EGCG-5-HT binary oxidation self-polymerization nanoparticles as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine and pharmacology, and particularly relates to EGCG-5-HT binary oxidation self-agglutination nanoparticles as well as a preparation method and application of the EGCG-5-HT binary oxidation self-agglutination nanoparticles. The EGCG (epigallocatechin gallate)-5-HT binary oxidation auto-agglutination nanoparticles disclosed by the invention are obtained by carrying out oxidation auto-agglutination on EGCG and 5-HT in an alkaline environment. The total concentration of the EGCG and the 5-HT in the reaction liquid is 1.0 to 3.0 mg / mL. The nanoparticles provided by the invention have an anti-oxidation effect, and can inhibit pyroptosis and regulate and control a cell cycle; an NF-kappa B signal channel is inhibited, and the anti-inflammatory effect is achieved; an MAPK signal channel is activated, and cell apoptosis is inhibited; a PI3K-Akt signal channel is activated, and cell proliferation and mucous membrane repair are promoted. When the compound is applied to preparation of IBD medicines, multi-mechanism synergistic treatment of anti-inflammatory, anti-oxidation and mucous membrane repair of IBD is realized; the invention also provides a preparation method of the nanoparticle, and the preparation method is mild in condition and simple.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an EGCG-5-HT binary oxide self-polymerizing nanoparticle, its preparation method, and its application. Background Technology

[0002] Inflammatory bowel disease (IBD) is a chronic digestive system disease. Immune metabolic dysregulation of intestinal macrophages plays a crucial role in IBD development, with a large number of M1 macrophages infiltrating the lesion site. These macrophages continuously produce reactive oxygen species (ROS) and reactive nitrogen species (RONS), while releasing pro-inflammatory cytokines and chemokines, inducing necrosis of intestinal epithelial cells (IECs), disrupting the integrity of the intestinal barrier, and forming a vicious cycle of inflammation-intestinal mucosal damage, continuously weakening digestive system function.

[0003] Currently, anti-inflammatory drugs are widely used in the medical treatment of IBD, such as salicylates (e.g., 5-aminosalicylic acid), corticosteroids (e.g., hydrocortisone), monoclonal antibodies (e.g., infritillaria), and JAK-STAT inhibitors (e.g., tofacitinib). However, these drugs have significant limitations: they can only reduce inflammation and cannot repair damaged intestinal mucosa, and long-term use can cause significant side effects. 5-Aminosalicylic acid, as a first-line clinical drug, is easily inactivated by intestinal flora through acetylation; corticosteroids are mostly used for acute inflammation relief in IBD, but long-term use is ineffective and can easily cause serious side effects such as Cushing's syndrome; monoclonal antibodies have common defects in biological agents, easily inducing anti-drug antibodies (ADA) during maintenance therapy, affecting the neutralization of inflammatory cytokines; the safety of long-term use of JAK-STAT inhibitors is questionable, and serious cardiovascular events are prone to occur during treatment. Although multi-drug combination therapy in clinical practice has to some extent compensated for the shortcomings of monotherapy, it is still limited to "anti-inflammatory small molecule drugs + immunosuppressants" to enhance inflammation suppression and relieve symptoms, and the efficacy for IBD is unsatisfactory.

[0004] Epigallocatechin gallate (EGCG) is the most abundant polyphenolic active ingredient in green tea and has shown broad application potential in the biomedical field in recent years. Its mechanisms of action are diverse, mainly involving antioxidation, anti-inflammation, anti-infection, anti-tumor, neuroprotection, and metabolic regulation. However, it faces several challenges in practical applications, such as low oral bioavailability, short half-life, rapid metabolism, and poor stability.

[0005] Serotonin (5-HT) plays a variety of physiological functions in animals, participating in neural regulation, behavioral control, and metabolic processes. It also acts on intestinal 5-HT receptors to promote cell proliferation and differentiation and repair damaged intestinal mucosa. However, excessively high concentrations in the body can trigger 5-HT syndrome, causing adverse reactions such as myoclonus, mania, sweating, and confusion. Finding a suitable route of administration and dosage for 5-HT has become a challenge. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the above-mentioned defects of the prior art and provide an EGCG-5-HT binary oxide self-polymerized nanoparticle; its application in the preparation of inflammatory bowel disease (IBD) drugs opens up a new idea for the treatment of IBD through a multi-mechanism of "anti-inflammatory-antioxidant-mucosal repair"; the present invention also provides a method for preparing EGCG-5-HT binary oxide self-polymerized nanoparticles, which is mild and simple.

[0007] The EGCG-5-HT binary oxidative self-polymerizing nanoparticles of this invention are obtained by oxidative self-polymerization of EGCG and 5-HT in an alkaline environment.

[0008] The mass ratio of EGCG to 5-HT is 1:4. The total concentration of EGCG and 5-HT in the reaction solution is 1.0~3.0 mg / mL.

[0009] The obtained nanoparticles have a particle size between 80 and 250 nm, and the total number ratio of EGCG to 5-HT molecules is 1:10 to 10:1.

[0010] The alkaline environment has a pH of 7.5 to 9.5, and the stabilizer polysorbate-80 is added during the oxidation process.

[0011] Stabilizers and manganese chloride are added during the oxidation process.

[0012] The preparation method of the aforementioned EGCG-5-HT binary oxide self-polymerizing nanoparticles includes the following steps:

[0013] S1. Add 5-HT solution dropwise to water and stir until well mixed;

[0014] S2. Add the polysorbate-80 solution dropwise to the solution in step S1 and stir until homogeneous;

[0015] S3. Add the EGCG solution dropwise into the mixture from step S2 and stir until homogeneous;

[0016] S4. Add a buffer solution with pH 7.5~9.5 dropwise to the mixture from step S3 and stir until homogeneous.

[0017] S5. Dialyze the reaction solution obtained in step S4 to obtain EGCG-5-HT binary oxide self-polymerized nanoparticles. The dialysis treatment involves dispensing the reaction solution into dialysis bags and dialyzing for 0.5 to 3.0 hours per milliliter of reaction solution.

[0018] The stirring in steps S1, S2, S3, and S4 was carried out in a water bath at 10~30℃, and the stirring reaction time was 1.0~6.0h.

[0019] Between steps S4 and S5, add the following step: add manganese chloride solution to the mixture from step S4, incubate in a water bath at 10-30°C, and stir until homogeneous. The final molar concentration of MnCl2 in the reaction solution is 0-0.5 mmol / L. If MnCl2 is present in the reaction solution, it acts only as a catalyst to accelerate the oxidative self-polymerization reaction of EGCG and 5-HT, and does not participate in the formation of nanoparticles; if MnCl2 is not present in the reaction solution, EGCG and 5-HT can still oxidatively self-polymerize to form nanoparticles.

[0020] The buffer solution was Tris, i.e., tris(hydroxymethyl)aminomethane buffer. The final concentration of 5-HT in the reaction solution was 1.0–3.0 mg / mL, the final concentration of polysorbate-80 in the reaction solution was 1.0–3.0 mg / mL, the final concentration of EGCG in the reaction solution was 0.1–0.7 mg / mL, and the final molar concentration of Tris in the reaction solution was 5.0–20.0 mmol / L.

[0021] The application of the EGCG-5-HT binary oxide self-polymerizing nanoparticles is as follows: they are used to prepare drugs for inflammatory bowel disease (IBD). These drugs can be administered as injectable, topical, or oral formulations. The EGCG-5-HT binary oxide self-polymerizing nanoparticles can also be loaded with other therapeutic drugs, such as antibodies and organic acids. Specifically, these drugs can be used to prepare antioxidants, drugs that regulate macrophage polarization and have anti-inflammatory effects, drugs that inhibit apoptosis, drugs that inhibit pyroptosis, and drugs that regulate the cell cycle, promote cell proliferation, and accelerate cell renewal.

[0022] The EGCG-5-HT binary oxide self-polymerized nanoparticles provided by this invention, in addition to their own anti-inflammatory, antioxidant, apoptosis-inhibiting, and cell proliferation-promoting properties, can also serve as effective carriers for other drugs, such as antibody-loaded drugs, to achieve multifunctional therapeutic effects, enhance the efficacy of prepared drugs, and show great potential in the preparation of IBD therapeutic drugs.

[0023] This invention reveals that the EGCG-5-HT binary oxide self-polymerized nanoparticles possess broad-spectrum free radical scavenging capabilities and can be used in the preparation of drugs for various inflammatory diseases.

[0024] The EGCG-5-HT binary oxidized self-polymerized nanoparticles of the present invention significantly downregulate the proportion of M1 type macrophages in RAW264.7 macrophages stimulated by lipopolysaccharide (LPS), promote the conversion of M1 type to M2 type, and have a protective effect on cells. They can be used to prepare anti-inflammatory drugs that regulate macrophage polarization.

[0025] The EGCG-5-HT binary oxidative self-polymerizing nanoparticles of the present invention significantly inhibit DSS+IL-1β-induced apoptosis in NCM460 cells, have a protective effect on cells, and can be used in the preparation of drugs to inhibit apoptosis.

[0026] The EGCG-5-HT binary oxide self-polymerized nanoparticles of the present invention can significantly inhibit pyroptosis of NCM460 cells caused by various factors, and have a protective effect on cells. They can be used to prepare drugs that inhibit pyroptosis.

[0027] The EGCG-5-HT binary oxidative self-polymerizing nanoparticles of the present invention significantly increase the proportion of G2 / M phase cells in NCM460 cells after DSS+IL-1β treatment, thus protecting the cells. They can be used to regulate the cell cycle, promote cell proliferation, and accelerate the preparation of drugs for cell renewal.

[0028] Specifically, the preparation method of the EGCG-5-HT binary oxide self-polymerizing nanoparticles includes the following steps:

[0029] S1. Add 5-HT solution dropwise into ultrapure water, and heat in a constant temperature water bath at 10~30℃ with vigorous stirring.

[0030] S2. Add the polysorbate-80 solution dropwise into the solution described in step S1, and then heat in a constant temperature water bath at 10~30℃ with vigorous stirring.

[0031] S3. Add the EGCG solution dropwise into the mixture described in step S2, and stir vigorously in a constant temperature water bath at 10~30℃.

[0032] S4. Add a buffer solution with pH 7.5~9.5 dropwise to the mixture described in step S3, and heat in a constant temperature water bath at 10~30℃ with vigorous stirring.

[0033] S5. Dialyze the reaction solution obtained in step S4 to obtain EGCG-5-HT binary oxide self-polymerized nanoparticles.

[0034] Alternatively, in step S5, add the MnCl2 solution dropwise to the reaction solution obtained in step S4, and then heat in a constant temperature water bath at 10~30℃ with vigorous stirring.

[0035] S6. Dialyze the reaction solution obtained in step S5 to obtain EGCG-5-HT binary oxide self-polymerized nanoparticles.

[0036] This invention provides EGCG-5-HT binary oxide self-polymerizing nanoparticles with pH-responsive surface charge reversal properties, which significantly prolong their residence time at IBD inflammatory sites. These nanoparticles possess broad-spectrum free radical scavenging capabilities. In vitro cell experiments confirmed their anti-inflammatory and antioxidant activities, promoting cell proliferation and migration, inhibiting inflammation-induced apoptosis, regulating the cell cycle, and accelerating cell renewal. Animal experiments demonstrated their systemic therapeutic efficacy and good biosafety for IBD, and elucidated their molecular biological mechanism of action. Furthermore, these nanoparticles can also serve as carriers for other drugs.

[0037] Epigallocatechin gallate (EGCG) is the most effective active ingredient among tea polyphenols. Its abundant hydroxyl groups endow it with powerful antioxidant capabilities, neutralizing free radicals and reducing oxidative stress. Simultaneously, it exerts anti-inflammatory effects by regulating signaling pathways such as NF-κB, protecting cells, reducing intestinal inflammation, and comprehensively intervening in the complex pathological process of IBD. Serotonin (5-HT) is an important biogenic amine neurotransmitter. As an endogenous substance in the intestinal microenvironment, it regulates epithelial cell proliferation and repair through a complex cell signaling network, maintains intestinal physical barrier homeostasis, regulates intestinal peristalsis, promotes intestinal nerve maturation, and maintains mucosal integrity.

[0038] The binary oxidative self-polymerized nanoparticles formed by EGCG and 5-HT can exert the synergistic effect of these two active ingredients: they efficiently scavenge free radicals, exhibiting significant anti-inflammatory and antioxidant activities; at the same time, they also possess excellent abilities to protect the intestinal mucosa, such as promoting cell proliferation and migration, and inhibiting apoptosis and pyroptosis. These nanoparticles demonstrate significant efficacy, high biosafety, and a clear molecular biological mechanism in the synergistic treatment of IBD through "anti-inflammatory-antioxidant-mucosal repair," providing a new approach for the clinical drug treatment of IBD.

[0039] Compared with the prior art, the beneficial effects of the present invention are:

[0040] (1) The EGCG-5-HT binary oxide self-polymerizing nanoparticles of the present invention have polysorbate-80 added as a stabilizer, which makes the nanoparticles more uniformly dispersed and smaller in size, avoiding aggregation and precipitation between particles; the particle size is between 80 and 250 nm, the structure is simple, the dispersibility is good, the water solubility is good, and the biocompatibility is good.

[0041] (2) The preparation method of the EGCG-5-HT binary oxide self-polymerized nanoparticles of the present invention has excellent characteristics such as mild reaction conditions, simple preparation process, clear nanoparticle composition, inexpensive and readily available reagents, and the ability to prepare in batches, making it easy to promote and apply on a large scale.

[0042] (3) The EGCG-5-HT binary oxide self-polymerized nanoparticles prepared in this invention are applied in the preparation of IBD drugs. By inhibiting the NF-κB signaling pathway and working synergistically with their free radical scavenging ability, they achieve anti-inflammatory and antioxidant functions. These nanoparticles activate 5-HT... 1A Upon receptor activation, the nanoparticles not only cascade and activate the MAPK signaling pathway, reducing the cleavage of the DNA repair enzyme PARP and inhibiting apoptosis, but also upregulate the PI3K-Akt signaling pathway, promoting cell proliferation. This nanoparticle achieves the goal of synergistic treatment of IBD through multiple mechanisms of "anti-inflammatory, anti-oxidative, and mucosal repair," making it a potential multi-target, highly efficient nanomedicine for IBD treatment. Attached Figure Description

[0043] Figure 1 This is a schematic diagram of the synthesis process of EGCG-5-HT binary oxide self-polymerized nanoparticles (TPSNs) in Example 1.

[0044] Figure 2 This is a schematic diagram of the synthesis process of EGCG-5-HT binary oxide self-polymerized nanoparticles (TPSNs) in Example 2.

[0045] Figure 3 This is a schematic diagram of the synthesis process of EGCG-5-HT binary oxide self-polymerized nanoparticles (TPSNs) in Example 3.

[0046] Figure 4 (A) is a particle size distribution diagram of TPSNs in Example 1; Figure 4 (B) is a graph showing the average DLS particle size and PDI of TPSNs in Example 1; Figure 4 (C) is the Zeta potential diagram of TPSNs in Example 1.

[0047] Figure 5 (A) is a particle size distribution diagram of TPSNs in Example 2; Figure 5 (B) is a graph showing the average DLS particle size and PDI of TPSNs in Example 2; Figure 5 (C) is the Zeta potential diagram of TPSNs in Example 2.

[0048] Figure 6 (A) is a particle size distribution diagram of TPSNs in Example 3; Figure 6 (B) is a graph showing the average DLS particle size and PDI of TPSNs in Example 3; Figure 6 (C) is the Zeta potential diagram of TPSNs in Example 3.

[0049] Figure 7 (A) is a transmission electron microscope image of TPSNs from Example 1; Figure 7 (B) is a scanning electron microscope image of the TPSNs solution of Example 1 after heating and drying; Figure 7(C) is a scanning electron microscope image of the TPSNs solution after freeze-drying in Example 1.

[0050] Figure 8 The image shows the results of elemental qualitative analysis of the TPSNs in Example 1 using TEM.

[0051] Figure 9 The UV-Vis spectra of EGCG, 5-HT, and TPSNs from Example 1 are shown.

[0052] Figure 10 The infrared absorption spectrum (FT-IR) of TPSNs for EGCG, 5-HT, a physical mixture of EGCG and 5-HT, and Example 1.

[0053] Figure 11 (A) is the full X-ray photoelectron spectroscopy (XPS) spectrum of the TPSNs in Example 1; Figure 11 (B) is the high-resolution C 1s spectrum of the above TPSNs; Figure 11 (C) is the high-resolution O 1s spectrum of the above TPSNs; Figure 11 (D) is the high-resolution N 1s spectrum of the above TPSNs.

[0054] Figure 12 This is a gel permeation chromatography (GPC) chromatogram of TPSNs from Example 1.

[0055] Figure 13 The organic elemental analysis spectrum of TPSNs in Example 1; Figure 13 (A) is the CHNS model map; Figure 13 (B) is the O mode map.

[0056] Figure 14 The graph shows the changes in particle size and PDI of TPSNs from Example 1 in ultrapure water (A), HEPES (pH 7.4) (B), Tris (pH 7.4) (C), PBS (pH 7.4) (D), 1640 medium (E), and MEM medium (10% FBS) (F) from 0 to 48.0 h.

[0057] Figure 15 (A) Time-scavenging rate curves of DPPH free radicals scavenged using a series of concentration solutions of 0-500 μg / mL prepared with TPSNs from Example 1; Figure 15 (B) shows the UV-Vis spectra of the above-mentioned TPSNs at different concentrations reacting with DPPH solution for 40 min.

[0058] Figure 16 (A) To remove TPSNs prepared using a series of concentrations from 0 to 500 μg / mL Time-scavenging rate curve of free radicals; Figure 16 (B) represents the different concentrations of TPSNs mentioned above and UV-Vis spectrum of solution after 40 min of reaction.

[0059] Figure 17 (A) To inhibit the use of TPSNs prepared in Example 1 with a series of concentrations from 0 to 500 μg / mL Quantitative analysis results of capabilities (graph); Figure 17 (B) represents the effects of different concentrations of TPSNs on... Cleared UV-Vis spectrum.

[0060] Figure 18 (A) To inhibit the use of TPSNs prepared in Example 1 with a series of concentrations from 0 to 500 μg / mL Quantitative analysis results of capabilities (graph); Figure 18 (B) represents the effects of different concentrations of TPSNs on... Cleared UV-Vis spectrum.

[0061] Figure 19 (A) is Groups and Group, Figure 19 (B) is Groups and Group, Figure 19 (C) is Groups and Group, Figure 19 (D) is Groups and The electron spin resonance (ESR) spectra of the group, TPSNs were prepared in Example 1.

[0062] Figure 20 (A) is a ROS-ID fluorescent probe, Figure 20 (B) is a DCFH-DA fluorescent probe. Figure 20 (C) is a DAF-FM DA fluorescent probe. Figure 20 (D) is a DHE fluorescent probe. Figure 20 (E) is a fluorescence image of HPF fluorescent probe loaded onto different groups of RAW264.7 macrophages.

[0063] Figure 21 Immunofluorescence imaging of CD86 and CD206, polarization markers of RAW264.7 macrophages in each group.

[0064] Figure 22 (A) Flow cytometry analysis of CD86 and CD206, polarization markers of RAW264.7 macrophages in each group; Figure 22 (B) refers to the CD86 groups mentioned above.+ Quantitative analysis of the proportion of CD86-positive macrophages; Figure 22 (C) represents the CD206 groups mentioned above. + (CD206 positive) Quantitative analysis of the proportion of macrophages.

[0065] Figure 23 (A) is a flow cytometry analysis of apoptosis in NCM460 cells in each group; Figure 23 (B) refers to the Annexin V groups mentioned above. + / PI + The results of quantitative analysis of NCM460 cell populations are shown in the figure.

[0066] Figure 24 (A) is a flow cytometry analysis of NCM460 cells in the Ctrl group and the TPSNs (prepared from TPSNs prepared in Example 1) group; Figure 24 (B) is a graph showing the results of the quantitative analysis of the cell cycle in the two groups mentioned above; Figure 24 (C) shows the cell cycle flow cytometry analysis of NCM460 cells in the DI group and the DI+TPSNs (prepared from TPSNs prepared in Example 1) group; Figure 24 (D) is a graph showing the results of the quantitative analysis of the cell cycle in the two groups mentioned above.

[0067] Figure 25 (A) is a graph showing the changes in body weight of C57BL / 6J mice in each group; Figure 25 (B) is a graph showing the changes in the Disease Activity Index (DAI) of mice in each group; Figure 25 (C) is a representative photograph of anal bleeding in mice of each group on day 12.

[0068] Figure 26 (A) is a representative photograph of the colon length of mice in each group; Figure 26 (B) is a graph showing the statistical analysis results of colon length in each group of mice.

[0069] Figure 27 H&E staining images of colon tissue from each group of mice.

[0070] Figure 28 PAS and AB-PAS staining images of colon tissue from mice in each group.

[0071] Figure 29 Immunohistochemical staining images of ZO-1 and Occludin in the colon tissue of mice in each group.

[0072] Figure 30 Immunofluorescence analysis of MUC-2 expression levels in colon tissue of mice in each group.

[0073] Figure 31Immunofluorescence analysis of Caspase 3 expression levels in colon tissues of mice in each group.

[0074] Figure 32 The graph shows the concentrations of pro-inflammatory cytokines IL-6, TNF-α, IL-1β, IL-18 and anti-inflammatory cytokines IL-10 in the serum of mice in each group.

[0075] Figure 33 This is a graph showing the gene transcription levels of pro-inflammatory cytokines IL-6, TNF-α, and IL-1β in the colon tissue of mice in each group.

[0076] Figure 34 (A) is a graph showing the ALT and AST levels in the serum of mice in each group (liver function indicators); Figure 34 (B) is a graph showing the levels of Cre and BUN in the serum of mice in each group (a renal function indicator).

[0077] Figure 35 H&E staining images of the heart, liver, spleen, lungs and kidneys of mice in each group.

[0078] Figure 36 This diagram shows the expression levels of p-IKKα / β, p-p65, and p-IκBα, key regulatory proteins in the NF-κB signaling pathway.

[0079] Figure 37 Image of PI (red) uptake by primary macrophages in each group.

[0080] Figure 38 The figure shows the results of detecting the concentrations of pro-inflammatory cytokines IL-1α (A, B, C) and IL-1β (D, E, F) in the supernatant of primary macrophage culture medium in each group using an ELISA kit.

[0081] Figure 39 Immunofluorescence imaging of GSDMD in primary macrophages from each group.

[0082] Figure 40 5-HT for NCM460 cells 1A Receptor immunofluorescence imaging.

[0083] Figure 41 This diagram shows the expression levels of p-MEK and p-ERK1 / 2, key regulatory proteins in the MAPK signaling pathway, and p-AKT and p-mTOR, key regulatory proteins in the PI3K-Akt signaling pathway.

[0084] Figure 42 A graph showing the cleavage levels of the DNA repair enzyme PARP in each group of NCM460 cells. Detailed Implementation

[0085] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0086] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art; unless otherwise specified, the reagents used in the embodiments are all commercially available.

[0087] Unless otherwise specified, the percentage sign "%" in this invention refers to the mass percentage; however, the percentage of a solution, unless otherwise specified, refers to the number of grams of solute contained in 100 mL of solution.

[0088] The weight parts mentioned in this invention can be weight units known in the art such as μg, mg, g, kg, or multiples thereof, such as 1 / 10, 1 / 100, 10 times, 100 times, etc.

[0089] The names and manufacturers of the main reagents used in the following examples are shown in Table 1.

[0090] Table 1. Main Reagent Names and Manufacturers

[0091]

[0092]

[0093]

[0094]

[0095] For details of the instruments and manufacturers used in the following embodiments, please refer to Table 2.

[0096] Table 2. Main Instrument Names and Manufacturers

[0097]

[0098]

[0099] Example 1

[0100] The preparation method of the aforementioned EGCG-5-HT binary oxide self-polymerizing nanoparticles (TPSNs) includes the following steps:

[0101] (1) Under constant temperature water bath conditions of 20℃, add 20.34mL of ultrapure water to a 100mL beaker, add 4.80mL of 10.0mg / mL 5-HT solution, and stir evenly.

[0102] (2) Take 600 μL of 100.0 mg / mL polysorbate-80 solution and add it dropwise to the solution in step (1) above, and stir until homogeneous.

[0103] (3) Take 1.20 mL of 10.0 mg / mL EGCG solution and add it dropwise into the mixed solution in step (2) above, and stir well.

[0104] (4) Take 3.00 mL of 100.0 mmol / L pH 8.0 Tris buffer solution and add it to the mixed solution in step (3) above, and stir well.

[0105] (5) Take 60 μL of 100.0 mmol / L MnCl2 solution and add it dropwise into the mixed solution of step (4) above, and stir until homogeneous.

[0106] (6) The reaction solution from step (5) above was stirred vigorously for 2.0 h. The reaction solution was divided into 5 dialysis bags, each containing 6.00 mL, and dialyzed for 6.0 h to obtain EGCG-5-HT binary oxide self-polymerized nanoparticles (TPSNs). The TPSNs were stored at 4°C for later use.

[0107] The above schematic diagram illustrates the synthesis process of EGCG-5-HT binary oxide self-polymerizing nanoparticles (TPSNs). Figure 1 As shown.

[0108] Example 2

[0109] The preparation method of the aforementioned EGCG-5-HT binary oxide self-polymerizing nanoparticles (TPSNs) includes the following steps:

[0110] (1) Under constant temperature water bath conditions of 20℃, add 20.34mL of ultrapure water to a 100mL beaker, add 4.80mL of 10.0mg / mL 5-HT solution, and stir evenly.

[0111] (2) Take 600 μL of 100.0 mg / mL polysorbate-80 solution and add it dropwise to the solution in step (1) above, and stir until homogeneous.

[0112] (3) Take 1.20 mL of 10.0 mg / mL EGCG solution and add it dropwise into the mixed solution in step (2) above, and stir well.

[0113] (4) Take 3.00 mL of 100.0 mmol / L pH9.0 Tris buffer solution and add it to the mixed solution in step (3) above, and stir well.

[0114] (5) Take 60 μL of 100.0 mmol / L MnCl2 solution and add it dropwise into the mixed solution in step (4) above, and stir until homogeneous.

[0115] (6) The reaction solution from step (5) above was stirred vigorously for 2.0 h. The reaction solution was divided into 5 dialysis bags, each containing 6.00 mL, and dialyzed for 6.0 h to obtain EGCG-5-HT binary oxide self-polymerized nanoparticles (TPSNs). The TPSNs were stored at 4°C for later use.

[0116] The above schematic diagram illustrates the synthesis process of EGCG-5-HT binary oxide self-polymerizing nanoparticles (TPSNs). Figure 2 As shown.

[0117] Example 3

[0118] The preparation method of the aforementioned EGCG-5-HT binary oxide self-polymerizing nanoparticles (TPSNs) includes the following steps:

[0119] (1) Under constant temperature water bath conditions of 20℃, add 20.34mL of ultrapure water to a 100mL beaker, add 4.80mL of 10.0mg / mL 5-HT solution, and stir evenly.

[0120] (2) Take 600 μL of 100.0 mg / mL polysorbate-80 solution and add it dropwise to the solution in step (1) above, and stir until homogeneous.

[0121] (3) Take 1.20 mL of 10.0 mg / mL EGCG solution and add it dropwise into the mixed solution in step (2) above, and stir well.

[0122] (4) Take 3.00 mL of 100.0 mmol / L pH 8.0 Tris buffer solution and add it to the mixed solution in step (3) above, and stir well.

[0123] (5) Stir the reaction solution from step (4) above vigorously for 2.0 h. Divide the reaction solution into 5 dialysis bags, each containing 6.00 mL, and dialyze for 6.0 h to obtain EGCG-5-HT binary oxide self-polymerized nanoparticles (TPSNs). Store at 4°C for later use.

[0124] The above schematic diagram illustrates the synthesis process of EGCG-5-HT binary oxide self-polymerizing nanoparticles (TPSNs). Figure 3 As shown.

[0125] Experimental Example 1

[0126] Characterization and colloidal stability study of EGCG-5-HT binary oxide self-polymerizing nanoparticles (TPSNs)

[0127] I. Particle size and Zeta potential

[0128] Detection method: Take 100 μL of the TPSNs solution prepared in Examples 1, 2 and 3, dissolve it in 900 μL of ultrapure water, mix well, and obtain the sample solution to be tested. Transfer all of them to the sample cell and place them in a Malvern ZS-90 nanoparticle size and ZETA potential analyzer. The particle size is detected by dynamic light scattering (DLS) method and the ZETA potential is measured in buffer solutions with different pH values. The test temperature is 25℃, and each sample is tested in parallel 3 times.

[0129] The results of Example 1 are as follows Figure 4 (A) Figure 4 (B) Figure 4 As shown in (C), the TPSNs of Example 1 have an average DLS particle size of approximately 97 nm and a uniform particle size distribution, with a PDI < 0.1. The potential in 10.0 mmol / L pH 5.5 HEPES buffer is 4.81 ± 0.55 mV; the potential in 10.0 mmol / L pH 7.4 HEPES buffer is -10.80 ± 0.20 mV, exhibiting a potential reversal phenomenon of "positive for acid, negative for base".

[0130] The results of Example 2 are as follows Figure 5 (A) Figure 5 (B) Figure 5 As shown in (C), the TPSNs of Example 2 have an average DLS particle size of approximately 156 nm and a uniform particle size distribution, with a PDI < 0.2. The potential in 10.0 mmol / L pH 5.5 HEPES buffer is 2.37 ± 0.17 mV; the potential in 10.0 mmol / L pH 7.4 HEPES buffer is -12.87 ± 0.57 mV, exhibiting a potential reversal phenomenon of "positive for acid, negative for base".

[0131] The results of Example 3 are as follows Figure 6 (A) Figure 6 (B) Figure 6 As shown in (C), the TPSNs of Example 3 have an average DLS particle size of approximately 247 nm with a uniform particle size distribution and a PDI < 0.2. The potential is 0.57 ± 0.05 mV in 10.0 mmol / L pH 5.5 HEPES buffer and -16.03 ± 0.38 mV in 10.0 mmol / L pH 7.4 HEPES buffer, exhibiting a potential reversal phenomenon of "positive for acid and negative for base".

[0132] II. Appearance and Micromorphological Characterization

[0133] Detection methods: (1) Take 100 μL of the TPSNs solution prepared in Example 1, add it dropwise to the surface of the copper mesh covered with carbon film using a pipette, place the copper mesh in an electric heating drying oven to dry, repeat 5 times, and examine the morphology of the sample using a spherical aberration corrected transmission electron microscope (TEM). (2) Take 20 μL of the TPSNs solution prepared in Example 1, add it dropwise to the surface of the silicon wafer, place the silicon wafer in an electric heating drying oven to dry, and examine the morphology of the sample using a scanning electron microscope (SEM). (3) Take 1.50 mL of the TPSNs solution prepared in Example 1, freeze-dry it using a vacuum freeze dryer, attach the freeze-dried sample to the sample stage, and examine the morphology of the freeze-dried sample using a scanning electron microscope (SEM).

[0134] Figure 7 (A) is a transmission electron microscope image of TPSNs in Example 1, with a scale bar of 100 nm; Figure 7 (B) is a scanning electron microscope image of the TPSNs solution of Example 1 after heating and drying, with a scale bar of 200 nm; Figure 7 (C) is a scanning electron microscope image of the TPSNs solution of Example 1 after freeze-drying, with a scale bar of 200 nm. As can be seen from the image, the TPSNs of the present invention are uniform particles under an electron microscope, with spherical or near-spherical structures, regular and smooth edges, and good dispersibility.

[0135] III. Elemental Analysis of the Outer Surface of TPSNs

[0136] Measurement methods: (1) The elemental composition of the sample in method (1) of the "II. Appearance and Micromorphology Characterization" section is qualitatively analyzed by the elemental mapping analysis function of TEM. (2) The elemental composition of the sample in method (2) of the "II. Appearance and Micromorphology Characterization" section is quantitatively analyzed by the energy dispersive spectrometer (EDS) equipped with SEM.

[0137] Figure 8The image shows the results of elemental qualitative analysis of the TPSNs from Example 1 using TEM, with a scale bar of 50 nm. As can be seen from the image, the high-angle annular dark-field imaging (HAADF) image shows the position and morphology of the TPSNs under the electron microscope. Within the outline of the TPSNs shown in the HAADF image, a large number of elemental clusters are observed in the carbon (C), oxygen (O), and nitrogen (N) images, indicating that the surface of the TPSNs is mainly composed of C, O, and N elements. No elemental clusters are observed in the manganese (Mn) and chlorine (Cl) images, indicating that Mn and Cl elements in MnCl2 were not detected, suggesting that MnCl2 only played a catalytic oxidation role and did not participate in the formation of nanoparticles. Table 3 shows the results of elemental quantitative analysis of the TPSNs from Example 1 using EDS. As shown in the table, the mass percentages (Wt%) of C, O, and N elements on the surface of TPSNs are 69.93%, 27.26%, and 2.82%, respectively; the atomic percentages (At%) are 75.35%, 22.05%, and 2.60%, respectively. These results indicate that TPSNs contain both EGCG and 5-HT components, and are nanoparticles with spherical or near-spherical structures formed by the co-oxidation and self-aggregation of both.

[0138] Table 3 Elemental quantitative analysis results of TPSNs

[0139]

[0140] IV. Ultraviolet-Vis Spectrum (UV-Vis)

[0141] EGCG, 5-HT, and the TPSNs prepared in Example 1 were subjected to UV-Vis spectroscopy scans, respectively. Figure 9 This is the UV-Vis spectrum. The graph shows that TPSNs have a UV absorption peak around 227 nm, which is consistent with... The presence of electronic transitions indicates that TPSNs retain the benzene ring structure of EGCG and the indole ring structure of 5-HT. TPSNs exhibit a UV absorption peak around 275 nm, but its intensity is weaker than that of 5-HT at this point. This is due to the bonding between the phenolic hydroxyl groups and the nitrogen atoms on the indole rings of TPSNs and other molecules. Therefore, TPSNs are produced by the co-oxidative self-polymerization of EGCG and 5-HT.

[0142] V. Infrared Absorption Spectroscopy (FT-IR)

[0143] FT-IR spectroscopy was performed on EGCG, 5-HT, a physical mixture of EGCG and 5-HT, and the TPSNs prepared in Example 1. Figure 10The figure shows the FT-IR spectrum. As can be seen from the figure, the characteristic peaks of the infrared spectrum of TPSNs are similar to those of the physical mixture of EGCG and 5-HT, indicating that it is composed of EGCG and 5-HT. Comparing the infrared spectra of TPSNs with those of EGCG and 5-HT, respectively, the absorption peak intensity is weakened due to the oxidative self-polymerization reaction, further indicating that EGCG and 5-HT co-oxidatively self-polymerize to form TPSNs.

[0144] VI. X-ray photoelectron spectroscopy (XPS)

[0145] X-ray photoelectron spectroscopy (XPS) was performed on the TPSNs prepared in Example 1. Figure 11 (A) The XPS full spectrum shows that TPSNs contain C, O, and N elements. From... Figure 11 (B) The high-resolution C 1s spectrum shows that 284.80 eV is... Peak, 286.29 eV Peak, 288.78 eV Peak. By Figure 11 (C) The high-resolution O 1s spectrum shows that the peak at 532.31 eV is assigned to... The peak at 532.85 eV is assigned to... Peak. In the high-resolution spectrum of O 1s The significantly increased proportion of peaks indicates that EGCG and 5-HT undergo a co-oxidative self-polymerization reaction to form TPSNs. Figure 11 (D) High-resolution N 1s spectra show that TPSNs possess the 5-HT structure. Peak (399.63 eV) and The peak (401.67 eV) and the peak at 398.22 eV, indicating that 5-HT undergoes oxidative polymerization, are also observed. Peaks. XPS analysis suggests that EGCG and 5-HT undergo co-oxidative self-polymerization to form TPSNs.

[0146] VII. Gel Permeation Chromatography (GPC)

[0147] The homogeneity and molecular weight of the TPSNs prepared in Example 1 were analyzed using gel permeation chromatography (GPC). Figure 12 The figure shows the GPC plot, and Table 4 shows the GPC characterization results. The figure shows that TPSNs exhibit a single peak and a relatively symmetrical distribution, indicating that they are homogeneous nanoparticles. The table shows that TPSNs have a molecular weight distribution of Mw = 3306 g / mol and a molecular weight distribution of Mn = 2723 g / mol, with a relatively low polydispersity index, close to that of ideal polymers (Mw / Mn = 1), indicating that TPSNs have a relatively concentrated molecular weight distribution and high purity.

[0148] Table 4. GPC characterization results of TPSNs

[0149]

[0150] VIII. Quantitative Analysis of Organic Elements

[0151] The TPSNs prepared in Example 1 were subjected to quantitative analysis of organic elements using an organic elemental analyzer. Figure 13 The organic elemental analysis spectrum of TPSNs is shown in Table 5, and the quantitative analysis results of organic elements are presented. As shown in the graph and table, C: 58.57%, H: 7.003%, N: 2.15%, O: 29.689%, and S is absent. Within the allowable error range, this result is similar to the quantitative elemental analysis results of the TPSNs surface by EDS in section "III. Elemental Analysis of the Outer Surface of Nanoparticles" (mass percentages: C: 69.93%, N: 2.82%, O: 27.26%). This indicates that EGCG and 5-HT jointly constitute TPSNs, and both components are uniformly distributed on the surface and inside the nanoparticles.

[0152] Calculations show that the theoretical elemental content of 5 EGCG molecules and 2 5-HT molecules is C: 59.05%, H: 4.35%, N: 2.12%, and O: 34.49%. Within the allowable error range, this theoretical calculation value is similar to the quantitative detection results of organic elements in TPSNs of this invention, confirming that the total ratio of EGCG to 5-HT molecules in the entire TPSNs system is 5:2.

[0153] Table 5. Quantitative analysis results of organic elements in TPSNs

[0154]

[0155] IX. Colloidal Stability Study

[0156] The TPSNs prepared in Example 1 were dispersed in ultrapure water, HEPES buffer (10.0 mmol / L, pH 7.4), Tris buffer (10.0 mmol / L, pH 7.4), PBS buffer (10.0 mmol / L, pH 7.4), 1640 medium and MEM medium (containing 10% FBS), and placed at room temperature (25°C). Samples were taken at 0, 2.0, 6.0, 12.0, 18.0, 24.0, 36.0 and 48.0 h to determine the changes in TPSN particle size and PDI.

[0157] Stability results are as follows Figure 14 As shown in the figure, the particle size and PDI of TPSNs did not change significantly within 48.0 h, indicating that TPSNs have good stability in ultrapure water, HEPES buffer, Tris buffer, PBS buffer, 1640 medium and MEM medium from 0 to 48.0 h under room temperature conditions.

[0158] Experiment Example 2

[0159] Investigation of the scavenging activity of TPSNs prepared in Example 1 against RONS

[0160] I. Study on the free radical scavenging performance of DPPH

[0161] According to the instructions of the DPPH free radical scavenging ability test kit, the DPPH powder was dissolved and diluted to the working concentration with a universal diluent. 190 μL of DPPH working solution was mixed thoroughly with 10 μL of TPSNs (prepared using TPSNs prepared in Example 1) at concentrations of 20, 50, 100, 200 and 500 μg / mL, and a blank group (reaction system without TPSNs) was set up. The reaction solution was added to a 96-well plate, and the absorbance value (OD) at 515 nm at each set time point was detected using an ELISA reader. The scavenging rate was calculated according to the following formula (1), and a time-scavenging rate curve was plotted. After 40 min of reaction, the UV-Vis spectra of each group of samples were collected at 400~800 nm.

[0162] (1)

[0163] Figure 15 (A) Time-scavenging rate curves of DPPH free radicals scavenged using a series of concentration solutions of 0-500 μg / mL prepared with TPSNs from Example 1; Figure 15 (B) shows the UV-Vis spectra of the above-mentioned TPSNs reacting with DPPH solution at different concentrations for 40 min, with the inset showing photographs of the reaction solutions at different concentrations at the end of the reaction. Figure 15 (A) indicates that the longer the reaction time, the higher the DPPH scavenging rate of the same concentration of TPSNs. From Figure 15 (B) It can be seen that the higher the concentration of TPSNs, the lower the absorbance at 515 nm, the lighter the purple color of the solution at the end of the reaction, and the higher the DPPH scavenging rate. The results show that TPSNs scavenge DPPH rapidly and effectively in a time- and concentration-dependent manner.

[0164] two, Free radical scavenging performance study

[0165] Follow the instructions for the Total Antioxidant Capacity Assay Kit (ABTS method). Vortex mix 100 μL of ABTS solution with 100 μL of oxidant solution and incubate at room temperature in the dark for 24.0 h to fully eliminate background interference. Dilute the reacted ABTS working stock solution 40 times to prepare the ABTS working solution, store at room temperature in the dark, and use for later use.

[0166] In a 96-well plate, 200 μL of ABTS working solution and 10 μL of TPSNs (prepared using TPSNs prepared in Example 1) with concentrations of 20, 50, 100, 200, and 500 μg / mL were added to each well, and a blank group (reaction system without TPSNs) was set up. The OD value at 734 nm at each set time point was detected using an ELISA reader, and the clearance rate was calculated according to the following formula (2), and a time-clearance rate curve was plotted. After 40 min of reaction, the UV-Vis spectra of each group of samples in the range of 400–1000 nm were collected.

[0167] (2)

[0168] Figure 16 (A) To remove TPSNs prepared using a series of concentrations from 0 to 500 μg / mL Time-scavenging rate curve of free radicals; Figure 16 (B) represents the different concentrations of TPSNs mentioned above and UV-Vis spectra of the solution after 40 min of reaction, with insets showing photographs of reaction solutions at different concentrations at the end of the reaction. Figure 16 (A) It can be seen that the longer the reaction time, the greater the effect of the same concentration of TPSNs on... The higher the clearance rate, the better. Figure 16 (B) It can be seen that the higher the concentration of TPSNs, the lower the absorbance at 734 nm, and the lighter the blue-green color of the solution at the end of the reaction. The higher the clearance rate, the better. Results show that TPSNs have a higher clearance rate. The removal is rapid and effective, and is time- and concentration-dependent.

[0169] III. Hydroxyl radicals ( Cleaning performance study

[0170] Follow the instructions for the hydroxyl radical test kit. Mix 200 μL of substrate application solution with 200 μL of TPSNs (prepared using TPSNs prepared in Example 1) at concentrations of 50, 100, 200, and 500 μg / mL. Add 400 μL of reagent solution and immediately place the mixture in a 37°C water bath for 1 min. Immediately add the colorimetric reagent to terminate the reaction. Measure the OD value at 550 nm using a microplate reader. Calculate the hydroxyl radical inhibition capacity according to the following formula (3). Collect the UV-Vis spectra of each group of samples from 400 to 800 nm.

[0171] (3)

[0172] Figure 17(A) To inhibit the use of TPSNs prepared in Example 1 with a series of concentrations from 0 to 500 μg / mL Quantitative analysis results of capabilities (graph); Figure 17 (B) represents the effects of different concentrations of TPSNs on... The UV-Vis spectrum of the purified solution is shown, with insets depicting photographs of reaction solutions at different concentrations at the end of the reaction. The graph shows that higher TPSN concentrations result in lower absorbance at 550 nm and a lighter purple-red color at the end of the reaction. The higher the clearance ability, the better. Results show that TPSNs have a higher clearance ability. It has a strong scavenging ability, which is concentration-dependent.

[0173] IV. Superoxide anion free radicals ( Cleaning performance study

[0174] The procedure was performed according to the instructions of the kit for inhibiting and generating superoxide anion radicals. The application solution was added sequentially to 50 μL of TPSNs (prepared using TPSNs prepared in Example 1) solutions with concentrations of 20, 50, 100, 200, and 500 μg / mL. After vortexing, the mixture was placed in a 37°C water bath for 40 min. After the reaction was complete, the colorimetric reagent was added, and the mixture was allowed to stand at room temperature for 10 min. The OD value at 550 nm was measured using a microplate reader. The ability to inhibit superoxide anion radicals was calculated according to the following formula (4), and UV-Vis spectra of each group of samples were collected from 400 to 800 nm.

[0175] (4)

[0176] Figure 18 (A) To inhibit the use of TPSNs prepared in Example 1 with a series of concentrations from 0 to 500 μg / mL Quantitative analysis results of capabilities (graph); Figure 18 (B) represents the effects of different concentrations of TPSNs on... The UV-Vis spectrum of the purified solution is shown, with insets depicting photographs of reaction solutions at different concentrations at the end of the reaction. The graph shows that higher TPSN concentrations result in lower absorbance at 550 nm and a lighter purple-red color at the end of the reaction. The higher the clearance ability, the better. Results show that TPSNs have a higher clearance ability. It has a strong scavenging ability, which is concentration-dependent.

[0177] V. Electron Spin Resonance (ESR) Experimental Evaluation of RONS Sweep Performance

[0178] (1) Scavenging hydroxyl radicals ( )

[0179] Process according to the following groups: Group: Blank control. Take 100 μL of 5.0 mg / mL FeSO4 solution, add 10 μL of pure DMPO and 80 μL of ultrapure water, then add 10 μL of 30% H2O2, mix well and react for 5 min, then take a sample for instrument detection. Group: Except that 80 μL of ultrapure water was replaced with 80 μL of 300.0 mg / mL TPSNs (prepared using the TPSNs prepared in Example 1), the experimental procedures were the same. The groups are the same.

[0180] (2) Scavenging superoxide anion free radicals ( )

[0181] Process according to the following groups: Group: Blank control. All reagents were prepared with 40.0 mmol / L pH=7.4 PBS buffer. 20 μL of 10.0 mmol / L xanthine solution and 20 μL of 1 U / mL xanthine oxidase solution were mixed, and then 10 μL of 200.0 mmol / L BMPO solution and 50 μL PBS buffer were added. The mixture was stirred and reacted for 10 min. Samples were then taken for instrumental analysis. Group: Except that the 50 μL PBS buffer was replaced with 50 μL of 300.0 mg / mL TPSNs (prepared using the TPSNs prepared in Example 1), the experimental procedures were the same. The groups are the same.

[0182] (3) Remove singlet oxygen ( )

[0183] Process according to the following groups: Group: Blank control. Take 50 μL of 2.0 mg / mL TiO2 nanopowder aqueous solution, add 50 μL of 100.0 mmol / L TEMP solution, then add 100 μL of ultrapure water, mix well, load the sample into a capillary tube, irradiate with a 300W xenon lamp for 10 min, and collect data. Group: Except that 100 μL of ultrapure water was replaced with 100 μL of 300.0 mg / mL TPSNs (prepared using the TPSNs prepared in Example 1), the experimental procedures were the same. The groups are the same.

[0184] (4) Scavenging nitrogen oxide free radicals ( )

[0185] Process according to the following groups: Group: Blank control. All reagents were prepared with 40.0 mmol / L PBS buffer at pH 7.4. Take 20 μL of 10.0 mmol / L SNAP solution, 20 μL of 2.0 mmol / L Carboxy-PTIO solution and 20 μL of PBS buffer, react for 10 min, and then take samples for instrument detection. Group: Except that the 20 μL PBS buffer was replaced with 20 μL of 300.0 mg / mL TPSNs (prepared using the TPSNs prepared in Example 1), the experimental procedures were the same. The groups are the same.

[0186] Figure 19 (A) is Groups and Group, Figure 19 (B) is Groups and Group, Figure 19 (C) is Groups and Group, Figure 19 (D) is Groups and The electron spin resonance (ESR) spectra of the group, TPSNs prepared in Example 1. As shown in the figure, Group, Group, Groups and The groups present typical ESR spectra of each free radical; Group, Group, Group, Compared with the groups mentioned above, the height of the peaks in this group was significantly reduced, indicating that the content of the corresponding free radical adducts in the reaction system was reduced, which indirectly shows the reduction in the content of each free radical, proving that TPSNs have a good scavenging ability for common free radicals.

[0187] The above experimental results all confirm that TPSNs have broad-spectrum RONS scavenging activity at the in vitro level.

[0188] VI. Study on intracellular RONS clearance capacity

[0189] (1) Modeling: Mouse mononuclear macrophage leukemia cells (abbreviated as: macrophage RAW264.7) were selected as model cells, and a cell model with high RONS levels was induced by LPS. The scavenging activity of TPSNs on different types of intracellular RONS was investigated by using RONS probes that selectively respond to different RONS.

[0190] (2) RONS probe selection: ROS-ID oxidative stress probes reflect the overall oxidative stress level, representing the total free radical level; 2',7'-dichlorofluorescein diacetate (DCFH-DA), diaminofluorescein-FM-diacetate (DAF-FM DA), ethidium dihydrogen fluorescein (DHE), and hydroxyphenylfluorescein (HPF) represent specific H2O2, , and Fluorescent probe.

[0191] (3) Study on the activity of TPSNs in clearing intracellular RONS, the specific steps are as follows: RAW264.7 macrophages were divided into groups of 5 × 10⁶ cells per well. 4 Cells were seeded at a density of [number] cells / well in 24-well plates and incubated in a cell culture incubator for 12.0 h to allow for full cell adhesion. The culture medium was then aspirated, and the cells were washed twice with PBS. Cells were then grouped and treated as follows: Ctrl group: blank control, incubated with 500 μL of culture medium for 48.0 h. TPSNs (prepared using TPSNs prepared in Example 1) group: incubated with 500 μL of medium for 24.0 h, the medium was discarded; then treated with 500 μL of medium containing 150 μg TPSNs for 24.0 h. LPS group: Incubated with 500 μL of medium for 24.0 h, then the medium was discarded; then stimulated with 500 μL of medium containing 5 μg LPS for 24.0 h. LPS+TPSNs (prepared using TPSNs prepared in Example 1) group: After stimulation with 500 μL of medium containing 5 μg LPS for 24.0 h, the medium was discarded; then treated with 500 μL of medium containing 150 μg TPSNs for 24.0 h. After incubation, the medium in each group was discarded, washed twice with PBS, and 500 μL of redox-sensitive fluorescent probes (1:1000 ROS-ID probe diluted with phenol red-free RPMI-1640 basal medium, 10 μmol / L DCFH-DA, 5 μmol / L DAF-FM DA, 5 μmol / L DHE and 20 μmol / L HPF) were added. The mixture was incubated in the dark for 60 min, the probe staining solution was discarded, the mixture was washed three times with PBS, and observed under a fluorescence microscope.

[0192] Figure 20 (A) is a ROS-ID fluorescent probe, Figure 20 (B) is a DCFH-DA fluorescent probe. Figure 20 (C) is a DAF-FM DA fluorescent probe. Figure 20 (D) is a DHE fluorescent probe. Figure 20(E) is a fluorescence image of HPF fluorescent probe loaded onto different groups of RAW264.7 macrophages. Figure 20 (A) to Figure 20 (E) represent intracellular General RONS, H2O2, and , and Horizontal plane, scale bar 200 μm. As shown in the figure, the fluorescence imaging results of the TPSNs group and the Ctrl group are consistent, indicating that TPSNs do not stimulate macrophages to produce free radicals. The LPS group exhibits very strong fluorescence, indicating that macrophages produce excessive free radicals, suggesting that LPS modeling was successful. Compared with the LPS group, the fluorescence intensity of the LPS+TPSNs group is significantly reduced, indicating that after LPS stimulation, the addition of TPSNs can significantly eliminate excessive free radicals in inflammatory cells, and has a positive effect on intracellular General RONS, H2O2, and... , and Both have good cleaning effects.

[0193] The combined results of in vitro and cellular free radical scavenging experiments demonstrate that TPSNs possess broad-spectrum free radical scavenging capabilities in vitro.

[0194] Experimental Example 3

[0195] The regulatory effect of TPSNs prepared in Example 1 on macrophage polarization was investigated: macrophages were prevented from polarizing to the pro-inflammatory M1 type and macrophages were promoted to polarize to the anti-inflammatory M2 type.

[0196] I. Study on the expression level of macrophage polarization markers

[0197] RAW264.7 macrophages in the logarithmic growth phase were harvested at a concentration of 5 × 10⁻⁶ cells. 4 Seeds were planted at a density of cells / well in 24-well plates and incubated in a cell culture incubator for 12.0 h to allow for full cell adhesion. The culture medium was then aspirated, and the cells were washed twice with PBS. Cells were then grouped and treated as follows: Ctrl group: blank control, incubated with 500 μL of culture medium for 48.0 h. TPSNs (prepared using TPSNs prepared in Example 1) group: incubated with 500 μL of medium for 24.0 h, the medium was discarded; then treated with 500 μL of medium containing 150 μg TPSNs for 24.0 h. LPS group: Incubated with 500 μL of medium for 24.0 h, then the medium was discarded; then stimulated with 500 μL of medium containing 5 μg LPS for 24.0 h. LPS+TPSNs (prepared using TPSNs prepared in Example 1) group: After stimulation with 500 μL of medium containing 5 μg LPS for 24.0 h, the medium was discarded; then treated with 500 μL of medium containing 150 μg TPSNs for 24.0 h. After incubation, the medium in each group was discarded, and the cells were washed twice with PBS. 1.00 mL of 4% paraformaldehyde was added for fixation at room temperature for 10 min. After fixation, the paraformaldehyde was discarded, and the cells were washed twice with PBS. 1.00 mL of 0.2% Triton X-100 was added, and the cells were permeabilized at room temperature for 15 min, followed by washing twice with PBS. Then, 1.00 mL of 1% BSA solution was added, and the cells were blocked at room temperature for 30 min. CD86 antibody solution and CD206 antibody solution diluted in Staining Buffer were added to the cells, and the cells were incubated at 4°C for 12.0 h. After incubation, the cells were labeled. Alexa Fluor 594-bonded fluorescent secondary antibody was added to cells containing CD86 antibody, and FITC-bonded Goat-anti-Rabbit fluorescent secondary antibody was added to cells containing CD206 antibody. Finally, anti-fluorescence attenuation mounting medium (containing DAPI, which labels the cell nuclei) was added and the cells were stored at 4°C for observation using a Cytation 5 multi-well microplate reader.

[0198] Figure 21 Immunofluorescence imaging of CD86 and CD206, polarization markers of RAW264.7 macrophages in each group, with a scale bar of 100 μm. As shown in the figures, both columns of DAPI exhibit bright blue fluorescence, marking the position of RAW264.7 macrophages under the microscope. The red fluorescence in the CD86 column and the green fluorescence in the CD206 column must correspond to the blue fluorescence in the corresponding DAPI column to confirm that the polarization marker is produced by macrophages. Comparing the TPSNs group and the Ctrl group, there was no difference in CD86 and CD206 expression, indicating that TPSNs do not induce CD86 and CD206 expression on the macrophage surface. The LPS group showed bright red fluorescence under the microscope for CD86, indicating that LPS can stimulate high expression of CD86 on the macrophage surface. A weak green fluorescence was also observed in this group, suggesting a slight upregulation of CD206, indicating successful LPS modeling. Compared with the LPS group, the expression of CD86 protein labeled with red fluorescence was significantly reduced in the LPS+TPSNs group, while the expression of CD206 labeled with green fluorescence was significantly increased. This demonstrates that TPSNs can downregulate the expression of CD86, a marker of M1 macrophages, while upregulating the expression of CD206, a marker of M2 macrophages, and has a significant regulatory effect on macrophage polarization.

[0199] II. Study on the polarization ratio of M1 / M2 macrophages

[0200] RAW264.7 macrophages in the logarithmic growth phase were harvested at a concentration of 3 × 10⁻⁶ cells. 5 Seeds were planted at a density of cells / well in 6-well plates and incubated in a cell culture incubator for 12.0 h to allow for full cell adhesion. The culture medium was then aspirated, and the cells were washed twice with PBS. Cells were then grouped and treated as follows: Ctrl group: blank control, with 2.00 mL of culture medium added. TPSNs (prepared using TPSNs prepared in Example 1): 2.00 mL of culture medium containing 600 μg TPSNs was added. LPS group: Add 2.00 mL of culture medium containing 20 μg LPS. LPS+TPSNs (prepared using TPSNs prepared in Example 1) group: 2.00 mL of culture medium containing 20 μg LPS and 600 μg TPSNs was added. All groups were incubated for 24.0 h. After incubation, the culture medium was discarded, and the cells were washed three times with PBS. 300 μL of phenol red-free and EDTA-free trypsin digestion buffer was added to each well, and digestion was carried out at 37°C for 5 min. An equal volume of complete culture medium was added to terminate the digestion. The cells were centrifuged at 2500 rpm for 5 min at 4°C, resuspended in 600 μL of PBS, and washed twice with PBS. The cells were resuspended in 150 μL of Cell Staining Buffer, and 5 μL of flow cytometry antibody was added to each tube. The cells were incubated at 4°C for 30 min. After incubation, the cells were centrifuged at 2500 rpm for 5 min at 4°C, and the supernatant was removed. The cells were resuspended in 300 μL of Cell Staining Buffer and analyzed by flow cytometry.

[0201] Figure 22 (A) Flow cytometry analysis of CD86 and CD206, polarization markers of RAW264.7 macrophages in each group; Figure 22 (B) refers to the CD86 groups mentioned above. + Quantitative analysis of the proportion of CD86-positive macrophages; Figure 22 (C) represents the CD206 groups mentioned above. + (CD206 positive) Quantitative analysis of macrophage percentage; experimental data are expressed as mean ± standard deviation (n=3, , , As shown in the figure, compared with the Ctrl group, the CD86 of the TPSNs group and the Ctrl group are different. + and CD206 + The macrophage ratio was not different, indicating that TPSNs do not increase CD86. + and CD206 + Macrophage ratio. Comparing the LPS group and the Ctrl group, the CD86 levels were... + The difference in the proportion of macrophages was statistically significant. ), both CD206 + The difference in the proportion of macrophages was statistically significant. This indicates that LPS can significantly improve CD86 + The proportion of macrophages can also affect CD206. + The proportion of macrophages increased. Compared with the LPS group, the LPS+TPSNs group had a lower CD86 content. + and CD206 + The differences in the proportion of macrophages were all statistically significant. This demonstrates that TPSNs can significantly downregulate the M1 type (CD86). + The proportion of macrophages was significantly increased, while the proportion of M2-type (CD206) cells was also significantly increased. + The ratio of macrophages has a very strong regulatory effect on macrophage polarization.

[0202] The above results indicate that TPSNs can regulate macrophage polarization, effectively preventing macrophages from polarizing to the pro-inflammatory M1 type, while simultaneously inducing macrophages to polarize to the anti-inflammatory M2 type, which is beneficial for controlling the development of inflammatory responses and reducing the secretion of inflammatory cytokines.

[0203] Experiment Example 4

[0204] The inhibitory effect of TPSNs prepared in Example 1 on apoptosis was investigated.

[0205] NCM460 cells in the logarithmic growth phase were harvested at a concentration of 3 × 10⁻⁶. 5 Seeds were planted at a density of cells / well in 6-well plates and incubated in a cell culture incubator for 12.0 h to allow for full cell adhesion. The culture medium was then aspirated, and the cells were washed twice with PBS. Cells were then grouped and treated as follows: Ctrl group: blank control, with 2.00 mL of culture medium added. TPSNs (prepared using TPSNs prepared in Example 1): 2.00 mL of culture medium containing 600 μg TPSNs was added. DI group: Add 2.00 mL of culture medium containing 1% DSS and 40 ng IL-1β protein. DI+TPSNs (prepared using TPSNs prepared in Example 1): 2.00 mL of culture medium containing 1% DSS, 40 ng IL-1β protein, and 600 μg TPSNs was added. All groups were incubated for 24.0 h. After incubation, the culture medium was discarded, and the cells were washed twice with PBS. Cells were digested with 1.00 mL of phenol red-free and EDTA-free trypsin. Cells were transferred to EP tubes, centrifuged at 500×g for 5 min at 4°C, resuspended in 1× binding buffer, and Annexin V-FITC and propidium iodide (PI) dye were added for flow cytometry analysis.

[0206] Figure 23 (A) is a flow cytometry analysis of apoptosis in NCM460 cells in each group; Figure 23 (B) refers to the Annexin V groups mentioned above. + / PI + The results of quantitative analysis of NCM460 cell populations are shown in the figure; experimental data are expressed as mean ± standard deviation (n=3, (ns indicates no specific difference). As shown in the figure, compared to the Ctrl group, the Annexin V of the TPSNs group was significantly different. + / PI + There was no significant difference in cell cluster proportions (ns), indicating that TPSNs do not induce apoptosis. Annexin V in the DI group + / PI + The cell cluster ratio was 29.9%, indicating that the combined action of DSS and the pro-inflammatory cytokine IL-1β significantly increased the level of cell apoptosis, demonstrating the successful establishment of the DI model. Compared with the DI group, the DI+TPSNs group showed significantly higher levels of Annexin V. + / PI + The difference in cell cluster proportions was statistically significant. Furthermore, the percentage of apoptosis in the DI group decreased significantly from 29.9% to 7.12% in the DI+TPSNs group, demonstrating that TPSNs have a very strong anti-apoptotic ability.

[0207] Experimental Example 5

[0208] The effects of TPSNs prepared in Example 1 on regulating the cell cycle of inflammatory cells, promoting cell proliferation, and accelerating cell renewal were investigated.

[0209] NCM460 cells in the logarithmic growth phase were harvested at a concentration of 3 × 10⁻⁶. 5 Seeds were planted at a density of cells / well in 6-well plates and incubated in a cell culture incubator for 12.0 h to allow for full cell adhesion. The culture medium was then aspirated, and the cells were washed twice with PBS. Cells were then grouped and treated as follows: Ctrl group: blank control, with 2.00 mL of culture medium added. TPSNs (prepared using TPSNs prepared in Example 1): 2.00 mL of culture medium containing 600 μg TPSNs was added. DI group: Add 2.00 mL of culture medium containing 1% DSS and 40 ng IL-1β protein. DI+TPSNs (prepared using TPSNs prepared in Example 1): 2.00 mL of culture medium containing 1% DSS, 40 ng IL-1β protein, and 600 μg TPSNs was added. All groups were incubated for 24.0 h. After incubation, the culture medium was discarded, and the cells were washed twice with PBS. Cells were digested with 1.00 mL of phenol red-free and EDTA-free trypsin, transferred to EP tubes, centrifuged at 500 × g for 5 min at 4 °C, resuspended in 75% ethanol, and fixed at 4 °C for 24.0 h. The cell suspension was centrifuged at 500 × g for 5 min at 4 °C, resuspended in PBS, and propidium iodide (PI) was added for flow cytometry analysis.

[0210] Figure 24 (A) is a flow cytometry analysis of NCM460 cells in the Ctrl group and the TPSNs (prepared from TPSNs prepared in Example 1) group; Figure 24 (B) shows the results of the quantitative analysis of cell cycle in the two groups above; experimental data are expressed as mean ± standard deviation (n=3, ns indicates no significant difference). As can be seen from the figure, there is no significant difference (ns) in cell cycle between the TPSNs group and the Ctrl group, indicating that TPSNs does not affect the cell cycle of normal cells. Figure 24 (C) shows the cell cycle flow cytometry analysis of NCM460 cells in the DI group and the DI+TPSNs (prepared from TPSNs prepared in Example 1) group; Figure 24 (D) is a graph showing the results of the two groups of cell cycle quantitative analysis above; DI: DSS+IL-1β, experimental data are expressed as mean ± standard deviation (n=3, , (ns indicates no significant difference). As shown in the figure, there was no significant difference (ns) in the proportion of cells in the G0-G1 phase between the DI+TPSNs group and the DI group, indicating that TPSNs do not affect inflammatory cells in the G0-G1 phase; the difference in the proportion of cells in the S phase between the two groups was statistically significant. The proportion of cells in the DI group decreased significantly from 31.29% to 4.4% in the DI+TPSNs group, indicating that TPSNs can significantly downregulate the proportion of inflammatory cells in the S phase; the difference in the proportion of cells in the G2 / M phase between the two groups was statistically significant. The proportion of inflammatory cells in the G2 / M phase significantly increased from 5.80% in the DI group to 35.10% in the DI+TPSNs group, indicating that TPSNs can significantly upregulate the proportion of inflammatory cells in the G2 / M phase. These results suggest that TPSNs primarily regulate the cell cycle of inflammatory cells by downregulating the proportion of cells in the S phase and upregulating the proportion of cells in the G2 / M phase; by promoting the transition of inflammatory cells from the S phase to the G2 / M phase, they promote cell division and proliferation, thereby accelerating cell renewal.

[0211] Experimental Example 6

[0212] Investigating the therapeutic effect of TPSNs prepared in Example 1 on a DSS-induced IBD mouse model

[0213] Normally fed C57BL / 6J mice were divided into 4 groups of 5 mice each: Ctrl group: drank ordinary purified water and received intraperitoneal injections of the same volume of physiological saline as the drug treatment group. TPSNs (prepared from TPSNs prepared in Example 1) group: drank ordinary purified water and received intraperitoneal injection of 5 mg / kg (dose / body weight) of TPSNs solution. DSS group: Drink 2.5% DSS solution for 7 days, and receive intraperitoneal injection of the same volume of physiological saline as the drug treatment group. DSS+TPSNs (prepared from TPSNs prepared in Example 1) group: drank 2.5% DSS solution for 7 days and received intraperitoneal injection of 5 mg / kg (dose / body weight) of TPSNs solution.

[0214] Mice were intraperitoneally injected with 2.5% DSS solution at the same time on days 0, 3, 6, and 9. The DSS group and the DSS+TPSNs group received 2.5% DSS solution for 7 days, after which the solution was replaced with ordinary purified water. Daily changes in mouse body weight and disease activity index (DAI) were recorded for each group. On day 12, mice were sacrificed and dissected, blood samples were collected, and the entire colon from the cecum to the anus was retrieved and its length measured. Serum inflammatory cytokines (IL-6, TNF-α, IL-1β, IL-18, and IL-10) and the transcriptional levels of pro-inflammatory cytokines in colon tissue were detected. Colon tissue was fixed, paraffin-embedded, sectioned, and subjected to H&E staining, PAS and AB-PAS staining, immunohistochemical staining, and immunofluorescence staining. Pathological changes were observed under a microscope to evaluate the therapeutic effect of TPSNs on DSS-induced colitis in normal mice.

[0215] Figure 25 (A) is a graph showing the changes in body weight of C57BL / 6J mice in each group; Figure 25 (B) is a graph showing the changes in the Disease Activity Index (DAI) of mice in each group; Figure 25(C) Representative photographs of anal bleeding in mice of each group on day 12; experimental data are expressed as mean ± standard deviation (n=5, As shown in the figure, the differences in body weight and DAI between the DSS+TPSNs group and the DSS group were statistically significant. The results indicate that TPSNs can slow down DSS-induced weight loss in mice and reduce DAI scores; there was a lot of blood around the anus in the DSS group, while there was no blood around the anus in the DSS+TPSNs group, indicating that TPSNs can reduce anal bleeding caused by DSS.

[0216] Figure 26 (A) is a representative photograph of the colon length of mice in each group; Figure 26 (B) is a graph showing the statistical analysis results of colon length in each group of mice; experimental data are expressed as mean ± standard deviation (n=5, , (ns indicates no significant difference). As shown in the figure, the difference in colon length between the DSS group and the Ctrl group was statistically significant. This indicates that the DSS model was successfully established; compared with the Ctrl group, there was no significant difference (ns) in colon length between the TPSNs group and the TPSNs group, indicating that TPSNs does not affect the colon length of normal mice; compared with the DSS group, the DSS+TPSNs group showed a highly statistically significant difference in colon length. This indicates that TPSNs can alleviate DSS-induced colonic shortening.

[0217] Figure 27 The images show H&E staining of colon tissue from mice in each group. The scale bars above and below are 1 mm and 100 μm, respectively. As can be seen from the images, the inflammatory cell infiltration and ulceration in the colon tissue of the DSS+TPSNs group were significantly lower than those in the DSS group, indicating that TPSNs can effectively alleviate DSS-induced pathological damage to the mouse colon.

[0218] Figure 28 The images show PAS and AB-PAS staining patterns of colon tissue from each group of mice. The scale bars above and below are 100 μm and 50 μm, respectively. As shown in the figures, compared to the DSS group, the DSS+TPSNs group exhibited a significant increase in the number of goblet cells and the content of neutral mucin, while the content of acidic mucin was decreased. This demonstrates that TPSNs can alleviate DSS-induced intestinal inflammation and intestinal epithelial barrier damage in mice.

[0219] Figure 29Immunohistochemical staining images of ZO-1 and Occludin in the colon tissue of mice in each group are shown, with a scale bar of 50 μm. As can be seen from the figure, the expression levels of ZO-1 and Occludin in the DSS+TPSNs group were significantly higher than those in the DSS group, indicating that TPSNs have a good protective effect on the intestinal mucosal mechanical barrier.

[0220] Figure 30 Immunofluorescence analysis of MUC-2 expression levels in colon tissue of mice in each group is shown in the figure, with a scale bar of 100 μm. As can be seen from the figure, the DAPI images all exhibit bright blue fluorescence, marking the position of goblet cells under the microscope; the red fluorescence in the MUC-2 image must correspond to the blue fluorescence position in the corresponding DAPI image to prove that MUC-2 is produced by goblet cells. The red fluorescence in the DSS+TPSNs group was significantly higher than that in the DSS group, indicating that TPSNs increased MUC-2 expression, strengthened the protective effect of the intestinal chemical barrier, and reduced the damage to the intestinal mucosa caused by DSS.

[0221] Figure 31 Immunofluorescence analysis of Caspase 3 expression levels in colon tissues of mice in each group is shown, with a scale bar of 20 μm. As can be seen from the figures, the DAPI maps all exhibit bright blue fluorescence, marking the location of intestinal epithelial cells under a microscope. The green fluorescence of the Caspase 3 map must correspond to the blue fluorescence location of the corresponding DAPI map to prove that Caspase 3 is produced by intestinal epithelial cells. The green fluorescence of the DSS+TPSNs group was significantly lower than that of the DSS group, indicating that TPSNs can reduce excessive apoptosis of intestinal epithelial cells and protect the intestinal barrier function by lowering Caspase 3 expression levels.

[0222] Figure 32 The graph shows the concentrations of pro-inflammatory cytokines IL-6, TNF-α, IL-1β, IL-18, and anti-inflammatory cytokine IL-10 in the serum of mice in each group; experimental data are expressed as mean ± standard deviation (n=3, , , , As shown in the figure, the differences in serum inflammatory cytokine concentrations between the DSS+TPSNs group and the DSS group were statistically significant. and This indicates that TPSNs can significantly reduce the levels of pro-inflammatory cytokines IL-6, TNF-α, IL-1β and IL-18 in mouse serum, and have the activity of promoting the secretion of anti-inflammatory cytokine IL-10.

[0223] Figure 33This is a graph showing the gene transcription levels of pro-inflammatory cytokines IL-6, TNF-α, and IL-1β in the colon tissue of mice in each group; experimental data are expressed as mean ± standard deviation (n=5, , , As shown in the figure, compared with the DSS group, there were significant differences in the gene transcription levels of pro-inflammatory cytokines IL-6, TNF-α, and IL-1β in the colon tissue of the DSS+TPSNs group. , and This indicates that TPSNs can significantly reduce the gene transcription levels of pro-inflammatory cytokines IL-6, TNF-α, and IL-18 in mouse colon tissue.

[0224] The above results indicate that TPSNs have a significant therapeutic effect on the DSS-induced IBD model in mice.

[0225] Experimental Example 7

[0226] Investigating the biosafety of the TPSNs prepared in Example 1 in vivo.

[0227] After centrifuging the whole blood sample from mice in "Experimental Example 6" to obtain serum, the liver and kidney function indicators in the serum, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (Cre), and blood urea nitrogen (BUN), were detected using a fully automated biochemical analyzer. The heart, liver, spleen, lungs, and kidneys of mice were stained with H&E to evaluate the in vivo biosafety of TPSNs.

[0228] Figure 34 (A) is a graph showing the ALT and AST levels in the serum of mice in each group (liver function indicators); Figure 34 (B) Graphs showing the levels of Cre and BUN in the serum of mice in each group (indicators of renal function); experimental data are expressed as mean ± standard deviation (n=5, , ns indicates no special difference). Figure 35 The images show H&E staining of the heart, liver, spleen, lungs, and kidneys of mice in each group, with a scale bar of 100 μm. As can be seen from the images, compared with the Ctrl group, the TPSNs group showed no significant abnormalities in serum liver and kidney function indicators, and no significant pathological changes in any organ.

[0229] The above results demonstrate that TPSNs have good biocompatibility in vivo.

[0230] Experimental Example 8

[0231] Investigating the anti-inflammatory mechanism of TPSNs prepared in Example 1: Study on the NF-κB signaling pathway

[0232] The phosphorylation levels of key regulatory proteins in the NF-κB signaling pathway were detected using Western blotting to determine the regulatory role of TPSNs in this pathway. Furthermore, the anti-inflammatory effects of polyEGCG nanoparticles (TPNs), poly5-HT nanoparticles (PSTs), and TPSNs were compared.

[0233] RAW264.7 macrophages in the logarithmic growth phase were harvested at a concentration of 5 × 10⁻⁶ cells. 5 Seeds were planted at a density of cells / well in 6-well plates and incubated in a cell culture incubator for 12.0 h to allow for full cell adhesion. The culture medium was then aspirated, and the cells were washed twice with PBS. Cells were then grouped and treated as follows: 1) Mock portion: None group: blank control, with 2.00 mL of culture medium added. TPNs (solely synthesized polyEGCG nanoparticles) group: Add 2.00 mL of culture medium containing 600 μg TPNs. PST (isolated poly5-HT nanoparticles) group: Add 2.00 mL of culture medium containing 600 μg PST. TPSNs (prepared from TPSNs prepared in Example 1): 2.00 mL of culture medium containing 600 μg TPSNs was added. 2) LPS portion: Group None: Add 2.00 mL of culture medium containing 20 μg LPS. TPNs (isolated polyEGCG nanoparticles): Add 2.00 mL of culture medium containing 20 μg LPS and 600 μg TPNs. PST (isolated poly5-HT nanoparticles) group: 2.00 mL of culture medium containing 20 μg LPS and 600 μg PST was added. TPSNs (prepared from TPSNs prepared in Example 1): 2.00 mL of culture medium containing 20 μg LPS and 600 μg TPSNs was added. All groups were incubated for 12.0 h. After incubation, the culture medium was discarded, and the samples were washed three times with pre-cooled PBS. Protein samples were extracted and quantified, followed by SDS-PAGE gel electrophoresis and transfer, blocking and antibody incubation, and protein band development to complete the experiment.

[0234] Figure 36This is a graph showing the expression levels of p-IKKα / β, p-p65, and p-IκBα, key regulatory proteins of the NF-κB signaling pathway. As shown in the graph: 1) LPS portion: The expression levels of p-IKKα / β, p-p65, and p-IκBα proteins in macrophages in the None group were significantly higher than those in the Mock portion of the None group, indicating successful modeling. LPS activates the NF-κB signaling pathway from top to bottom and induces inflammation. 2) Mock portion: Compared with the None group, the TPNs, PST, and TPSNs groups showed no difference in the expression levels of p-IKKα / β, p-p65, and p-IκBα proteins, and none of them were expressed, indicating that TPNs, PST, and TPSNs do not activate the NF-κB signaling pathway and do not induce inflammation. 3) LPS portion: The expression levels of p-IKKα / β, p-p65, and p-IκBα proteins in the TPNs group were significantly lower than those in the None group, and almost no expression was observed. This indicates that TPNs can significantly inhibit the NF-κB signaling pathway and exert a strong anti-inflammatory effect of EGCG. Compared with the None group, there was no difference in the expression levels of the three proteins mentioned above in the PST group, indicating that PST cannot inhibit the NF-κB signaling pathway and has no anti-inflammatory effect. The expression levels of the three proteins in the TPSNs group were significantly lower than those in the None group, but not as significantly downregulated as in the TPNs group. This indicates that the EGCG component in TPSNs can inhibit the NF-κB signaling pathway and exert a strong anti-inflammatory effect. TPSNs are composed of EGCG and 5-HT, which results in a weaker anti-inflammatory effect compared to TPNs composed of pure EGCG.

[0235] Experimental Example 9

[0236] Study on the inhibitory effect of TPSNs prepared in Example 1 on pyroptosis

[0237] The complex inflammatory signaling network and oxidative stress environment at the site of IBD lesions lead to widespread pyroptosis, necessitating the evaluation of the anti-pyroptosis capabilities of TPSNs.

[0238] 1. Pyroptosis leads to the formation of pores in the cell membrane and the leakage of pro-inflammatory cytokines.

[0239] (1) Activation methods of the pyroptosis pathway

[0240] 1) Classic pyroptosis pathway activation method one (LPS+nigericin): LPS and nigericin are used to induce pyroptosis in primary macrophages.

[0241] 2) Classic pyroptosis pathway activation method two (LPS+ATP): Using LPS and adenosine triphosphate (ATP) to induce pyroptosis in primary macrophages.

[0242] 3) Non-classical pyroptosis pathway activation method (LPS transfection): Using LPS transfection to induce pyroptosis in primary macrophages.

[0243] (2) Qualitative study of cell membrane pores: propidium iodide (PI) staining

[0244] Normal cells cannot pass through PI because their cell membranes are intact. However, in pyroptosis-affected cells, pores form on the cell membrane surface, allowing PI to penetrate and bind to DNA, emitting red fluorescence. Based on the fluorescence, the degree of cell membrane damage can be assessed, providing a visual indicator for evaluating the level of pyroptosis.

[0245] Primary macrophages were used at a rate of 4 × 10 5 Cells were seeded at a density of [number] cells / well in 24-well plates and incubated in a cell culture incubator for 12.0 h to allow for full cell adhesion. The culture medium was then aspirated, and the cells were washed twice with PBS. Cells were then treated according to the following groups: 1) Classical pyroptosis pathway activation method one (LPS + nigericin): Mock group: blank control, incubated with 500 μL of culture medium for 4.5 h. LPS+nigericin group: positive control. Stimulated with 500 μL of medium containing 50 ng LPS for 3.0 h, the medium was discarded; then incubated with 500 μL of medium for 0.5 h, the medium was discarded; finally treated with 500 μL of medium containing 5 nmol nigericin for 1.0 h. LPS + nigericin + TPSNs (prepared from TPSNs prepared in Example 1) group: Stimulated with 500 μL of medium containing 50 ng LPS for 3.0 h, the medium was discarded; then incubated with 500 μL of medium containing 150 μg TPSNs for 0.5 h, the medium was discarded; finally treated with 500 μL of medium containing 5 nmol nigericin for 1.0 h. 2) Non-classical pyroptosis pathway (LPS transfection): Mock group: blank control, incubated with 500 μL Opti-MEM medium for 9.5 h, the medium was discarded; then incubated with 500 μL MEM complete medium containing 10% FBS for 16.0 h. LPS transfection group: positive control. Pretreated with 500 μL of Opti-MEM medium containing 500 ng Pam3CSK4 for 3.0 h, then the medium was discarded; then incubated with 500 μL of Opti-MEM medium for 0.5 h, then the medium was discarded; 500 μL of Opti-MEM medium containing 1 μg LPS and 5 μL P3000™ was mixed with 500 μL of Opti-MEM medium containing 5 μL Lipofectamine™ 3000, and incubated at room temperature for 15 min to prepare 1.00 mL of "liposome-LPS" complex Opti-MEM medium. 500 μL of "liposome-LPS" complex Opti-MEM medium was added to primary macrophages and incubated for 6.0 h, then the medium was discarded; finally, incubated with 500 μL of MEM complete medium containing 10% FBS for 16.0 h. LPStransfection + TPSNs (prepared from TPSNs prepared in Example 1) group: except that the incubation time of 500 μL Opti-MEM medium for 0.5 h was replaced with 500 μL Opti-MEM medium containing 150 μg TPSNs for 0.5 h, the experimental procedures were the same as those in Example 1. The LPStransfection+none group is the same.

[0246] After incubation, the culture medium was discarded from each group, and the cells were washed twice with PBS. The cells were then stained with 500 μL of 1 μg / mL PI solution at room temperature for 20 min. The staining solution was discarded, and the cells were washed with PBS. Observation was performed using a Cytation 5 multi-well microplate reader.

[0247] Figure 37 The images show the uptake of PI (red) by primary macrophages in each group, with a scale bar of 100 μm. As can be seen from the figures, the red fluorescence intensity in the LPS+nigericin+TPSNs group was reduced compared to the LPS+nigericin group, and the red fluorescence intensity in the LPS transfection+TPSNs group was reduced compared to the LPS transfection group, suggesting improved cell membrane integrity, i.e., a reduced level of pyroptosis. This indicates that TPSNs can reverse the formation of cell membrane pores and inhibit pyroptosis.

[0248] (3) ELISA analysis of pro-inflammatory cytokine leakage

[0249] Primary macrophages were used at a rate of 4 × 10 5Cells were seeded at a density of [number] cells / well in 24-well plates and incubated in a cell culture incubator for 12.0 h to allow for full cell adhesion. The culture medium was then aspirated, and the cells were washed twice with PBS. Cells were then treated according to the following groups: 1) Classical pyroptosis pathway activation method one (LPS + nigericin): Ctrl group: blank control, incubated with 500 μL of culture medium for 4.5 h. Model group: positive control. Stimulated with 500 μL of medium containing 50 ng LPS for 3.0 h, the medium was discarded; then incubated with 500 μL of medium for 0.5 h, the medium was discarded; finally treated with 500 μL of medium containing 5 nmol nigericin for 1.0 h. TPSNs (prepared from TPSNs prepared in Example 1): Stimulated with 500 μL of medium containing 50 ng LPS for 3.0 h, then the medium was discarded; then incubated with 500 μL of medium containing 150 μg TPSNs for 0.5 h, then the medium was discarded; finally, treated with 500 μL of medium containing 5 nmol nigericin for 1.0 h. 2) Classical pyroptosis pathway activation method two (LPS+ATP): Ctrl group: blank control, incubated with 500 μL of culture medium for 4.5 h. Model group: positive control. Stimulated with 500 μL of medium containing 50 ng LPS for 3.0 h, the medium was discarded; then incubated with 500 μL of medium for 0.5 h, the medium was discarded; finally treated with 500 μL of medium containing 2.5 nmol ATP for 1.0 h. TPSNs (prepared from TPSNs prepared in Example 1): Stimulated with 500 μL of medium containing 50 ng LPS for 3.0 h, then the medium was discarded; then incubated with 500 μL of medium containing 150 μg TPSNs for 0.5 h, then the medium was discarded; finally, treated with 500 μL of medium containing 2.5 nmol ATP for 1.0 h. 3) Non-classical pyroptosis pathway (LPS transfection): Ctrl group: blank control, incubated with 500 μL Opti-MEM medium for 9.5 h, then the medium was discarded; then incubated with 500 μL MEM complete medium containing 10% FBS for 16.0 h. Model group: positive control. Pretreated with 500 μL of Opti-MEM medium containing 500 ng Pam3CSK4 for 3.0 h, then the medium was discarded; then incubated with 500 μL of Opti-MEM medium for 0.5 h, then the medium was discarded; 500 μL of Opti-MEM medium containing 1 μg LPS and 5 μL LP3000™ was mixed with 500 μL of Opti-MEM medium containing 5 μL Lipofectamine™ 3000, and incubated at room temperature for 15 min to prepare 1.00 mL of "liposome-LPS" complex Opti-MEM medium. 500 μL of "liposome-LPS" complex Opti-MEM medium was added to primary macrophages and incubated for 6.0 h, then the medium was discarded; finally, incubated with 500 μL of MEM complete medium containing 10% FBS for 16.0 h. TPSNs (prepared from TPSNs prepared in Example 1): Except that the incubation time of 500 μL Opti-MEM medium for 0.5 h was replaced with 500 μL Opti-MEM medium containing 150 μg TPSNs for 0.5 h, the experimental procedures were the same as those in Example 1. The Model group is the same.

[0250] After incubation, the cell culture medium of each group was collected, the supernatant was obtained by centrifugation, and the concentration of pro-inflammatory cytokines IL-1α and IL-1β was detected by ELISA kit.

[0251] Figure 38 The results of detecting the concentrations of pro-inflammatory cytokines IL-1α (A, B, C) and IL-1β (D, E, F) in the supernatant of primary macrophage culture medium using ELISA kits are shown in the figure. Experimental data are expressed as mean ± standard deviation (n=3, , , , As shown in the figure, the concentrations of pro-inflammatory cytokines IL-1α and IL-1β in the Ctrl group were very low; after activation of primary macrophage pyroptosis via both classical and non-classical pyroptosis pathways, the concentrations of IL-1α and IL-1β in the Model group were significantly increased; compared with the Model group, the differences in IL-1α and IL-1β concentrations between the TPSNs group and the Model group were statistically significant. , , and This indicates that TPSNs can prevent the leakage of pro-inflammatory cytokines and have the activity of inhibiting pyroptosis.

[0252] II. Research on the causes of cell membrane pore formation: GSDMD-NT oligomerization

[0253] Gasdermin D (GSDMD) is an executive protein that plays a crucial role in pyroptosis. When cells are subjected to dangerous stimuli, caspases or granzymes cleave GSDMD, releasing its active N-terminal domain (GSDMD-NT). GSDMD-NT binds to phosphatidylinositol and cardiolipin on the inner side of the cell membrane and oligomerizes, forming pores with a diameter of 10-20 nm in the cell membrane. This promotes the release of intracellular pro-inflammatory cytokines, leading to osmotic imbalance and ultimately triggering pyroptosis. Labeling GSDMD-NT with fluorescent groups and visualizing this process using immunofluorescence imaging can serve as a basis for assessing the level of pyroptosis.

[0254] Primary macrophages were used at a rate of 5 × 10 4 Seeds were placed at a density of cells / well in confocal microplates and incubated in a cell culture incubator for 12.0 h to allow for full cell adhesion. The culture medium was then aspirated, and the cells were washed twice with PBS. Cells were then grouped and treated as follows: Mock group: blank control, incubated with 500 μL of culture medium for 4.5 h. LPS+nigericin group: positive control. Stimulated with 500 μL of medium containing 50 ng LPS for 3.0 h, the medium was discarded; then incubated with 500 μL of medium for 0.5 h, the medium was discarded; finally treated with 500 μL of medium containing 5 nmol nigericin for 1.0 h. The LPS+nigericin+TPSNs group (prepared from TPSNs prepared in Example 1) was stimulated with 500 μL of medium containing 50 ng LPS for 3.0 h, and the medium was discarded. Then, the cells were incubated with 500 μL of medium containing 150 μg TPSNs for 0.5 h, and the medium was discarded. Finally, the cells were treated with 500 μL of medium containing 5 nmol nigericin for 1.0 h. After incubation, the medium was discarded from each group, and the cells were washed twice with PBS. 1.00 mL of 4% paraformaldehyde was added for fixation at room temperature for 10 min. After fixation, the paraformaldehyde was discarded, and the cells were washed twice with PBS. 1.00 mL of 1% BSA solution was added for blocking at room temperature for 30 min. GSDMD antibody solution diluted in Staining Buffer was added to each of the above groups, and the cells were incubated at 4°C for 12.0 h. After incubation, the cells were labeled with Alexa Fluor 594-bonded Goat-anti-Rabbit fluorescent secondary antibody and anti-fluorescence decay mounting medium (containing DAPI, which is used to label cell nuclei) was added. The cells were stored at 4°C and observed using a laser confocal microscope.

[0255] Figure 39Immunofluorescence imaging of GSDMD in primary macrophages from each group is shown, with a scale bar of 20 μm. As can be seen, a distinct red fluorescent band appears at the edge of the primary macrophage membrane in the LPS+nigericin group, with several discontinuous breaks within the band, indicating that GSDMD-NT accumulates on the cell membrane surface and forms pores, suggesting pyroptosis. The red fluorescent band disappears at the edge of the primary macrophage membrane in the LPS+nigericin+TPSNs group, indicating that TPSNs can inhibit the oligomerization of GSDMD-NT on the inner side of the cell membrane, suppressing pore formation and reversing pyroptosis.

[0256] The above results indicate that TPSNs inhibit pyroptosis by suppressing the oligomerization of GSDMD-NT on the inner side of the cell membrane, reversing the formation of cell membrane pores, and preventing the leakage of pro-inflammatory cytokines, thereby achieving an anti-pyroptosis effect.

[0257] Experimental Example 10

[0258] Investigating the mechanism of action of TPSNs prepared in Example 1 in promoting intestinal barrier repair: a study on the MAPK signaling pathway, PI3K-Akt signaling pathway, and the cleavage of the DNA repair enzyme PARP.

[0259] I. 5-HT on the surface of NCM460 cells 1A Research on receptor distribution

[0260] NCM460 cells in the logarithmic growth phase were harvested at a concentration of 5 × 10⁻⁶ cells / year. 4 Seeds were placed at a density of cells / well in confocal microplates and incubated in a cell culture incubator for 12.0 h to allow for full cell adhesion. The culture medium was then aspirated, and the cells were washed twice with PBS. 1.00 mL of 4% paraformaldehyde was added, and the cells were fixed at room temperature for 10 min. The paraformaldehyde was then aspirated, and the cells were washed twice with PBS. 1.00 mL of 1% BSA solution was added, and the cells were blocked at room temperature for 30 min. 5-HT diluted in Staining Buffer was then added... 1A Receptor antibody solution was added to cells and incubated at 4°C for 12.0 h. After incubation, cells were labeled with Alexa Fluor 594-bonded fluorescent secondary antibody and anti-fluorescence attenuation mounting medium (containing DAPI, which is used to label cell nuclei) was added. Cells were stored at 4°C and observed using a laser confocal microscope.

[0261] Figure 40 5-HT for NCM460 cells 1A Immunofluorescence imaging of the receptor, scale bar 20 μm. As shown in the figure, bright red fluorescence was detected on the surface of NCM460 cells, indicating that 5-HT... 1A The receptor is highly expressed on the cell surface, meaning that a large number of 5-HT receptors are distributed on the cell surface. 1AThe receptor indicates that NCM460 cells can be used to study the effect of TPSNs on 5-HT. 1A Receptor activation status.

[0262] II. TPSNs activate 5-HT 1A Research on receptors, which activate the MAPK and PI3K-Akt signaling pathways in an intracellular cascade, inhibiting apoptosis and promoting proliferation.

[0263] To confirm TPSNs pass 5-HT 1A The receptor cascade activates the MAPK and PI3K-Akt signaling pathways, and the phosphorylation levels of key regulatory proteins in these two pathways were detected by Western blotting. 5-HT was used. 1A The receptor-specific inhibitor WAY-100635, when used to treat cells in the same manner, inhibited 5-HT. 1A Receptors were used for reverse verification. Furthermore, the inhibitory effects of poly-EGCG nanoparticles (TPNs), poly-5-HT nanoparticles (PSTs), and TPSNs on apoptosis inhibition and proliferation promotion were compared.

[0264] NCM460 cells in the logarithmic growth phase were harvested at a concentration of 5 × 10⁻⁶ cells / year. 5 Seeds were planted at a density of cells / well in 6-well plates and incubated in a cell culture incubator for 12.0 h to allow for full cell adhesion. The culture medium was then aspirated, and the cells were washed twice with PBS. Cells were then grouped and treated as follows: 1) Mock portion: Nono group: blank control, with 2.00 mL of culture medium added. TPNs (solely synthesized polyEGCG nanoparticles) group: Add 2.00 mL of culture medium containing 600 μg TPNs. PST (isolated poly5-HT nanoparticles) group: Add 2.00 mL of culture medium containing 600 μg PST. TPSNs (prepared from TPSNs prepared in Example 1): 2.00 mL of culture medium containing 600 μg TPSNs was added. 2) WAY-100635 section: Nono group: blank control, with 2.00 mL of culture medium containing 10 nmol WAY-100635 added. TPNs (solely synthesized polyEGCG nanoparticles) group: Add 2.00 mL of culture medium containing 600 μg TPNs and 10 nmol WAY-100635. PST (isolated poly5-HT nanoparticles) group: Add 2.00 mL of culture medium containing 600 μg PST and 10 nmol WAY-100635. TPSNs (prepared from TPSNs prepared in Example 1): 2.00 mL of medium containing 600 μg TPSNs and 10 nmol WAY-100635 was added. All groups were incubated for 24.0 h. After incubation, the medium was discarded, and the samples were washed three times with pre-cooled PBS. Protein samples were extracted and quantified, followed by SDS-PAGE gel electrophoresis and transfer, blocking and antibody incubation, and protein band development to complete the experiment.

[0265] Figure 41 This is a graph showing the expression levels of p-MEK and p-ERK1 / 2, key regulatory proteins in the MAPK signaling pathway, and p-AKT and p-mTOR, key regulatory proteins in the PI3K-Akt signaling pathway. The graph shows: 1) Mock section: Compared with the None group, there was no difference in the expression levels of p-MEK, p-ERK1 / 2, p-AKT and p-mTOR proteins in NCM460 cells in the TPNs group, and none of them were expressed, indicating that TPNs cannot activate the MAPK signaling pathway and the PI3K-Akt signaling pathway. The expression levels of the four proteins mentioned above in the PST group were significantly higher than those in the None group, indicating that PST can activate the MAPK signaling pathway and exert the effect of 5-HT to inhibit cell apoptosis; activate the PI3K-Akt signaling pathway and exert the effect of 5-HT to promote cell proliferation, which helps in intestinal barrier repair. The expression levels of the four proteins mentioned above in the TPSNs group were significantly higher than those in the None group, but not as high as those in the PST group. This indicates that the 5-HT component in TPSNs can activate the MAPK signaling pathway and the PI3K-Akt signaling pathway, exerting a strong inhibitory effect on apoptosis and promoting cell proliferation, thus protecting the intestinal barrier. TPSNs, composed of EGCG and 5-HT, resulted in a weaker inhibitory effect on apoptosis and a weaker effect on cell proliferation compared to PST, which is composed of pure 5-HT. 2) WAY-100635 part: 5-HT added. 1A The receptor-specific inhibitor WAY-100635, Compared with the None group and the corresponding Mock group, no difference was found in the expression levels of p-MEK, p-ERK1 / 2, p-AKT, and p-mTOR proteins in the TPNs group, and none of them were expressed, indicating inhibition of 5-HT. 1A Even after receptor activation, TPNs still cannot activate the MAPK signaling pathway and the PI3K-Akt signaling pathway. Compared with the PST group, TPSNs group, and None group, there was no difference in the expression levels of the above four proteins, and none of them were expressed; compared with the corresponding groups in the Mock section, the expression levels of the above four proteins were not upregulated; indicating that inhibition of 5-HT 1AFollowing the receptor, neither the MAPK signaling pathway nor the PI3K-Akt signaling pathway was activated. This demonstrates that 5-HT... 1A The necessity of receptor activation of the MAPK and PI3K-Akt signaling pathways also clarifies that this receptor is a target of TPSNs and PST.

[0266] III. TPSNs activate 5-HT 1A Research on receptors, reducing the cleavage of the DNA repair enzyme PARP, and inhibiting apoptosis

[0267] The DNA repair enzyme PARP promotes the repair of damaged DNA and is a cleavage substrate of caspase, a core member of apoptosis. When the apoptotic program is initiated, activated caspase cleaves PARP, inactivating it and preventing the damaged DNA from being repaired, thus positively promoting the apoptosis process. Therefore, cleaved PARP is a marker of apoptosis.

[0268] When apoptosis occurs, it is necessary to confirm that TPSNs activate 5-HT. 1A The cleavage of the post-receptor DNA repair enzyme PARP was investigated using Western blotting to detect the expression level of cleaved PARP protein. 5-HT was used. 1A The receptor-specific inhibitor WAY-100635, when used to treat cells in the same manner, inhibited 5-HT. 1A Receptors were used for reverse verification. Furthermore, the inhibitory effects of polyEGCG nanoparticles (TPNs), poly5-HT nanoparticles (PSTs), and TPSNs on apoptosis were compared.

[0269] NCM460 cells in the logarithmic growth phase were harvested at a concentration of 5 × 10⁻⁶ cells / year. 5 Seeds were planted at a density of cells / well in 6-well plates and incubated in a cell culture incubator for 12.0 h to allow for full cell adhesion. The culture medium was then aspirated, and the cells were washed twice with PBS. Cells were then grouped and treated as follows: 1) Mock portion: None group: blank control, with 2.00 mL of culture medium added. LPS group: Add 2.00 mL of culture medium containing 20 μg LPS. LPS+TPNs (solely synthesized polyEGCG nanoparticles) group: Add 2.00 mL of culture medium containing 20 μg LPS and 600 μg TPNs. LPS+PST (isolated poly5-HT nanoparticles) group: Add 2.00 mL of culture medium containing 20 μg LPS and 600 μg PST. LPS+TPSNs (prepared from TPSNs prepared in Example 1): 2.00 mL of culture medium containing 20 μg LPS and 600 μg TPSNs was added. 2) WAY-100635 section: None group: blank control, with 2.00 mL of culture medium containing 10 nmol WAY-100635 added. LPS group: Add 2.00 mL of culture medium containing 20 μg LPS and 10 nmol WAY-100635. LPS+TPNs (solely synthesized polyEGCG nanoparticles) group: Add 2.00 mL of culture medium containing 20 μg LPS, 600 μg TPNs and 10 nmol WAY-100635. LPS+PST (isolated poly5-HT nanoparticles) group: 2.00 mL of culture medium containing 20 μg LPS, 600 μg PST and 10 nmol WAY-100635 was added. LPS+TPSNs (prepared from TPSNs prepared in Example 1) group: 2.00 mL of medium containing 20 μg LPS, 600 μg TPSNs, and 10 nmol WAY-100635 was added. All groups were incubated for 12.0 h. After incubation, the medium was discarded, and the samples were washed three times with pre-cooled PBS. Protein samples were extracted and quantified, followed by SDS-PAGE gel electrophoresis and transfer, blocking and antibody incubation, and protein band development to complete the experiment.

[0270] Figure 42 This is a graph showing the cleavage levels of the DNA repair enzyme PARP in each group of NCM460 cells. As shown in the graph, 1) Mock section: The expression level of cleaved PARP protein in NCM460 cells of the LPS group was significantly higher than that of the None group, indicating that the model was successful. LPS caused a large amount of DNA repair enzyme PARP to be cleaved, leading to apoptosis. Compared with the LPS group, the expression levels of the above proteins in the LPS+TPNs group, LPS+PST group, and LPS+TPSNs group decreased sequentially, with almost no expression observed in the LPS+TPSNs group. This indicates that TPNs, PST, and TPSNs can reduce the cleavage of the DNA repair enzyme PARP, and that the EGCG and 5-HT components in TPSNs play different physiological roles, synergistically inhibiting apoptosis and thus better promoting intestinal barrier repair. 2) WAY-100635 part: 5-HT added. 1A The receptor-specific inhibitor WAY-100635, The expression level of cleaved PARP protein in the LPS group was significantly higher than that in the None group, indicating that the target of LPS is not 5-HT.1A Once the receptor is successfully established and the model is established, the DNA repair enzyme PARP undergoes extensive cleavage, leading to apoptosis. Compared with the LPS group, the LPS+TPNs group showed decreased expression levels of cleaved PARP protein; compared with the corresponding group in the Mock section, the expression levels of the above protein were the same; indicating inhibition of 5-HT 1A Even after the receptor is activated, TPNs can still reduce the cleavage of the DNA repair enzyme PARP and inhibit apoptosis, indicating that the receptor is not a target of TPNs. Compared with the LPS group, the LPS+PST group and the LPS+TPSNs group showed the same cleaved PARP protein expression level; compared with the corresponding groups in the Mock section, the expression levels of the above proteins were significantly upregulated, indicating inhibition of 5-HT. 1A Following receptor activation, PST and TPSNs failed to reduce the cleavage of the DNA repair enzyme PARP and did not inhibit apoptosis. This demonstrates that TPSNs and PST activate 5-HT... 1A Receptors reduce the cleavage of the DNA repair enzyme PARP, thereby inhibiting apoptosis.

[0271] The above results indicate that TPSNs activate 5-HT 1A Upon receptor activation, the intracellular cascade activates the MAPK signaling pathway, reducing the cleavage of the DNA repair enzyme PARP to inhibit apoptosis; simultaneously, it activates the PI3K-Akt signaling pathway, promoting cell proliferation and thus accelerating intestinal barrier repair.

[0272] In summary, TPSNs demonstrate the following functions: (1) significantly scavenging free radicals and intracellular RONS; (2) promoting macrophage polarization towards the anti-inflammatory M2 type; (3) inhibiting pyroptosis, regulating the cell cycle, and accelerating cell renewal; (4) inhibiting the NF-κB signaling pathway, downregulating the phosphorylation levels of key regulatory proteins IKKα / β, p65, and IκBα, and exerting anti-inflammatory effects; (5) activating the MAPK signaling pathway, upregulating the phosphorylation levels of key regulatory proteins MEK and ERK, reducing the cleavage of the DNA repair enzyme PARP, and inhibiting apoptosis; (6) activating the PI3K-Akt signaling pathway, upregulating the phosphorylation levels of key regulatory proteins AKT and mTOR, promoting cell proliferation, and completing intestinal barrier repair. Ultimately, TPSNs achieve the goal of synergistic treatment of IBD through "anti-inflammatory-antioxidant-mucosal repair," and are potential multi-target, highly efficient nanomedicines for IBD treatment.

[0273] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. EGCG-5-HT binary oxidative self-polymerization nanoparticles, characterized in that: The EGCG-5-HT binary oxidized self-polymer nanoparticles are obtained by oxidation of EGCG and 5-HT in an alkaline environment.

2. The EGCG-5-HT binary oxidatively self-polymerizing nanoparticle of claim 1, wherein: The mass ratio of the EGCG to 5-HT is 1:

4.

3. The EGCG-5-HT binary oxidatively self-polymerizing nanoparticle of claim 2, wherein: The particle size of the obtained nanoparticles is between 80-250 nm.

4. The EGCG-5-HT binary oxidatively self-polymerizing nanoparticle of claim 3, wherein: The pH of the alkaline environment is 7.5-9.5, and a stabilizer polysorbate-80 is added in the oxidation process.

5. The EGCG-5-HT binary oxidatively self-polymerizing nanoparticle of claim 3, wherein: A stabilizer and manganese chloride are added in the oxidation process.

6. A method for preparing the EGCG-5-HT binary oxidatively self-polymerizing nanoparticle according to any one of claims 1 to 4, characterized in that The method comprises the following steps: S1, dropping the 5-HT solution into water and stirring uniformly; S2, dropping the polysorbate-80 solution into the solution of step S1 and stirring uniformly; S3, dropping the EGCG solution into the mixture of step S2 and stirring uniformly; S4, dropping the buffer solution with pH 7.5-9.5 into the mixture of step S3 and stirring uniformly; S5, dialyzing the reaction solution obtained in step S4 to obtain the EGCG-5-HT binary oxidized self-polymer nanoparticles.

7. The method for preparing EGCG-5-HT binary oxide self-polymerizing nanoparticles according to claim 6, characterized in that: The stirring of steps S1, S2, S3 and S4 is carried out in a water bath at 10-30℃.

8. The method for preparing EGCG-5-HT binary oxide self-polymerizing nanoparticles according to claim 6, characterized in that: The following step is added between step S4 and step S5: adding the manganese chloride solution into the mixture of step S4, stirring uniformly in a water bath at 10-30℃.

9. The method for preparing EGCG-5-HT binary oxide self-polymerizing nanoparticles according to claim 6, characterized in that: The buffer is Tris buffer.

10. Use of the EGCG-5-HT binary oxidatively self-polymerizing nanoparticle according to any one of claims 1 to 5, characterized in that: The method is used for preparing the drug for inflammatory bowel disease. The method is used for preparing the drug for inflammatory bowel disease.

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