Monoamine oxidation self-polymerization nanoparticles as well as preparation method and application thereof

Abstract

The invention relates to the field of nanometer material development and nanometer biological medicine and pharmacology, in particular to monoamine oxidation self-polymerization nanoparticles as well as a preparation method and application thereof. The preparation method of the monoamine oxidation self-polymerization nanoparticles comprises the following steps: sequentially adding a tween-80 solution, a 5-HT solution and an alkaline buffer solution into purified water in a water bath at 50-70 DEG C, stirring at 800-1200 rpm, and centrifuging to obtain the monoamine oxidation self-polymerization nanoparticles. The nanoparticles have free radical scavenging capacity, and the scavenging efficiency is in direct proportion to the concentration. After the 5-HT micromolecules are oxidized and self-polymerized to form the nanoparticles, the in-vivo cytotoxicity of the nanoparticles can be remarkably reduced, the biocompatibility is improved, and meanwhile, the nanoparticles have the capabilities of remarkably promoting cell proliferation and migration, inhibiting inflammation-induced cell apoptosis and regulating and controlling the cell cycle. The nanoparticles can also be used as a carrier to be combined with other drugs.

Description

Technical Field

[0001] This invention relates to the fields of nanomaterial development and nanobiomedicine, specifically to monoamine oxidative self-polymerized nanoparticles, their preparation methods, and applications. Background Technology

[0002] Inflammatory bowel disease (IBD) is a chronic digestive system disease characterized by recurrent inflammation and disruption of the intestinal barrier. It is characterized by a long course, persistent nature, and high relapse rate. The pathogenesis of IBD is complex, primarily resulting from the interaction between genetic defects, the intestinal immune system, and the gut microbiota, ultimately disrupting the integrity of the intestinal barrier. At IBD lesion sites, the levels of reactive oxygen species (ROS) and inflammatory factors are significantly elevated. Persistent oxidative stress and inflammatory responses activate mucosal immunity, exacerbating intestinal tissue damage and further worsening the condition.

[0003] Currently, drug intervention is the preferred strategy for treating IBD, including salicylates, corticosteroids, immunomodulators, and monoclonal antibodies targeting inflammatory cytokines. However, these drugs primarily suppress intestinal inflammation and relieve symptoms, lacking the ability to repair the intestinal barrier. Furthermore, their clinical application suffers from drawbacks such as low bioavailability, systemic toxicity at high doses, and cost-effectiveness, failing to fully meet patient needs. Therefore, developing safe drugs with multiple functions including antioxidant, anti-inflammatory, and intestinal mucosal repair is of significant clinical importance for the treatment of IBD.

[0004] Nanoparticle drug delivery systems have strong targeting and high bioavailability in the physiological environment, which can increase the local concentration of drugs, improve efficacy, and enhance the overall treatment experience for patients. They have great application prospects and show great potential in the treatment of IBD.

[0005] 5-Hydroxytryptamine (5-HT) is a biogenic amine neurotransmitter, belonging to the monoamine class of compounds, and plays a crucial role in maintaining the physiological function of the intestinal mucosal barrier. 90% of the body's 5-HT is synthesized by intestinal chromaffin cells. It acts on 5-HT receptors in intestinal cells, regulating intestinal motility, promoting intestinal nerve maturation, and promoting intestinal cell proliferation and differentiation. It also possesses antioxidant activity, capable of repairing damaged intestinal mucosal barriers and reducing inflammatory responses.

[0006] Based on the idea that substances produced by the body itself are safer and easier to absorb, 5-HT is made into oxidized self-polymerized nanoparticles, which can exert antioxidant, anti-inflammatory and intestinal mucosal repair functions. Through the "antioxidant-anti-inflammatory-mucosal repair" pathway, multi-level drug treatment of IBD is achieved, providing a new research direction for multi-mechanism synergistic treatment of IBD. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a monoamine oxidation self-polymerized nanoparticle with a stable structure.

[0008] This invention also provides a method for preparing 5-hydroxytryptamine (5-HT) by oxidizing and self-polymerizing it in an alkaline environment to form monoamine oxidized self-polymerized nanoparticles (Polyserotonin, PST). This method for preparing nanoparticles is simple and the conditions are controllable.

[0009] This invention also provides its applications. The monoamine oxidized self-polymerized nanoparticles prepared can scavenge free radicals, promote cell proliferation and migration, inhibit cell apoptosis, regulate cell cycle and accelerate cell renewal. They have multiple molecular biological mechanisms of "antioxidation-anti-inflammation-mucosal repair" and can be used in the preparation of IBD drugs, with huge market application potential.

[0010] The monoamine oxidative self-polymerized nanoparticles of the present invention are prepared by oxidative self-polymerization of 5-HT in a water bath at pH 8.0~10.0 and 50℃~70℃.

[0011] The method for preparing monoamine oxidized self-polymerized nanoparticles according to the present invention includes the following steps:

[0012] S1. Dissolve Tween-80 solution in purified water, and heat in a constant temperature water bath at 50℃~70℃, stirring at 800~1200rpm.

[0013] S2. Add the 5-HT solution dropwise into the aqueous solution described in step S1, and stir in a constant temperature water bath at 50℃~70℃ at 800~1200 rpm.

[0014] S3. Add the buffer solution with pH 8.0~10.0 dropwise into the mixture described in step S2, and stir at 800~1200 rpm in a constant temperature water bath at 50℃~70℃.

[0015] S4. Centrifuge the reaction solution obtained in step S3, collect the precipitate and wash it with purified water to obtain monoamine oxidized self-polymerized nanoparticles.

[0016] Preferably, the final concentration of Tween-80 in the reaction solution in step S1 is 3.0~5.0 mg / mL.

[0017] Preferably, in step S2, the final concentration of 5-HT in the reaction solution is 1.0~3.0 mg / mL.

[0018] Preferably, the buffer solution in step S3 is a Tris buffer solution.

[0019] Preferably, the Tris buffer solution has a final molar concentration of 5-15 mM in the reaction solution.

[0020] Preferably, the stirring reaction time in step S3 is 4.0~8.0h.

[0021] The application of the monoamine oxidized self-polymerized nanoparticles described in this invention is used to prepare drugs for treating inflammatory bowel disease (IBD).

[0022] Preferably, the monoamine oxidized self-polymerized nanoparticles can be used in injectable formulations, topical formulations, or oral formulations.

[0023] Preferably, the monoamine oxidized self-polymerized nanoparticles can be simultaneously loaded with other therapeutic agents, such as antibodies and organic acids.

[0024] The monoamine oxidative self-polymerized nanoparticles described above can be used to prepare antioxidant drugs, drugs that promote cell proliferation and migration, drugs that inhibit cell apoptosis, or drugs that promote cell proliferation and accelerate cell renewal by regulating the cell cycle.

[0025] Based on a general inventive concept, the present invention also provides an application of monoamine oxidation self-polymerized nanoparticles for scavenging free radicals.

[0026] Based on a general inventive concept, the present invention also provides an application of monoamine oxidized self-polymerized nanoparticles to reduce the cytotoxicity of 5-HT in vivo and improve biocompatibility.

[0027] Based on a general inventive concept, the present invention also provides an application of monoamine oxidation self-polymerized nanoparticles in promoting cell proliferation and migration.

[0028] Based on a general inventive concept, the present invention also provides an application of monoamine oxidation self-polymerized nanoparticles in inhibiting cell apoptosis.

[0029] Based on a general inventive concept, the present invention also provides an application of monoamine oxidation self-polymerized nanoparticles to regulate the cell cycle, thereby promoting cell proliferation and accelerating cell renewal.

[0030] The monoamine oxidized self-polymerized nanoparticles in this invention are formed by the rapid oxidation and polymerization of 5-HT in an alkaline environment, resulting in a series of oligomers. As the reaction time increases, the number of oligomers gradually increases, leading to aggregation. Furthermore, these nanoparticles follow the principle of minimum surface energy and self-assemble into monoamine oxidized self-polymerized nanospheres through non-covalent interactions.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0032] (1) The monoamine oxidative self-polymerized nanoparticles of the present invention are formed by the oxidative self-polymerization of 5-HT in an alkaline environment without the need for a catalyst; a constant temperature water bath of 50℃~70℃ results in a short reaction time and high efficiency, and a large number of nanoparticles can be generated within 4.0~8.0h; under stirring at 800~1200rpm, there is no need to use a microporous filter membrane to control the particle size, and nanoparticles with a particle size of about 157nm and uniform distribution can be obtained; Tween-80 is only used as a stabilizer to keep the nanoparticles in a uniform and stable state, and to avoid aggregation and precipitation between particles; the nanoparticles have a simple structure, good water solubility, and good biocompatibility.

[0033] (2) The preparation method of the monoamine oxidized self-polymerized nanoparticles described in this invention is simple, the conditions are controllable, and the composition is well-defined. These oxidized self-polymerized nanoparticles have the excellent characteristics of simple preparation, stable structure, and inexpensive and readily available reaction reagents, and can be prepared in batches, making them easy to promote and apply on a large scale.

[0034] (3) In addition to its own antioxidant properties, ability to reduce 5-HT cytotoxicity, promote cell proliferation and migration, inhibit cell apoptosis and regulate cell cycle, the monoamine oxidative self-polymerized nanoparticles of the present invention can also serve as effective carriers for other drugs, such as loading antibodies and organic acid drugs, to achieve multiple therapeutic mechanisms and enhance the efficacy of the prepared drugs. They have shown great application potential in the preparation of IBD treatment drugs.

[0035] (4) The present invention creatively discovers that the monoamine oxidized self-polymerized nanoparticles have free radical scavenging ability and can be used in the preparation of various inflammatory disease drugs. At the same time, it provides an innovative application of monoamine oxidized self-polymerized nanoparticles in the preparation of IBD drugs: the monoamine oxidized self-polymerized nanoparticles can scavenge free radicals and have antioxidant activity.

[0036] (5) The monoamine oxidation self-polymerized nanoparticles provided by the present invention can reduce the toxicity of 5-HT to cells and improve biocompatibility.

[0037] (6) The monoamine oxidized self-polymerized nanoparticles of the present invention have been shown to have a significant ability to promote cell proliferation and migration through cell plate cloning experiments, cell scratch experiments and cell migration experiments. They have a protective effect on cells and can be used to prepare drugs that promote cell proliferation and migration.

[0038] (7) The monoamine oxidized self-polymerized nanoparticles provided by the present invention can significantly inhibit cell apoptosis induced by DSS+IL-1β, and have a protective effect on cells. They can be used to prepare drugs that inhibit cell apoptosis.

[0039] (8) The monoamine oxidized self-polymerized nanoparticles described in this invention can significantly increase the proportion of G2 / M phase cells in cells treated with DSS+IL-1β, and have a protective effect on cells. They can be used to prepare drugs that promote cell proliferation and accelerate cell renewal by regulating the cell cycle.

[0040] (9) The monoamine oxidation self-polymerized nanoparticles provided by the present invention achieve the goal of “antioxidation-anti-inflammatory-mucosal repair” in the treatment of IBD, and are a highly efficient multi-mechanism synergistic nanomedicine for the treatment of IBD. Attached Figure Description

[0041] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0042] Figure 1 This is a schematic diagram of the synthesis process of monoamine oxidative self-polymerized nanoparticles (PST) in Example 1.

[0043] Figure 2 (A) is a photograph of the PST solution in Example 1; Figure 2 (B) is the PST particle size distribution diagram of Example 1; Figure 2 (C) is the PST average DLS particle size and PDI diagram of Example 1; Figure 2 (D) is the Zeta potential diagram of PST in Example 1.

[0044] Figure 3 (A) is a PST transmission electron microscope image of Example 1, with a scale bar of 200 nm; Figure 3 (B) is a scanning electron microscope image of the PST solution of Example 1 after heating and drying, with a scale bar of 100 nm; Figure 3 (C) is a scanning electron microscope image of the PST solution after freeze-drying in Example 1, with a scale bar of 200 nm.

[0045] Figure 4 The image shows the results of elemental qualitative analysis of PST in Example 1 using TEM, with a scale bar of 20 nm.

[0046] Figure 5 The image shows the PST UV absorption spectrum of 5-HT and Example 1.

[0047] Figure 6 The PST infrared absorption spectrum of 5-HT, Example 1.

[0048] Figure 7The graph shows the changes in particle size and PDI of PST in Example 1 over 0 to 48.0 h in purified water (A), HEPES (pH 7.4) (B), Tris (pH 7.4) (C), 1640 medium (D), and MEM medium (10% FBS) (E).

[0049] Figure 8 (A) Time-scavenging rate curve of PST scavenging DPPH free radicals in Example 1 (100 μg / mL); Figure 8 (B) is the UV-Vis spectrum of the above PST and DPPH solution after incubation for 40 min; Figure 8 (C) is a graph showing the DPPH free radical scavenging results of PST at concentrations of 10, 50 and 100 μg / mL in Example 1 after 40 min of incubation.

[0050] Figure 9 (A) PST clearance at 100 μg / mL in Example 1 Time-scavenging rate curve of free radicals; Figure 9 (B) refers to the above PST and UV-Vis spectrum of solution incubation for 40 min; Figure 9 (C) PST at concentrations of 10, 50, and 100 μg / mL in Example 1 after incubation for 40 min. Free radical scavenging rate results graph.

[0051] Figure 10 (A) is a graph showing the survival rate of NCM460 cells after incubation with different concentrations of 5-HT for 24.0 h; Figure 10 (B) is a graph showing the survival rate of NCM460 cells after incubation with PST of different concentrations in Example 1 for 24.0 h.

[0052] Figure 11 Figure 1 shows the results of the plate colony formation experiment for NCM460 cells and Caco-2 cells in each group.

[0053] Figure 12 (A) Photographs of the scratch assay results of NCM460 cells in each group; Figure 12 (B) is a graph showing the quantitative analysis results of the unhealed areas of NCM460 cell scratches in each group; Figure 12 (C) Photographs of the scratch test results of Caco-2 cells in each group; Figure 12 (D) is a graph showing the quantitative analysis results of the unhealed areas of Caco-2 cell scratches in each group; the scale bar is 20 μm.

[0054] Figure 13 Images showing the migration results of NCM460 and Caco-2 cells in each group, with a scale bar of 100 μm.

[0055] Figure 14 (A) is a flow cytometry analysis result of apoptosis in NCM460 cells in each group; Figure 14 (B) refers to the Annexin V groups mentioned above. + / PI + The results of quantitative analysis of NCM460 cell populations are shown in the figure.

[0056] Figure 15 (A) is a graph showing the flow cytometry results of NCM460 cells in the Ctrl and PST groups; Figure 15 (B) is a graph showing the results of the quantitative analysis of the cell cycle in the two groups mentioned above; Figure 15 (C) is a flow cytometry analysis result of NCM460 cells in the DI group and DI+PST group; Figure 15 (D) is a graph showing the results of the quantitative analysis of the cell cycle in the two groups mentioned above; DI: DSS+IL-1β. Detailed Implementation

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] The main reagents used in the following examples and their manufacturers are listed in Table 1:

[0062] Table 1. Main Reagent Names and Manufacturers

[0063]

[0064] For details regarding the instruments and manufacturers used in the following embodiments, please refer to Table 2:

[0065] Table 2. Main Instrument Names and Manufacturers

[0066]

[0067] Example 1

[0068] The steps for preparing monoamine oxidized self-polymerized nanoparticles (PST) are as follows:

[0069] S1. Under constant temperature water bath conditions of 60℃, add 9.9mL of purified water to a 30mL glass screw-top bottle with a cap, then add 600μL of 100mg / mL Tween-80 solution and stir at 1000rpm.

[0070] S2. Take 3.0 mL of 10 mg / mL 5-HT solution and add it dropwise to the solution in step S1 above, and stir at 1000 rpm.

[0071] S3. Take 1.5 mL of 100 mM pH 9.0 Tris buffer salt and add it dropwise to the mixed solution in step S2 above. Stir at 1000 rpm for 6.0 h.

[0072] S4. Take the reaction solution obtained in step S3, centrifuge at 16,000 rpm for 15 min at 4°C using a low-temperature high-speed centrifuge, collect the precipitate, add purified water to wash and centrifuge, repeat twice, and finally re-dissolve the nanoparticle precipitate by sonication to obtain monoamine oxidized self-polymerized nanoparticles (PST), store at 4°C for later use.

[0073] The above schematic diagram illustrates the synthesis process of monoamine oxidative self-polymerized nanoparticles (PST). Figure 1 As shown.

[0074] Experimental Example 1

[0075] Characterization and colloidal stability study of monoamine oxidized self-polymerized nanoparticles (PST)

[0076] I. Particle size and Zeta potential

[0077] Detection method: Take 100 μL of the PST solution prepared in Example 1, dissolve it in 900 μL of purified water and mix well to obtain the sample solution to be tested. Transfer the entire solution to the sample cell and place it in a Malvern ZS-90 nanoparticle size and zeta potential analyzer. The particle size is detected by dynamic light scattering (DLS) and the zeta potential is measured in buffer solutions with different pH values. The test temperature is 25℃, and each sample is tested in triplicate.

[0078] Figure 2 (A) is a photograph of the PST solution in Example 1; Figure 2 (B) is the PST particle size distribution diagram of Example 1; Figure 2 (C) is the PST average DLS particle size and PDI diagram of Example 1; Figure 2(D) is the Zeta potential diagram of PST in Example 1. As shown in the figure, the PST in Example 1 is a transparent, brown, homogeneous, and stable solution. The average DLS particle size is approximately 157 nm with a uniform particle size distribution, and PDI < 0.2. The potential is 21.23 ± 0.42 mV in 10 mM pH 5.5 HEPES buffer and 11.10 ± 0.90 mV in 10 mM pH 7.4 HEPES buffer.

[0079] II. Appearance and Micromorphological Characterization

[0080] Detection methods: (1) Take 100 μL of the PST 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 PST 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 1.5 mL of the PST 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).

[0081] Figure 3 (A) is a PST transmission electron microscope image of Example 1, with a scale bar of 200 nm; Figure 3 (B) is a scanning electron microscope image of the PST solution of Example 1 after heating and drying, with a scale bar of 100 nm; Figure 3 (C) is a scanning electron microscope image of the PST solution of Example 1 after freeze-drying, with a scale bar of 200 nm. As can be seen from the image, the PST of the present invention consists of uniform particles under an electron microscope, with a relatively regular spherical structure, smooth edges, and good dispersibility.

[0082] III. Elemental Analysis of the PST Outer Surface

[0083] Measurement methods: (1) The elemental composition of the sample in method (1) of the "II. Appearance and Micromorphology Characterization" section is qualitatively analyzed by elemental mapping analysis of TEM. (2) The elemental composition of the sample in method (2) of the "II. Appearance and Micromorphology Characterization" section is quantitatively analyzed by energy dispersive spectroscopy (EDS) equipped with SEM.

[0084] Figure 4The image shows the results of elemental qualitative analysis of the PST from Example 1 using TEM, with a scale bar of 20 nm. The high-angle annular dark-field imaging (HAADF) image shows the location and morphology of the PST under the electron microscope. Within the PST outline shown in the HAADF image, a large number of elemental clusters appear in the carbon (C), oxygen (O), and nitrogen (N) images, indicating that the PST surface is mainly composed of C, O, and N elements. Table 3 shows the results of quantitative elemental analysis of the PST from Example 1 using EDS. The table shows that C, O, and N elements were detected in the EDS spectrum; the mass percentages (Weight%) of C, O, and N elements on the PST surface were 59.44%, 26.78%, and 13.79%, respectively; and the atomic percentages (Atomic%) were 65.06%, 22.00%, and 12.94%, respectively. These results fully demonstrate that 5-HT can successfully generate spherical PST structures through oxidative self-polymerization.

[0085] Table 3 Elemental quantitative analysis results of PST

[0086]

[0087] IV. Ultraviolet Absorption Spectrum

[0088] The ultraviolet absorption spectra of 5-HT and PST prepared in Example 1 were scanned. Figure 5 This is the ultraviolet absorption spectrum. As can be seen from the figure, 5-HT exhibits an ultraviolet absorption peak around 230 nm, which is consistent with... The transition is related to electronic kinetics and is generated by the conjugated double bond system on the indole ring. An absorption peak appears around 275 nm, possibly related to the benzene ring, the nitrogen atom on the indole ring, and the phenolic hydroxyl group on the benzene ring. The blue shift of the characteristic absorption peak in PST is due to the oxidative self-polymerization of 5-HT, which affects the planarity of the conjugated molecule, reducing the degree of conjugation and increasing the energy required for the transition. The significant weakening of the absorption peak at 275 nm in PST is likely due to the formation of bonds between the nitrogen atom on the indole ring and other molecules and the reduction in the number of phenolic hydroxyl groups after 5-HT oxidative self-polymerization. Therefore, PST is generated by 5-HT oxidative self-polymerization.

[0089] V. Infrared Absorption Spectroscopy

[0090] Infrared absorption spectroscopy was performed on 5-HT and the PST prepared in Example 1. Figure 6 This is an infrared absorption spectrum. The graph shows that 5-HT... The strong and broad absorption peak is The stretching vibration peak, and The peak at that point is the stretching vibration peak of the CH bond. and The nearby absorption peak is due to the bending vibration of NH. (PST) and The peak at that point represents the stretching vibration peak of the NH bond. and The peak at that point represents the stretching vibration peak of the CH bond. Furthermore, PST retains the basic framework of 5-HT, and the weakening of the absorption peak intensity of PST due to oxidative self-polymerization indicates that 5-HT oxidative self-polymerization forms PST.

[0091] VI. Colloidal Stability Study

[0092] The PST prepared in Example 1 was dispersed in purified water, HEPES buffer (10 mM, pH 7.4), Tris buffer (10 mM, 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 PST particle size and PDI.

[0093] The results of colloidal stability are as follows Figure 7 As shown in the figure, the particle size and PDI of PST did not change significantly within 48.0 h, indicating that PST has good stability in purified water, HEPES buffer, Tris buffer, 1640 medium and MEM medium from 0 to 48.0 h under room temperature conditions.

[0094] Experimental Example 2

[0095] Investigation of the ROS scavenging activity of PST prepared in Example 1

[0096] I. Study on the free radical scavenging performance of DPPH

[0097] According to the instructions of the DPPH free radical scavenging ability test kit, the DPPH powder was dissolved and diluted to the working concentration using a universal diluent. 190 μL of DPPH working solution was thoroughly mixed with 10 μL of 100 μg / mL PST solution, and a blank group (without PST system) 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 a microplate reader. The scavenging rate was calculated according to the following formula (1), and a time-scavenging rate curve was plotted. After incubation for 40 min, the UV-Vis spectra of each group of samples were collected at 400~800 nm. In addition, the scavenging rate of DPPH free radicals by 10 and 50 μg / mL PST was detected by the same operation as above, and the scavenging rate results were plotted.

[0098] (1)

[0099] Figure 8(A) Time-scavenging rate curve of PST scavenging DPPH free radicals in Example 1 (100 μg / mL); Figure 8 (B) is the UV-Vis spectrum of the above PST and DPPH solution after incubation for 40 min; Figure 8 (C) is a graph showing the DPPH radical scavenging rate of PST at concentrations of 10, 50, and 100 μg / mL in Example 1 after 40 min of incubation. The graph shows that PST can effectively scavenge DPPH radicals; the longer the incubation time, the higher the DPPH radical scavenging rate of PST; the DPPH radical scavenging rate is directly proportional to the PST concentration and exhibits a concentration-dependent relationship.

[0100] two, Free radical scavenging performance study

[0101] 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 incubated ABTS working stock solution 40 times to prepare the ABTS working solution, store at room temperature in the dark, and use for later use.

[0102] In a 96-well plate, 200 μL of ABTS working solution and 10 μL of 100 μg / mL PST solution were added to each well, mixed thoroughly, and a blank group (without PST system) was set up. The OD value at 734 nm at each set time point was detected using a microplate reader, and the clearance rate was calculated according to the following formula (2), and a time-clearance rate curve was plotted. After incubation for 40 min, the UV-Vis spectra of each group of samples were collected at 400~1000 nm. In addition, the effects of 10 and 50 μg / mL PST on the clearance rate were detected using the same procedure as above. Calculate the free radical scavenging rate and plot the scavenging rate results.

[0103] (2)

[0104] Figure 9 (A) PST clearance at 100 μg / mL in Example 1 Time-scavenging rate curve of free radicals; Figure 9 (B) refers to the above PST and UV-Vis spectrum of solution incubation for 40 min; Figure 9 (C) PST at concentrations of 10, 50, and 100 μg / mL in Example 1 after incubation for 40 min. Free radical scavenging rate results. The graph shows that PST can scavenge free radicals. Free radicals; the longer the incubation time, the better PST is at inhibiting the growth of free radicals. The higher the free radical scavenging rate; The free radical scavenging rate is directly proportional to the PST concentration and is concentration-dependent.

[0105] The above experimental results all confirm that PST has ROS scavenging activity at the in vitro level.

[0106] Experimental Example 3

[0107] In vivo cytotoxicity and biocompatibility studies of 5-HT and PST prepared in Example 1.

[0108] NCM460 cells in logarithmic growth phase were selected and cultured at a concentration of 5 × 10⁻⁶ cells. 3 Cells were seeded at a density of [number] cells / well in 96-well plates and incubated for 12.0 h to allow for full cell adhesion. The culture medium was then aspirated, and the cells were washed twice with PBS. Grouping and treatment: 5-HT group: The sample group was prepared by adding 100 μL of RPMI-1640 complete culture medium containing 0, 5, 10, 20, 50, 100, 250 and 500 μg / mL of 5-HT. PST group: The sample group was prepared by adding 100 μL of RPMI-1640 complete culture medium containing the corresponding concentration of PST mentioned above. Blank group: Add 100 μL of RPMI-1640 complete culture medium. Negative control group: 100 μL of RPMI-1640 complete medium was added. The cells were incubated in a cell culture incubator for 24.0 h, the medium was discarded, and the cells were rinsed twice with PBS. 100 μL of RPMI-1640 basal medium containing 10% CCK-8 was added to each well of the sample group and blank group, and 100 μL of RPMI-1640 basal medium was added to each well of the negative control group. The cells were incubated for another 0.5 h. The OD value at 450 nm of each sample was detected using a microplate reader, and the cell viability was calculated according to formula (3).

[0109] (3)

[0110] Figure 10 (A) is a graph showing the survival rate of NCM460 cells after incubation with different concentrations of 5-HT for 24.0 h; Figure 10(B) This figure shows the survival rate of NCM460 cells incubated with different concentrations of PST in Example 1 for 24.0 h. As can be seen from the figure, within the concentration range of 0–10 μg / mL, the survival rate of NCM460 cells in both the 5-HT and PST groups was higher than 90%, indicating that 5-HT and PST are safe for cells in vivo within this concentration range. However, when the concentration was between 20–500 μg / mL, the survival rate of cells in the 5-HT group decreased rapidly and in a concentration-dependent manner, indicating that 5-HT has high toxicity to cells in vivo within this concentration range. In contrast, the survival rate of cells in the corresponding concentrations of PST was higher than that of the 5-HT group and both were higher than 90%, indicating that PST has good safety for cells in vivo within this concentration range. This demonstrates that inducing small molecule oxidative self-polymerization to form nanomaterials can significantly reduce the cytotoxicity of small molecule monomers in vivo and improve their biocompatibility.

[0111] Experiment Example 4

[0112] The study investigated the significant cell proliferation and migration-promoting effects of the PST prepared in Example 1.

[0113] I. Cell Plate Colony Formation Assay

[0114] Log-phase NCM460 cells and Caco-2 cells were selected and cultured at a ratio of 1×10⁻⁶. 3 Cells were seeded at a density of [number] cells / well in 6-well plates and incubated for 12.0 h to allow for full cell adhesion. The culture medium was then aspirated, and the cells were washed twice with PBS. Grouping and treatment: Ctrl group: blank control, with 2.0 mL of culture medium added and incubated for 14 days. PST group: Add 2.0 mL of culture medium containing 300 μg / mL PST and incubate for 14 days. Discard the culture medium, wash twice with PBS, add crystal violet staining solution, and stain at room temperature for 10 min. After washing off the staining solution with PBS, observe and photograph under an inverted microscope.

[0115] Figure 11 The figures show the results of the colony formation experiments of NCM460 cells and Caco-2 cells in each group. As can be seen from the figures, compared with the Ctrl group, the number of colonies formed in the PST group of both NCM460 and Caco-2 cells was significantly increased, indicating that PST significantly promoted cell proliferation.

[0116] II. Cell Scratch Test

[0117] Log-phase NCM460 cells and Caco-2 cells were selected and cultured at a ratio of 5 × 10⁻⁶ cells / year. 5 Cells were seeded at a density of [number] cells / well in 6-well plates and incubated for 12.0 h to allow for full cell adhesion. The culture medium was then aspirated, and the cells were washed twice with PBS. Grouping and treatment: Ctrl group: Use a 200μL Tip tip to make a scratch, observe and photograph under an inverted microscope; add 2.0mL of culture medium, incubate for 24.0h, discard the culture medium, observe and photograph under an inverted microscope; add another 2.0mL of culture medium, incubate for 24.0h, discard the culture medium, and observe and photograph under an inverted microscope again. PST group: Scratch marks were made with a 200μL tip and observed and photographed under an inverted microscope; 2.0mL of medium containing 300μg / mLPST was added and incubated for 24.0h, the medium was discarded, and the samples were observed and photographed under an inverted microscope; then 2.0mL of medium containing 300μg / mLPST was added again and incubated for 24.0h, the medium was discarded, and the samples were observed and photographed under an inverted microscope again.

[0118] Figure 12 (A) Photographs of the scratch assay results of NCM460 cells in each group; Figure 12 (B) is a graph showing the quantitative analysis results of the unhealed areas of NCM460 cell scratches in each group; Figure 12 (C) Photographs of the scratch test results of Caco-2 cells in each group; Figure 12 (D) is a graph showing the quantitative analysis results of the unhealed area of ​​Caco-2 cell scratches in each group; the scale bar is 20 μm, and the experimental data are expressed as mean ± standard deviation (n=3, ).from Figure 12 (A) and Figure 12 (C) shows that in the scratch assays of NCM460 and Caco-2 cells, compared with the Ctrl group, the PST group showed a significant tendency for cell healing at both 24.0 and 48.0 h, while the Ctrl group did not show any tendency for cell healing; indicating that PST significantly promoted the migration of NCM460 and Caco-2 cells. Figure 12 (B) and Figure 12 (D) indicates that the scratch assay results for NCM460 cells and Caco-2 cells were the same. After incubation for 24.0 h and 48.0 h, the area of ​​unhealed scratches in the PST group was significantly reduced compared to the Ctrl group, and the difference was statistically significant. This indicates that PST has a significant cell migration-promoting effect.

[0119] III. Cell Migration Experiment

[0120] Log-phase NCM460 cells and Caco-2 cells were selected and cultured at a ratio of 5 × 10⁻⁶ cells / year. 4 Cells were seeded at a density of 1 cell / well in the upper chamber of a Transwell chamber, with 200 μL of complete culture medium added to each well. The cells were incubated for 12.0 h to allow for full cell adhesion. The culture medium was then aspirated and replaced with 200 μL of basal culture medium. The cells were then divided into groups for further treatment. Ctrl group: Add 1.0 mL of complete culture medium containing 20% ​​FBS to the lower chamber. PST group: Add 1.0 mL of complete culture medium containing 20% ​​FBS and 300 μg / mL PST to the lower chamber. Incubate for 48.0 h, discard the culture medium, add crystal violet staining solution, and stain at room temperature for 10 min; wipe the cells inside the chamber with a cotton swab, and observe and photograph under an inverted microscope.

[0121] Figure 13 Images show the migration results of NCM460 and Caco-2 cells in each group; scale bar is 100 μm. As shown in the figures, compared with the Ctrl group, PST significantly promoted the migration of NCM460 and Caco-2 cells to the outer membrane of the cytoplasm, indicating that PST has a significant effect on promoting cell migration.

[0122] The above experiments all demonstrate that PST has a significant ability to promote cell proliferation and migration.

[0123] Experimental Example 5

[0124] The inhibitory effect of PST prepared in Example 1 on cell apoptosis was investigated.

[0125] NCM460 cells in logarithmic growth phase were selected and cultured at a concentration of 3 × 10⁻⁶ cells. 5 Cells were seeded at a density of [number] 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. Grouping and treatment: Ctrl group: blank control, with 2.0 mL of culture medium added. PST group: Add 2.0 mL of culture medium containing 300 μg / mL PST. DI group: Add 2.0 mL of culture medium containing 1% DSS and 20 ng / mL IL-1β. DI+PST group: 2.0 mL of culture medium containing 1% DSS, 20 ng / mL IL-1β, and 300 μg / mL PST was added. All groups were incubated for 24.0 h, the culture medium was discarded, and the cells were washed twice with PBS. Cells were digested with 1.0 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.

[0126] Figure 14 (A) is a flow cytometry analysis result of apoptosis in NCM460 cells in each group; Figure 14 (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 significant difference). As shown in the figure, compared to the Ctrl group, the Annexin V values ​​of the PST group and the Ctrl group were significantly different. + / PI + There was no significant difference in cell cluster proportions (ns), indicating that PST does not induce apoptosis. Annexin V in the DI group. + / PI + The cell cluster ratio was 29.9%, indicating that the combined effects 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+PST group showed significantly higher levels of Annexin V. + / PI + The difference in cell cluster proportions was statistically significant. Furthermore, the percentage of apoptosis decreased significantly from 29.9% in the DI group to 4.09% in the DI+PST group, demonstrating that PST has a very strong anti-apoptotic ability.

[0127] Experimental Example 6

[0128] The effects of PST prepared in Example 1 on regulating the cell cycle of inflammatory cells, promoting cell proliferation, and accelerating cell renewal were investigated.

[0129] NCM460 cells in logarithmic growth phase were selected and cultured at a concentration of 3 × 10⁻⁶ cells. 5 Cells were seeded at a density of [number] 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. Grouping and treatment: Ctrl group: blank control, with 2.0 mL of culture medium added. PST group: Add 2.0 mL of culture medium containing 300 μg / mL PST. DI group: Add 2.0 mL of culture medium containing 1% DSS and 20 ng / mL IL-1β. DI+PST group: 2.0 mL of culture medium containing 1% DSS, 20 ng / mL IL-1β, and 300 μg / mL PST 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 1.0 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 again in PBS, and propidium iodide (PI) was added for flow cytometry analysis.

[0130] Figure 15(A) is a flow cytometry analysis of NCM460 cells in the Ctrl and PST groups; Figure 15 (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 PST group and the Ctrl group, indicating that PST does not affect the cell cycle of normal cells. Figure 15 (C) shows the cell cycle flow cytometry analysis of NCM460 cells in the DI group and the DI+PST group; Figure 15 (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+PST group and the DI group, indicating that PST does not affect inflammatory cells in the G0-G1 phase; however, the difference in the proportion of cells in the S phase between the two groups was statistically significant. The proportion of cells in the S phase decreased from 31.29% in the DI group to 13.64% in the DI+PST group, indicating that PST can significantly downregulate the proportion of cells damaged 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 cells in the G2 / M phase increased from 5.80% in the DI group to 31.43% in the DI+PST group, indicating that PST significantly upregulated the proportion of inflammatory cells in the G2 / M phase. These results suggest that PST primarily regulates 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, it promotes cell division and proliferation, thereby accelerating cell renewal.

[0131] In summary, this demonstrates that PST can (1) remove DPPH, (1) It inhibits free radicals and has antioxidant effects; (2) It reduces the cytotoxicity of 5-HT in vivo and improves its biocompatibility; (3) It promotes cell proliferation and migration and repairs damaged intestines; (4) It inhibits cell apoptosis; (5) It regulates the cell cycle, accelerates cell renewal, and promotes intestinal barrier repair. In the end, PST achieved the goal of multi-level treatment of IBD through "antioxidation-anti-inflammation-mucosal repair", and is a multi-mechanism synergistic and efficient nanomedicine with therapeutic effects on IBD.

[0132] 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. A monoamine oxidized self-polymerizing nanoparticle, characterized in that, It is prepared by oxidative self-polymerization of 5-hydroxytryptamine (5-HT) in a water bath at pH 8.0~10.0 and 50℃~70℃.

2. A method for preparing monoamine oxidative self-polymerized nanoparticles, characterized in that, Includes the following steps: S1. Dissolve Tween-80 solution in purified water, and heat in a constant temperature water bath at 50℃~70℃, stirring at 800~1200rpm. S2. Add the 5-HT solution dropwise into the aqueous solution described in step S1, and stir in a constant temperature water bath at 50℃~70℃ at 800~1200 rpm. S3. Add the buffer solution with pH 8.0~10.0 dropwise into the mixture described in step S2, and stir at 800~1200 rpm in a constant temperature water bath at 50℃~70℃. S4. Centrifuge the reaction solution obtained in step S3, collect the precipitate and wash it with purified water to obtain monoamine oxidized self-polymerized nanoparticles.

3. The method for preparing monoamine oxidative self-polymerized nanoparticles according to claim 2, characterized in that, In step S1, the final concentration of Tween-80 in the reaction solution is 3.0~5.0 mg / mL.

4. The method for preparing monoamine oxidative self-polymerized nanoparticles according to claim 2, characterized in that, In step S2, the final concentration of 5-HT in the reaction solution is 1.0~3.0 mg / mL.

5. The method for preparing monoamine oxidative self-polymerized nanoparticles according to claim 2, characterized in that, The buffer solution used in step S3 is Tris buffer solution.

6. The method for preparing monoamine oxidative self-polymerized nanoparticles according to claim 5, characterized in that, The Tris buffer solution has a final molar concentration of 5-15 mM in the reaction solution.

7. The method for preparing monoamine oxidative self-polymerized nanoparticles according to claim 2, characterized in that, The stirring reaction time in step S3 is 4.0~8.0h.

8. An application of the monoamine oxidized self-polymerized nanoparticles according to claim 1, characterized in that, The monoamine oxidized self-polymerized nanoparticles are used in the preparation of drugs for treating inflammatory bowel disease, and are used as injectable, topical, or oral formulations, or simultaneously loaded with other therapeutic drugs.

9. The application of the monoamine oxidized self-polymerized nanoparticles according to claim 8, characterized in that, Used to prepare antioxidant drugs, drugs that promote cell proliferation and migration, drugs that inhibit apoptosis, or drugs that promote cell proliferation and accelerate cell renewal by regulating the cell cycle.

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