 min.

[0148] (10) After centrifugation, carefully remove the centrifuge tubes, carefully discard the supernatant, add about 500 μL of 75% ethanol to each tube along the tube wall, invert 3 times, put them back into the centrifuge, and centrifuge at 4°C and 14000 rpm for 3 min.

[0149] (11) Repeat step (10) to wash the RNA again (at this time, observe the size of the RNA precipitate at the bottom, and the amount of DEPC water to be added will be determined based on the size of the precipitate).

[0150] (12) After centrifugation, carefully remove the centrifuge tube, carefully pour out the supernatant, put the centrifuge tube back into the centrifuge, and centrifuge for 30 seconds at 4°C and 14000 rpm.

[0151] (13) After the empty centrifugation is complete, remove the centrifuge tube, carefully aspirate the last remaining liquid at the bottom with a pipette, open the cap and place it in a 42°C metal thermostat for 2 minutes.

[0152] (14) Add an appropriate amount of DEPC water according to the size of the RNA precipitate, dissolve the RNA by blowing, and detect the RNA concentration using a Nanodrop visible spectrophotometer.

[0153] 4. Reverse transcription

[0154] The Nanjing Novizan reagent kit (NO:R223-01) was used with primers as described in Table 1; the gene primers used in this part of the experiment were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0155] Table 1 Primer sequences

[0156]

[0157] (1) Take a 200 μL centrifuge tube without enzyme and the Novizan reverse transcription reagent stored at -30℃. Add the samples according to Table 2. After adding all the samples into the centrifuge tube, vortex for 10 seconds, briefly separate the samples, and then place them in the PCR instrument. Set the program to run at 42℃ for 2 minutes.

[0158] Table 2 Reverse Transcription Reaction System I

[0159]

[0160] (2) After the 42℃ operation is completed, take out the centrifuge tube and add samples according to the following sample addition table 3.

[0161] Table 3 Reverse Transcription Reaction System II

[0162]

[0163]

[0164] (3) After adding to the centrifuge tube, vortex for 10 seconds, briefly separate, place in the PCR instrument, and run the program in Table 4.

[0165] Table 4 Reverse Transcription Procedure

[0166]

[0167] (4) After the program finishes running, the RNA is completely reverse transcribed into cDNA. Take out the cDNA, add 20 μL of DEPC water to dilute it, and store it at 4℃ for later use (Note: cDNA can be stored at 4℃ for several weeks. If you want to store it for a long time, please place it at -20℃).

[0168] 5. Real-time quantitative PCR

[0169] (1) Configure N+1 reaction systems as needed (N is the actual number of reaction systems for this gene). After mixing the total system, take out a 96-well qPCR reaction plate, add 18 μL of the system to the corresponding well, and then add 2 μL of cDNA. The specific system loading is shown in Table 5.

[0170] Table 5 Reaction System

[0171]

[0172] After adding samples to all wells, seal the wells with film, place them in a centrifuge, and centrifuge at 1000 rpm for 30 seconds.

[0173] (2) After centrifugation, take out the 96-well qPCR reaction plate and put it into the CFX96 Real-Time PCR System instrument for qPCR reaction. The program is shown in Table 6.

[0174] Table 6 PCR reaction procedure

[0175]

[0176]

[0177] (3) After the program finishes running, use BioRad CFX Manager software to export the Cq value, following step 2. -△△Ct (△Ct value = Ct) 目的 -Ct内参 △△Ct value = △Ct 实验组 -△Ct 对照组 The relative expression levels of genes were calculated, with the GAPDH gene used as a control gene, to detect the mRNA expression levels of inflammatory factors in cells (results are shown in...). Figure 3 As shown in Figures E, F, and G, RT-qPCR results indicated that the relative mRNA expression levels of inflammatory factors IL-6, IL-1β, and TNF-α in RAW264.7 cells of the BiHA nanoparticle + LPS group were significantly lower than those in the LPS-stimulated group (P < 0.05). BiHA nanoparticles can inhibit cellular inflammation.

[0178] V. BiHA's ability to scavenge reactive oxygen species (ROS)

[0179] 1. In vitro ROS measurement

[0180] (1) The scavenging activity of BiHA against ROS was determined by measuring the fluorescence signal of 2'-7' dichlorofluorescein diacetate (DCFH-DA, Sigma-Aldrich, catalog number: D6883) formed by H2O2 oxidation of 2'-7' dichlorofluorescein diacetate in the presence of BiHA.

[0181] (2) Using PBS as the control group, 50 μM DCFH-DA and 1 mM H2O2 were co-incubated at 37°C for 1 h in the presence of 1×PBS or BiHA (5 mg / mL).

[0182] (3) Use the fluorescence module of the SpectraMax iD3 multi-functional microplate reader to detect fluorescence intensity (excitation wavelength, 490nm, emission wavelength, 520nm). Figure 5 The DCF fluorescence intensity results showed that the BiHA nanoparticle + H2O2 group had a significantly lower DCF fluorescence intensity than the H2O2-stimulated group alone (P < 0.05). These results indicate that BiHA nanoparticles can scavenge ROS induced by H2O2.

[0183] 2. Cellular ROS Measurement

[0184] (1) Take a 24-well plate, place a round cell spreader in each well, and seed 5 × 10⁶ cells. 4 One RAW24.7 cell was placed back into the incubator for further culture.

[0185] (2) After culturing the cells for 18 hours, RAW 264.7 cells were treated with LPS (10 μg / mL). The treatment group was given 5-10 μL BiHA (1 mg / mL) nanoparticles and cultured for another 4 hours.

[0186] (3) Discard the solution and stain the cells with 50 μM DCFH-DA at 37°C for 15 min.

[0187] (4) Then collect the samples, add 500 μL of 1×PBS buffer to the corresponding wells, shake gently a few times, discard the PBS, and repeat the washing three times.

[0188] (5) Add 200 μL of 4% paraformaldehyde and fix at room temperature for 1 h.

[0189] (6) Take out the fixed smear, discard the 4% paraformaldehyde, add 500μL 1×PBS buffer, shake gently a few times, discard the PBS, and repeat the washing three times.

[0190] (7) Add 300 μL of DAPI staining solution and stain at room temperature in the dark for 15 min.

[0191] (8) Add 500 μL of 1×PBS buffer, shake gently a few times, discard the PBS, and repeat the washing process three times, leaving a small amount of PBS in the well plate.

[0192] (9) Take a clean glass slide, add a drop of anti-fluorescence quenching agent, pick up the slide with tweezers, carefully use the edge to contact the filter paper to absorb the moisture, invert it onto the anti-fluorescence quenching agent, drip neutral resin around the edge, let it stand at room temperature in the dark to solidify, and store it in the dark after it has stabilized.

[0193] (10) Laser confocal observation (see...) Figure 5 The results showed that the ROS level of the RAW264.7 cell group after LPS stimulation was 4.13 times that of the normal group. However, the ROS level of the RAW264.7 cell group after LPS+BiHA treatment was significantly lower than that of the RAW264.7 cell group after LPS stimulation, and was comparable to that of the normal group. Figure 5 The B-values ​​showed a significant difference (P < 0.05).

[0194] VI. BiHA Nanoparticles for the Treatment of Mitochondrial Damage

[0195] The reagent kits used in this experiment were provided by Aladdin, catalog number: M273063-100T

[0196] 1. Preparation of JC-1 staining working solution

[0197] The required amount of JC-1 staining working solution per well of a six-well plate is 1 mL. The amount of JC-1 staining working solution for other culture dishes is calculated accordingly. Take an appropriate amount of JC-1 (200×) and dilute it with 8 mL of ultrapure water for every 50 μL of JC-1 (200×). Shake vigorously to fully dissolve and mix the JC-1. Then add 2 mL of JC-1 staining buffer (5×) and mix well. This is the JC-1 staining working solution. Store at 4°C protected from light for later use.

[0198] 2. Cell plating and drug treatment

[0199] (1) Take a 24-well plate, place a round cell spreader in each well, and seed 5 × 10⁶ cells. 4 One RAW24.7 cell was placed back into the incubator for further culture.

[0200] (2) After culturing the cells for 18 hours, RAW 264.7 cells were treated with LPS (10 μg / mL). The treatment group was given 5-10 μL BiHA (1 mg / mL) nanoparticles and cultured for another 4 hours.

[0201] 3. JC-1 staining of cells

[0202] (1) Aspirate the culture medium, wash the cells once with 1×PBS, and add 1mL of DMEM cell culture medium.

[0203] (2) Add 1 mL of JC-1 staining working solution, mix thoroughly, and incubate at 37°C for 30 min in a cell culture incubator.

[0204] (3) During incubation, prepare an appropriate amount of 1×JC-1 staining buffer by adding 4mL of distilled water to every 1mL of 5×JC-1 staining buffer, and store it in a refrigerator at 4℃.

[0205] (4) After incubation at 37℃, discard the supernatant and wash three times with 1×JC-1 staining buffer.

[0206] 4. Cell fixation and observation

[0207] (1) Add 200 μL of 4% paraformaldehyde and fix at room temperature for 1 h.

[0208] (2) Take out the fixed smear, discard 4% paraformaldehyde, add 500μL 1×PBS buffer, shake gently a few times, discard PBS, and repeat the washing three times.

[0209] (3) Add 300 μL of DAPI staining solution and stain at room temperature in the dark for 15 min.

[0210] (4) Add 500 μL of 1×PBS buffer, shake gently a few times, discard the PBS, and repeat the washing process three times, leaving a small amount of PBS in the well plate.

[0211] (5) Take a clean glass slide, add a drop of anti-fluorescence quenching agent, pick up the slide with tweezers, carefully use the edge to contact the filter paper to absorb the moisture, invert it onto the anti-fluorescence quenching agent, drip neutral resin around the edge, let it stand at room temperature in the dark to solidify, and store it in the dark after it has stabilized.

[0212] (6) Laser confocal observation (see...) Figure 5 The results showed that the mitochondrial membrane potential of RAW264.7 cells decreased after LPS stimulation, but the mitochondrial membrane potential of the RAW264.7 group treated with LPS+BiHA recovered to the level of the normal group. Figure 4 The C-statistics showed a significant difference (P < 0.05).

[0213] VII. In vivo imaging in mice after oral administration of Cy5.5-labeled BiHA nanoparticles (Cy5.5@BiHA)

[0214] 1. Cy5.5 labeled BiHA

[0215] Take 0.8 mL (10 mg / mL) of BiHA, add 8 mg of EDC, add 22 mg of Sulfo-NHS, and add 3 mL of 1×MES buffer (0.1 M 2-[morpholino]ethanesulfonic acid, MES, 0.5 M NaCl, pH 6.0). Mix thoroughly and react at 37 °C for 30 min. Increase the pH of the buffer to above 7.0 using concentrated PBS or other non-amine buffers such as sodium bicarbonate. Add 0.5 mL (1 mg / mL) of Cy5.5 to the above solution, mix thoroughly, and incubate at room temperature for 2 h. Centrifuge at 14000 rpm in a 3K ultrafiltration tube for 15 min, collect the supernatant, resuspend in 3 mL of 1×PBS, and store at 4 °C protected from light.

[0216] 2. In vivo imaging of mice

[0217] (1) Take out two mice, weigh the mice, and calculate the dosage of anesthetic. The dosage of anesthetic is 8 μL / g (e.g., for a 20g mouse, 160 μL of 1% sodium pentobarbital needs to be injected).

[0218] (2) Pick up the mouse, expose its abdomen, and puncture the upper part of the thigh to inject an anesthetic into the abdominal cavity. After the injection, put the mouse back in the cage and wait for 3-5 minutes.

[0219] (3) After the mouse is completely comatose, the hair on the mouse's abdomen is shaved (to prevent interference with live imaging).

[0220] (4) Take 150 μL of Cy5.5-labeled BiHA and administer it into the mice by gavage, then return them to their cages.

[0221] (5) After gavage administration of Cy5.5-labeled BiHA for 6h, 12h, 24h, 48h, 72h, and 96h, the mice were re-anesthetized. After the mice were in a coma, they were placed in the IVIS Lumina in vivo imaging system for imaging. The excitation wavelength was selected as 660nm and the absorption wavelength was selected as 710nm.

[0222] The results showed that after gavage administration of Cy5.5@BiHA, a relatively strong fluorescent signal appeared in the abdomen of mice. Figure 6 In the case of Cy5.5@BiHA, the fluorescence signal gradually weakened over time, and the fluorescence signal could still be detected in the abdomen after 96 hours, suggesting that Cy5.5@BiHA can remain at the site of inflammation for a long time.

[0223] 3. Mouse tissue imaging

[0224] Mice were separately taken and administered 150 μL of Cy5.5@BiHA by gavage. The mice were then dissected at 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h. Organ tissues and serum were collected from the mice, including the heart, lungs, liver, stomach, spleen, kidneys, and all intestinal segments (see [link to original text]). Figure 6 (B) Using ChemiDoc MP The Imaging System performs fluorescence imaging on the collected organ tissues (excitation wavelength 715 nm).

[0225] The results showed that the fluorescence signal was still mainly present in the intestinal lumen, such as the colon and small intestine, with weak fluorescence visible in the stomach (see...). Figure 6 (B) The tissue imaging results were largely consistent with the in vivo imaging data, indicating that Cy5.5@BiHA has excellent imaging properties for sites of colonic inflammation in mice.

[0226] 8. BiHA Nanoparticle Therapy for DSS-Induced Ulcerative Colitis in Mice

[0227] 1. Mouse model construction

[0228] Thirty-six 9-week-old male mice were randomly divided into 6 groups and experimental chronic colitis was induced by DSS in 3 cycles. Specifically, colitis was induced by adding 2% (w / v) DSS (MW 36-50 kDa) to the mice's drinking water for 7 days, followed by normal drinking water for 9 days during the recovery period.

[0229] 2. BiHA gavage therapy

[0230] Control group mice received normal water throughout the process. The treatment period was designated as day 1 to day 48, during which BiHA 24 mg / kg was administered by gavage every two days. Animal weight, fecal consistency, and colonic bleeding were examined every two days. Disease activity score (DAI) was calculated based on the total score of weight loss, fecal consistency, and fecal blood volume. Figure 7 The body weight of mice in the α-DSS model decreased, but recovered somewhat after treatment with BiHA nanoparticles. Figure 7 The disease activity score of B showed that the DSS model mice had reduced body weight, severe hematochezia, and loose stools, and had a high DAI score. Compared with HA, BiHA nanoparticle treatment improved body weight, hematochezia, and stool consistency, and significantly reduced the DAI score.

[0231] Table 7 Disease Activity Scores

[0232]

[0233] 3. Removal of mouse colon

[0234] The specific steps are as follows:

[0235] (1) First, anesthetize the mice. Weigh the mice and calculate the dosage of anesthetic. The dosage of 1% sodium pentobarbital is 8 μL / g. Hold the mice and expose their abdomens. Make a puncture at the upper part of the thigh and inject the anesthetic into the abdominal cavity. After the injection, put the mice back in the cage and wait for them to become completely unconscious.

[0236] (2) Dissect the mouse, cut open the abdominal and thoracic cavities to fully expose all organs, observe changes in the color and shape of the colon, and take photos to record the findings. Figure 7 (F). Weigh the spleen ( Figure 7 In the DSS group, the spleen was enlarged and the spleen weight increased. In the DSS+BiHA nanoparticle group, the spleen returned to normal morphology and the weight returned to the level of normal mice.

[0237] (3) First, draw blood from the mouse heart using a 1mL syringe for subsequent testing. Then, remove the colon for further testing. Measure the colon length ( Figure 7 In the DSS group, the colon length of mice was significantly shorter than that of normal mice. After treatment with BiHA nanoparticles, the colon length returned to that of normal mice.

[0238] 4. MPO detection in mouse colon tissue

[0239] (1) Accurately weigh an appropriate amount of mouse colon tissue into a 1.5 mL centrifuge tube, and add 9 times the volume of anhydrous ethanol at a ratio of weight (g): volume (mL) = 1:9.

[0240] (2) The mixture was mechanically homogenized under ice-water bath conditions and ground thoroughly. Then it was centrifuged at 2500 rpm for 10 min, and the supernatant was collected for testing.

[0241] (3) The detection kit was purchased from Nanjing Jiancheng Biotechnology Institute (catalog number: A044-1-1). Results are shown below. Figure 7 In the middle E, it was observed that the MPO level in the colonic tissue of mice treated with BiHA by gavage was significantly lower than that in the DSS group (P<0.05). BiHA nanoparticles can alleviate DSS-induced oxidative stress in the mouse colon.

[0242] 5. Detection of inflammatory factors IL-6, IL-1β, and TNF-α in mouse colon tissue (qPCR)

[0243] (1) Cut an appropriate amount of mouse colon tissue into a 1.5 mL deenzyme-free centrifuge tube, add 300 μL of Trizol, and grind thoroughly at 4 °C. Then let stand on ice for 5 min.

[0244] (2) Add 1 / 5 volume of chloroform to each tube and immediately vortex for 15 seconds to mix thoroughly.

[0245] (3) Settle on ice again for 5 min, then centrifuge at 4°C, 14,000 rpm for 12 min.

[0246] (4) Pre-cool isopropanol and prepare the corresponding number of 1.5mL deenzyme centrifuge tubes and label them.

[0247] (5) Use a pipette to transfer an appropriate volume of supernatant (do not aspirate the white precipitate on the centrifuge tube wall or the Trizol reagent at the bottom of the tube) into the above-labeled de-enzyme centrifuge tube.

[0248] (6) Add an equal volume of isopropanol to precipitate the RNA, mix by inverting the container, and precipitate on ice for 10 min.

[0249] (7) Centrifuge at 4℃ and 14000rpm for 30min, and carefully discard the supernatant.

[0250] (8) Add 75% ethanol (prepared with DEPC water) to wash the precipitate, centrifuge at 14000 rpm for 5 min at 4℃, repeat the washing twice, carefully discard the supernatant, then centrifuge for 1 min, and dry the precipitate at 42℃.

[0251] (9) Add 10-20 μL DEPC water to dissolve the RNA.

[0252] (10) Reverse transcription and qPCR

[0253] I. Reverse transcription

[0254] Referring to the Nanjing Novizan reagent kit (NO:R223-01), the steps are as follows:

[0255] ①Removal of genomic DNA

[0256] Table 8 Reagents for removing genomic DNA

[0257]

[0258]

[0259] After gently mixing with a pipette, place the centrifuge tube into the PCR instrument and set the program to run at 42°C for 2 minutes.

[0260] ②Preparation of reverse transcription reaction system

[0261] Table 9 Reverse Transcription Reaction System

[0262]

[0263] Gently pipette the mixture to mix it thoroughly, then briefly centrifuge the centrifuge tube at 2000 rpm for 10 seconds before placing it in the PCR instrument for the next step.

[0264] ③Reverse transcription reaction

[0265] Table 10 Reverse Transcription Reaction Conditions

[0266]

[0267] After the PCR procedure is completed, the cDNA sample is stored in a -20°C freezer.

[0268] II. Real-time quantitative PCR (qRT-PCR)

[0269] The steps are as follows, referring to the Nanjing Novizan reagent kit (NO: Q311-02):

[0270] Table 11 Primer sequences

[0271]

[0272] ①Preparation of the reaction system

[0273] Table 12 Reaction System

[0274]

[0275] Mark the wells of the 96-well plate and add 20 μL of the above mixture to each well in sequence. After balancing the 96-well plate in a centrifuge, briefly centrifuge to collect the reaction solution.

[0276] ②PCR reaction (operated on a CFX96 Real-Time PCR System)

[0277] Table 13 PCR reaction procedure

[0278]

[0279] The relative expression levels of genes after the reaction were calculated according to 2. -△△Ct (△Ct value = Ct) 目的 -Ct 内参 , △△ Ct value = △ Ct 实验组 -△Ct 对照组 The relative expression levels of the genes were calculated, with the GAPDH gene used as a control gene, to detect the mRNA expression levels of inflammatory factors in mouse liver tissue (results are shown in...). Figure 7 As shown in H, I, and J), RT-qPCR results indicated that the relative mRNA expression levels of inflammatory factors IL-6, IL-1β, and TNF-α in the colonic tissue of DSS mice treated with BiHA nanoparticles were significantly lower than those in the DSS group (P < 0.05). BiHA nanoparticles can alleviate DSS-induced ulcerative colitis in mice.

[0280] 6. HE staining of mouse colon tissue

[0281] (1) After the mouse colon tissue was removed, 0.5-1 cm of the colon near the anus was cut off immediately and immersed in 4% paraformaldehyde fixative and stored at room temperature.

[0282] (2) HE staining of mouse colon tissue was performed by Wuhan Saiwei Medical Laboratory Co., Ltd. HE staining was used to observe the pathological condition of the colon tissue in each group of mice (see...). Figure 7 The results showed that oral administration of BiHA nanoparticles significantly reduced intestinal mucosal structure damage, crypt structure disorder, and inflammatory cell aggregation in the colon tissue of DSS mice, indicating that BiHA nanoparticles can alleviate DSS-induced ulcerative colitis in mice.

[0283] Cellular and animal experiments demonstrate that humic acid-bismuth nanoparticles are a promising diagnostic and therapeutic agent for ulcerative colitis, and are expected to be used in the treatment of inflammatory bowel disease.

[0284] The preparation of a humic acid-bismuth nanoparticle and its application in the diagnosis and treatment of inflammatory bowel disease were achieved through the preparation, characterization, safety evaluation, cell experiments, and animal experiments of the nanoparticle.

[0285] Through the validation of humic acid-bismuth nanoparticles and their functions, it was elucidated that oral administration of humic acid-bismuth nanoparticles can reduce inflammatory factors and reactive oxygen species, thereby treating and improving ulcerative colitis. Furthermore, after chemical conjugation of Cy5.5 fluorescent molecules, oral administration of the humic acid-bismuth nanoparticles, using in vivo and in vitro imaging in small animals, revealed that the humic acid-bismuth nanoparticles mainly accumulated in the inflamed areas of the colon in mice. The potential of this nanomaterial as a targeted therapeutic agent for inflammatory bowel disease has never been reported before.

[0286] The above are merely preferred embodiments of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. The application of a humic acid-bismuth nanomaterial in the preparation of drugs for treating diseases related to oxygen free radical damage, characterized in that, The preparation method of the humic acid-bismuth nanomaterial includes the following steps: Add a bismuth-soluble acidic solution dropwise to a humic acid solution; Add ferric iron, purify, centrifuge, freeze and vacuum dry the resulting precipitate to obtain humic acid-bismuth nanomaterials.

2. The application according to claim 1, characterized in that, The weight-average molecular weight of humic acid is 3,000-50,000.

3. The application according to claim 2, characterized in that, The weight-average molecular weight of humic acid is 20,000-50,000.

4. The application according to claim 1, characterized in that, Based on humic acid and bismuth, the mass ratio of humic acid solution to bismuth soluble acidic solution is (0.5:1) to (5:1).

5. The application according to claim 1, characterized in that, The trivalent iron is FeCl3·6H2O.

6. The application of a humic acid-bismuth nanomaterial in the preparation of a drug for treating inflammatory bowel disease, characterized in that, The humic acid-bismuth nanomaterial is the humic acid-bismuth nanomaterial of claim 1.

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