5-ASA-based anti-inflammatory / antioxidant carbon dot nanozymes, their preparation methods, and applications.

Abstract

This invention belongs to the field of antioxidant materials technology, specifically relating to a 5-ASA-based anti-inflammatory / antioxidant carbon dot nanozyme, its preparation method, and its application. Using 5-aminosalicylic acid and a primary amine compound as raw materials, a Schiff base reaction is carried out in a solvent system at room temperature to obtain the 5-ASA-based anti-inflammatory / antioxidant carbon dot nanozyme. This invention prepares a carbon dot nanozyme capable of effectively scavenging various free radicals. This nanozyme exhibits good therapeutic effects in a DSS-induced mouse model of acute colitis. The preparation method of this invention provides valuable reference for the mild preparation of carbon dot nanozymes with both therapeutic and high catalytic performance, offering a theoretical and technical basis for the application of drug-derived carbon dot nanozymes in anti-inflammatory therapy.

Description

Technical Field

[0001] This invention belongs to the field of antioxidant materials technology, specifically relating to an anti-inflammatory / antioxidant carbon dot nanozyme based on 5-ASA, its preparation method, and its application. Background Technology

[0002] The positive feedback loop of "oxidation-inflammation" makes the pathological process of colitis irreversible, ultimately leading to the continuous aggravation of ulcerative colitis (UC). This vicious cycle not only forms the pathophysiological basis of UC but also provides new treatment strategies for its prevention and treatment. Existing carbon dot nanozymes have limited functionality, possessing only ROS scavenging capabilities, making it difficult to effectively regulate the "oxidation-inflammation" pathological mechanism of UC. Therefore, designing carbon dot antioxidant nanozymes with anti-inflammatory functions has become a key direction in UC treatment research. Studies have shown that drug-derived carbon dots can inherit the efficacy of their precursors. Using clinical anti-inflammatory drugs for UC as precursors to prepare carbon dot nanozymes aims to retain the anti-inflammatory activity of the drugs while endowing them with ROS scavenging functions, achieving a dual therapeutic effect of "anti-inflammatory and antioxidant."

[0003] 5-ASA (5-amino-2-hydroxybenzoic acid) provides an ideal precursor for constructing multifunctional carbon dot antioxidant nanozymes for the treatment of ulcerative colitis (UC). The structure of a drug molecule directly affects its properties and functions. To retain anti-inflammatory activity, it is crucial that the structure of 5-ASA is not destroyed during the synthesis of carbon dot nanozymes. Choosing an appropriate preparation method is essential for preserving its structure. Since carbon dots were first reported in 2004, researchers have attempted various methods to synthesize them, with hydrothermal and carbonization methods widely used in the preparation of carbon dot nanozymes. However, high-temperature and high-pressure reaction conditions still have limitations in the preparation of carbon dot nanozymes: 1) They may lead to over-carbonization and severe dehydration of carbon dots, resulting in the loss of surface functional groups and weakening the enzyme activity; 2) They easily generate byproducts, making the purification process time-consuming and cumbersome, ultimately reducing the yield and purity of the final product; 3) They present significant challenges in terms of temperature control, equipment requirements, reaction monitoring, and energy consumption, hindering clinical application and translation; 4) They may damage the precursor molecular structure, preventing drug-derived carbon dot nanozymes from retaining the active drug structure. Therefore, it is of great significance to develop a simple, safe, efficient method for synthesizing carbon dot nanozymes that is suitable for room temperature conditions. Summary of the Invention

[0004] Targeting the pathogenesis of ulcerative colitis (UC) involving "oxidation and inflammation," and overcoming the complexity of existing carbon dot nanozyme synthesis methods, this invention provides an anti-inflammatory / antioxidant carbon dot nanozyme based on 5-ASA, its preparation method, and its applications, enabling the inheritance of the efficacy of prodrugs by drug-derived carbon dot nanozymes.

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

[0006] In a first aspect, the present invention provides a method for preparing an anti-inflammatory / antioxidant carbon dot nanozyme based on 5-ASA, comprising the following steps:

[0007] Using 5-aminosalicylic acid and primary amine compounds as raw materials, the 5-ASA antioxidant carbon dot nanozyme was obtained by reacting a Schiff base in a polar solvent at room temperature for a certain period of time.

[0008] 5-ASA, as an approved clinical drug, has a well-defined pharmacological basis and the following advantages: 1) Multifunctional active sites: The hydroxyl group can serve as a catalytic active site (such as SOD activity), while the amino group can enhance electron transfer ability through N doping, synergistically improving ROS scavenging efficiency; 2) Conjugated structure advantages: The benzene ring skeleton can promote the orderly arrangement of carbon nuclei through π-π stacking, forming a sp-rich structure. 2 The carbon dot structure features a hybrid carbon core and surface functional groups. This conjugated π-domain effectively promotes electron transfer during catalysis, significantly enhancing the catalytic activity of nanozymes. 3) Modified 5-ASA: 5-ASA-CDs are expected to overcome the shortcomings of 5-ASA, such as poor water solubility, easy acetylation, and lack of targeting to the intestine. Therefore, 5-ASA-derived carbon dot nanozymes are expected to overcome the inherent limitations of 5-ASA, achieving a synergistic "anti-inflammatory-antioxidant" effect, breaking through the limitations of traditional monotherapy, and showing greater clinical translational potential compared to carbon dot nanozymes from other sources.

[0009] Because the 5-ASA molecule contains a phenolic hydroxyl group, it can spontaneously oxidize under mild conditions to generate a quinone intermediate, which then undergoes a Schiff base reaction with a primary amine to form a structure with an imine (C=N) bond. Studies have shown that the Schiff base reaction strategy achieves room-temperature synthesis of carbon dots without introducing strong acids, strong oxidants, or strong bases. If this method can be used to synthesize 5-ASA-CDs nanozymes, it would satisfy both a safe and simple synthesis strategy and avoid the destruction of enzyme-active catalytic sites on the carbon dot surface by high temperatures. To achieve room-temperature synthesis of 5-ASA-CDs nanozymes, this invention introduces ethylenediamine (EDA) as a primary amine to react with 5-ASA in a Schiff base reaction. As a bifunctional primary amine, ethylenediamine exhibits high reactivity, excellent coordination ability, and structural designability when reacting with aldehydes to form Schiff bases, and it also shows good adaptability to solvents and temperatures, demonstrating irreplaceable advantages in Schiff base synthesis and the development of its functional materials.

[0010] In some specific embodiments, the primary amine compound is ethylenediamine.

[0011] In some specific embodiments, the solvent is ethanol or water.

[0012] In some specific embodiments, the mass-to-volume ratio of 5-aminosalicylic acid, ethylenediamine, and water is 140-160 mg: 20-40 μL: 8-12 mL.

[0013] In some specific embodiments, the reaction time is 0.5 hours to 6 hours.

[0014] In some specific embodiments, after the Schiff base reaction, the step further includes separating the 5-ASA antioxidant carbon dot nanozyme by dialysis and ultrafiltration.

[0015] In some specific embodiments, 5-ASA and ethylenediamine were mixed and stirred in a certain proportion for 2 h under normal temperature and pressure conditions. After purification, a 5-ASA antioxidant carbon dot nanozyme that can effectively scavenge a variety of free radicals was prepared. This nanozyme can effectively treat a DSS-induced mouse model of acute colitis.

[0016] Secondly, the present invention provides an anti-inflammatory / antioxidant carbon dot nanozyme based on 5-ASA, which is prepared by the preparation method described above.

[0017] Thirdly, the present invention provides the application of the 5-ASA-based anti-inflammatory / antioxidant carbon dot nanozyme in the preparation of drugs that scavenge free radicals.

[0018] Fourthly, the present invention provides the application of the 5-ASA-based anti-inflammatory / antioxidant carbon dot nanozyme in the preparation of a medicament for treating colitis.

[0019] Fifthly, the present invention provides a medicament for treating colitis, with the aforementioned 5-ASA-based anti-inflammatory / antioxidant carbon dot nanozyme as the active ingredient. The medicament is a water-soluble powder formulation, which, when used, is resuspended in water, administered by gavage, and applied to the intestines of mice.

[0020] The present invention has the following beneficial effects:

[0021] This invention, through the deep integration of materials science, enzymology and biomedical research methods, addresses the problem that simply clearing ROS or reducing inflammation is not enough to effectively treat UC. It achieves innovative breakthroughs in three aspects: material design, synthesis methods and treatment strategies, and promotes the transformation of drug-derived carbon dot antioxidant nanozymes from basic research to clinical application.

[0022] Innovative Material Design: This invention designs a carbon dot antioxidant nanozyme synthesized using 5-ASA as a precursor. This not only fully utilizes the structural characteristics of 5-ASA, resulting in a synthesized carbon dot nanozyme with strong antioxidant capabilities, but also retains the anti-inflammatory properties of 5-ASA itself. Furthermore, the structure of the carbon dot nanozyme overcomes the shortcomings of 5-ASA itself, such as poor water solubility, easy acetylation, and lack of targeting to the intestines—achieving three benefits in one. In summary, 5-ASA-CDs hold promise for overcoming the limitations of traditional monotherapy and have greater clinical translational potential compared to carbon dot nanozymes from other sources.

[0023] Innovative Synthesis Method: High-temperature, high-pressure synthesis conditions or room-temperature synthesis methods using strong acids and bases have limited applications in drug-derived carbon dot nanozymes due to their high energy consumption, demanding experimental requirements, cumbersome purification processes, potential functional group loss, weakened enzyme activity, and poor preservation of precursor structures. This invention utilizes the Schiff base reaction to achieve room-temperature synthesis of carbon dot nanozymes through simple stirring without introducing strong acids, strong oxidants, or strong bases. This nanozyme synthesis method is simple, low-cost, and conducive to large-scale production, providing technical support for the transition of drug-derived carbon dot nanozymes from research to clinical application.

[0024] Innovative Treatment Strategy: High levels of ROS and inflammatory events create multiple positive feedback regulatory signaling loops in the development of colitis, making the pathological process of ulcerative colitis (UC) difficult to reverse, ultimately leading to the continuous progression and aggravation of intestinal inflammation. This invention develops a carbon dot nanozyme with ROS scavenging capabilities using the anti-inflammatory drug 5-ASA, synergistically using inflammation suppression and ROS scavenging, which is expected to break the vicious cycle of "oxidation-inflammation." This "one drug, two effects" development strategy provides new ideas and directions for the treatment of UC and the development of carbon dot nanozymes.

[0025] The implementation of this invention not only provides an innovative treatment tool for colitis, but will also achieve breakthroughs in nanozyme catalysis theory, antioxidant therapy strategies and their clinical translation models, providing a replicable model for the development of drug-derived carbon dot nanozymes, and has important scientific significance and clinical application value. Attached Figure Description

[0026] Figure 1 Characterization of 5-ASA-CDs. A: Transmission electron microscopy (TEM) image of 5-ASA-CDs, with a high-resolution TEM image in the upper right corner. B: Particle size statistics of 5-ASA-CDs. C: Zeta potential results of 5-ASA-CDs. D: XRD results of 5-ASA-CDs. E: Raman spectrum of 5-ASA-CDs. F: Atomic force microscopy (AFM) results. G: Fluorescence emission and excitation spectra of 5-ASA-CDs. H: Quantum yield of 5-ASA-CDs. I: SOD enzyme activity assay results of 5-ASA-CDs.

[0027] Figure 2 Evaluation of the free radical scavenging ability of 5-ASA-CDs. A: Schematic diagram of the NBT method for detecting •O2. B: Calculation of the •O2 free radical scavenging rate (n=3) based on the absorption peak at 560 nm after 10 minutes of reaction between different concentrations of 5-ASA-CDs and NBT working solution. C: Determination of the •O2 free radical scavenging ability of different concentrations of 5-ASA-CDs using electron resonance spin method. D: Schematic diagram of the TMB decolorization method for detecting •OH. E: Calculation of the •OH scavenging rate (n=3) based on the absorption peak at 652 nm after 10 minutes of reaction between different concentrations of 5-ASA-CDs and TMB working solution. F: Determination of the •OH free radical scavenging ability of different concentrations of 5-ASA-CDs using electron resonance spin method. G: Schematic diagram of the DPPH free radical scavenging principle. H: Calculation of the DPPH free radical scavenging rate (n=3) based on the absorption peak at 515 nm after 10 minutes of reaction with DPPH working solution under different concentrations of 5-ASA-CDs. I: Electron resonance spin method for determining the DPPH• radical scavenging capacity of different concentrations of 5-ASA-CDs. J: ABTS + • Schematic diagram of the principle of ABTS for detecting free radical scavenging. K: ABTS under different concentrations of 5-ASA-CDs. + • ABTS was calculated from the absorption peak of the working solution at 734 nm after 10 minutes of reaction. + • Free radical scavenging rate (n = 3). L: ABTS determination of different concentrations of 5-ASA-CDs by electron resonance spin method. + • Free radical scavenging ability. M: Schematic diagram of the principle of PTIO• method for detecting free radical scavenging. N: PTIO• free radical scavenging rate calculated from the absorption peak at 557 nm after 10 minutes of reaction of PTIO• working solution under different concentrations of 5-ASA-CDs (n = 3). O: PTIO• free radical scavenging ability of different concentrations of 5-ASA-CDs determined by electron resonance spin method.

[0028] Figure 3 Evaluation of the efficacy of 5-ASA-CDs in treating an acute colitis model. A: Flowchart of the establishment and treatment of the acute colitis prevention model; B: Image of the mouse anus before the end of treatment; C: Representative endoscopic photograph of the mouse colon before the end of treatment; D: Photograph of a representative colon removed from the colitis mouse model after treatment; E: H&E staining image of the mouse colon after treatment; F: Statistical graph of mouse body weight during treatment (n = 5); G: Statistical graph of colon length in different groups at the end of treatment (n = 5); H: Statistical graph of disease activity index in mice during treatment; *, p<0.1; **, p<0.01; ***, p<0.001. Detailed Implementation

[0029] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0030] The CAS number of 5-aminosalicylic acid used in the following examples is [CAS number missing].

[0031] Example 1: Synthesis and characterization of 5-ASA antioxidant carbon dot nanozymes (5-ASA-CDs).

[0032] 1. Experimental Methods

[0033] ① Weigh 150 mg of 5-aminosalicylic acid and add it to 10 mL of ultrapure water. Stir at 500 rpm on a magnetic stirrer until completely dissolved, forming a homogeneous and clear solution. Add 30 μL of ethylenediamine; the solution immediately turns pale yellow. Place the mixture in a 25 mL round-bottom flask and stir magnetically at 800 rpm for 2 hours at room temperature (25 ± 2℃). The solution gradually turns brownish-yellow. Transfer the reaction solution to a 500 Da dialysis bag and dialyze it in 2 L of ultrapure water for 24 hours, changing the water every 4 hours. Alternatively, use a 3 kDa ultrafiltration tube and a 4000× [unclear - possibly a specific pressure or pressure]. g ① Centrifuge for 20 minutes; ② Observe the morphology and size of carbon dots using transmission electron microscopy and atomic force electron microscopy; ③ Characterize the particle size and zeta potential of carbon dots using dynamic light scattering and other methods; ④ Measure the ultraviolet absorption spectrum and fluorescence emission spectrum of carbon dots using ultraviolet absorption spectrometer and fluorescence spectrometer; ⑤ Analyze the surface functional groups and surface defects of carbon dots using Fourier transform infrared spectroscopy, nuclear magnetic resonance hydrogen spectroscopy and Raman spectroscopy.

[0034] 2. Experimental Results

[0035] In this embodiment, 5-ASA-CDs prepared using 5-aminosalicylic acid and ethylenediamine as precursors had an average particle size of 2.62 ± 0.47 nm and a Zeta potential of −20.07 ± 2.06 mV. TEM images showed that the carbon dots were uniformly distributed and monodisperse. High-resolution TEM showed that the lattice fringes of the carbon dots corresponded to the graphite (100) plane at 0.21 nm, which, together with the (002) peak of XRD, proved the presence of graphite cores in the 5-ASA-CDs. Figure 1 AD). Raman spectroscopy results also confirmed that the carbon dot had a high degree of graphitization, few defects, and good crystallinity. Figure 1 E). AFM images show that the nanoparticles are spherical and uniformly dispersed on the silicon wafer surface, with an average height of 3.1 ± 0.2 nm and a corrected lateral diameter of 3.5 ± 0.3 nm. Figure 1 F). The synthesized carbon dots exhibit fluorescence properties (Ex = 384 nm, Em = 492 nm) with a fluorescence quantum yield of 16% (F). Figure 1 G and H).

[0036] Example 2: Determination of the free radical scavenging ability of 5-ASA-CDs

[0037] 1. Experimental Methods

[0038] ① The SOD enzyme activity of carbon dots was determined using methods such as the SOD kit method, NBT method, superoxide anion scavenger DMPO, and electron resonance spintronics, and the corresponding enzyme kinetic constants were calculated; ② The ability of carbon dots to scavenge hydroxyl radicals was determined using hydroxyl radical scavenger DMPO and electron resonance spintronics; ③ The SOD enzyme activity of carbon dots was determined using DPPH• and ABTS methods. + • Evaluate the scavenging ability of carbon dots against free radicals and their overall antioxidant capacity in a free radical system.

[0039] 2. Experimental Results

[0040] This embodiment tested the free radical scavenging and antioxidant capacity of 5-ASA-CDs using multiple methods. The SOD enzyme activity of 5-ASA-CDs was detected by NBT and ESR methods. The experimental results showed that 5-ASA-CDs possess good SOD enzyme activity (…). Figure 1 I), and the SOD activity of this carbon dot is concentration-dependent ( Figure 2 AC. The TMB and ESR methods were used to detect the •OH radical scavenging ability of 5-ASA-CDs. The experimental results showed that the carbon dots exhibited excellent •OH scavenging ability in a concentration-dependent manner. Figure 2 DF). DPPH• radical scavenging experiments showed that the DPPH• radical scavenging rate continuously decreased with increasing 5-ASA-CDs concentration, indicating that this carbon point possesses excellent nitrogen radical scavenging ability. Figure 2 GI). As the concentration of 5-ASA-CDs increases, ABTS + The scavenging rate of PTIO• free radicals showed a continuous decreasing trend, indicating that this carbon dot has excellent antioxidant capacity. Figure 2 (JL). In summary, 5-ASA-CDs possess multiple free radical scavenging capabilities.

[0041] Example 3: Evaluation of the in vivo therapeutic effect of 5-ASA-CDs

[0042] 1. Experimental Methods

[0043] ① A mouse model of acute colitis induced by DSS was established. Oral administration was administered to the following groups: (I) Control group: normal drinking water + saline by gavage; (II) DSS group: 3% DSS-induced colitis in drinking water + saline by gavage; (III) 5-ASA-CDs group: normal drinking water + 5 mg / kg 5-ASA-CDs by gavage; (IV) 5-ASA + DSS group: 3% DSS in drinking water + 5 mg / kg 5-ASA by gavage; (V) 5-ASA-CDs + DSS group: 3% DSS in drinking water + 5 mg / kg 5-ASA-CDs by gavage. Mice were divided into 5 groups, with 6 mice in each group. The experimental period was 8 days. Mice in the DSS group and related treatment groups received 3% DSS solution in drinking water throughout the experiment (days 1-8) to induce acute colitis. Drug intervention groups (5-ASA, 5-ASA-CDs) were administered by gavage on days 1, 3, 5, and 7 at a dose of 5 mg / kg. The control group received normal drinking water and was administered physiological saline by gavage at the same time points. ② The body weight and fecal condition of mice in each group were observed and recorded daily; the disease activity index (DAI) of each group was calculated. ③ After drug administration, ulceration and inflammation in the colon of the mice were examined using a colonic endoscope. ④ Colonic tissue was collected from each group and its length was compared. ⑤ H&E staining was used to observe histopathological changes in the colonic tissue. In summary, the preventive effect of carbon nanozymes on colitis was evaluated at the animal level.

[0044] 2. Experimental Results

[0045] Based on the excellent free radical scavenging and antioxidant effects of 5-ASA-CDs, we established a DSS-induced mouse model of acute colitis and evaluated the preventive effect of 5-ASA-CDs. Healthy mice were randomly divided into 5 groups: Control group, DSS model group, 5-ASA-CDs group, DSS + 5-ASA group, and DSS + 5-ASA-CDs group. Mice in the Control group were fed normally, while mice in the DSS group were given drinking water containing 3% DSS continuously, and the drug was administered every other day. Mice were sacrificed on day 8. Figure 3 A). On day 5 of DSS induction, the mice began to lose weight and gradually developed bloody stools, indicating that the UC model was successfully established. Figure 3 B and Figure 3 F). Endoscopic results showed that the colonic vascular texture was clear in normal mice, and no abnormalities were observed in the colon; the colonic mucosal vascular texture disappeared in mice in the DSS model group, and ulcer lesions were visible in the colonic tissue; the colonic mucosal vascular texture was restored and the ulcers disappeared in mice treated with 5-ASA-CDs. Figure 3C). Regarding colon length, the average colon length in the healthy group of mice was 7.77 ± 0.47 cm, while the average colon length in the DSS group was 4.96 ± 0.29 cm. There was a statistically significant difference between the two groups (p<0.001). The colon length in the 5-ASA-CDs administration group was significantly increased. Figure 3 D and Figure 3 G). H&E staining of collected mouse colon tissue revealed epithelial damage, disappearance of crypts, and a reduction or even disappearance of goblet cells compared to the control group. Significant inflammatory cell infiltration was also observed. The 5-ASA treatment group showed some improvement, but significant inflammatory cell infiltration remained. In contrast, the 5-ASA-CDs group showed restored colonic epithelial structure and no inflammatory cell infiltration, indicating that 5-ASA-CDs can improve colonic inflammation. Figure 3 E). The results of the disease activity index further confirmed the therapeutic effect of 5-ASA-CDs on colitis ( Figure 3 In summary, 5-ASA carbon nanoparticles can effectively improve colitis in the DSS-induced UC model.

[0046] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0047] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

Claims

1. A method for the preparation of 5-ASA based anti-inflammatory / anti-oxidative carbon-dot nanoszyme, characterized by, The method comprises the following steps: The Schiff base reaction is carried out at room temperature in a solvent system using 5-aminosalicylic acid and a primary amine compound as raw materials to obtain the 5-ASA antioxidant carbon dot nanoscale enzyme.

2. The method for preparing an anti-inflammatory / antioxidant carbon dot nanozyme based on 5-ASA according to claim 1, characterized in that, The primary amine compound is ethylenediamine.

3. The method for preparing an anti-inflammatory / antioxidant carbon dot nanozyme based on 5-ASA according to claim 2, characterized in that, The solvent is a polar solvent.

4. The method for preparing an anti-inflammatory / antioxidant carbon dot nanozyme based on 5-ASA according to claim 3, characterized in that, The mass-volume ratio of the 5-aminosalicylic acid, ethylenediamine and water is 140-160 mg: 20-40 μL: 8-12 mL.

5. The method for preparing an anti-inflammatory / antioxidant carbon dot nanozyme based on 5-ASA according to claim 4, characterized in that, The reaction time is 0.5-6 hours.

6. The method of claim 5, wherein the 5-ASA based anti-inflammatory / anti-oxidative carbon dot nanoszyme is prepared by the method comprising the steps of: After the Schiff base reaction, the method further comprises the step of separating the 5-ASA antioxidant carbon dot nanoscale enzyme by dialysis or ultrafiltration.

7. An anti-inflammatory / anti-oxidative carbon-dot nanoszyme based on 5-ASA, characterized in that, The method is prepared by any one of claims 1-6.

8. Use of the 5-ASA-based anti-inflammatory / antioxidant carbon dot nanoscale enzyme of claim 7 in the preparation of a free radical scavenging drug.

9. Use of the 5-ASA-based anti-inflammatory / antioxidant carbon dot nanoscale enzyme of claim 7 in the preparation of a drug for treating colitis.

10. A medicament for treating colitis, characterized by, The 5-ASA-based anti-inflammatory / antioxidant carbon dot nanoscale enzyme of claim 7 is used as an effective ingredient.

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