Glycyrrhiza flavone carbon nanodots, preparation method and application thereof

Abstract

The application belongs to the technical field of medicine preparation, and particularly relates to a glycyrrhiza flavone carbon nanodot, a preparation method and application thereof. The glycyrrhiza flavone is dispersed in ultrapure water, stirred uniformly to form a glycyrrhiza flavone suspension; the glycyrrhiza flavone suspension is adjusted to a specified pH value by hydrochloric acid, and is reacted at a specified temperature for a specified time, and after the reaction is completed, the solution is naturally cooled to room temperature to obtain a brown-yellow solution; the brown-yellow solution is filtered, centrifuged, and the supernatant is dialyzed and then freeze-dried to obtain a solid powder, namely the glycyrrhiza flavone carbon nanodot. The GlyF-CDs are prepared by a solvothermal method, and have good repeatability and stability. The prepared GlyF-CDs have good water dispersibility and biocompatibility, and are highly stable within 7 days of storage at 4 DEG C in the dark within a physiological pH range (5-9), a physiological ionic strength (0-1.0 M NaCl).

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, specifically relating to a licorice flavonoid carbon nanodot, its preparation method, and its application. Background Technology

[0002] Colorectal cancer (CRC) is one of the most prevalent and deadly malignant tumors of the digestive system worldwide, and in recent years it has gradually become a significant public health issue affecting human health. According to relevant epidemiological statistics, in 2022, there were over 1.9 million new cases of colorectal cancer globally, with nearly 1 million deaths. In my country, the incidence of this cancer is second only to gastric cancer among digestive system malignancies, and it continues to rise. Currently, surgical resection remains the primary treatment method, often combined with chemotherapy, radiotherapy, and targeted therapy. However, commonly used chemotherapy drugs such as oxaliplatin and fluorouracil still suffer from low selectivity and significant toxic side effects in practical applications. Long-term use may also induce multidrug resistance in tumor cells, affecting treatment efficacy and reducing patients' quality of life. Therefore, exploring novel anti-colorectal cancer drugs with lower toxicity and more clearly defined targets has become one of the important directions in current oncology research.

[0003] Natural medicines provide abundant resources for the development of anti-tumor drugs. Among them, the active ingredients of traditional Chinese medicine have attracted increasing attention in tumor intervention research due to their multi-target and multi-pathway regulatory characteristics. Licorice, as one of the commonly used traditional Chinese medicinal herbs, has a long history of clinical application. Its main active ingredient, glycyrrhiza flavonoids (GlyF), includes various compounds such as glycyrrhizin, isoglycyrrhizin, and glycyrrhizin chalcone. Studies have shown that these components possess various biological activities, including anti-inflammatory, antioxidant, and tumor cell proliferation inhibition. Research results suggest that glycyrrhiza flavonoids may inhibit the proliferation of colon cancer cells by regulating signaling pathways such as PI3K / Akt and MAPK, and to some extent affect tumor angiogenesis and metastasis. However, due to the overall strong hydrophobicity of glycyrrhiza flavonoids, their water solubility and in vivo bioavailability are relatively low, which limits their further application to some extent.

[0004] With the development of nanotechnology, modifying natural active ingredients using nanomaterials has become an important research direction for improving the physicochemical properties of drugs. Carbon nanodots (CDs) are a new type of carbon-based nanomaterial. Since their discovery in 2004, they have attracted widespread attention in the biomedical field due to their typically smaller particle size (less than 10 nm), good water solubility, and excellent biocompatibility. Furthermore, carbon nanodots also possess good fluorescence properties and surface modifiability, thus showing potential applications in bioimaging, drug delivery, and disease diagnosis. In recent years, the synthesis of traditional Chinese medicine-based carbon nanodots using natural drug components as carbon sources has gradually become a research hotspot. These materials, to a certain extent, simultaneously retain the biological effects of the active ingredients in traditional Chinese medicine and the physicochemical properties of nanomaterials. However, how to provide a reproducible and stable preparation process to modify glycyrrhizin flavonoids using carbon nanomaterials, so that the synthesized glycyrrhizin-based carbon nanodots possess excellent biocompatibility and stability, remains a challenge. Summary of the Invention

[0005] To address the aforementioned issues, this application provides licorice flavonoid carbon nanodots, their preparation method, and their applications.

[0006] The technical solution to the above-mentioned technical problems in this application is as follows: A method for preparing licorice flavonoid carbon nanodots, comprising the following steps: S10. Disperse glycyrrhizin in ultrapure water and stir until homogeneous to form a glycyrrhizin suspension; S20. Adjust the pH of the licorice flavonoid suspension to a specified value with hydrochloric acid, react at a specified temperature for a specified time, and after the reaction is completed, allow it to cool naturally to room temperature to obtain a brownish-yellow solution; S30. The brownish-yellow solution is filtered, centrifuged, and the supernatant is dialyzed and freeze-dried to obtain a solid powder, namely licorice flavonoid carbon nanodots.

[0007] Preferably, in the above-mentioned method for preparing licorice flavonoid carbon nanodots, the purity of the licorice flavonoid is 80%-99%.

[0008] Preferably, in the above-mentioned method for preparing licorice flavonoid carbon nanodots, the concentration of the licorice flavonoid suspension is 0.04-0.12 g / mL.

[0009] Preferably, in the above-mentioned method for preparing licorice flavonoid carbon nanodots, the pH value is 5-9.

[0010] Preferably, in the above-mentioned method for preparing licorice flavonoid carbon nanodots, the specified temperature is 160-180 ℃.

[0011] Preferably, in the above method for preparing licorice flavonoid carbon nanodots, the specified time is 4-8 h.

[0012] A type of licorice flavonoid carbon nanodot is prepared by the method described above.

[0013] The above-mentioned application of licorice flavonoid carbon nanodots in the preparation of drugs for treating colon cancer.

[0014] Compared with the prior art, this application has at least the following advantages: This application discloses a method for preparing glycyrrhizin flavonoid carbon nanodots, comprising: S10. dispersing glycyrrhizin flavonoids in ultrapure water and stirring evenly to form a glycyrrhizin flavonoid suspension; S20. adjusting the pH of the glycyrrhizin flavonoid suspension to a specified pH value with hydrochloric acid, reacting at a specified temperature for a specified time, and naturally cooling to room temperature after the reaction to obtain a brownish-yellow solution; S30. filtering and centrifuging the brownish-yellow solution, dialyzing the supernatant, and freeze-drying to obtain a solid powder, namely glycyrrhizin flavonoid carbon nanodots. This application uses a solvothermal method to prepare GlyF-CDs, which has the advantages of relatively mild reaction conditions, simple operation, and good product dispersibility. In the repeatability test, the RSD of fluorescence intensity, quantum yield, and particle size of three batches of GlyF-CDs were 1.6%, 0.8%, and 3.1%, respectively, all <5%, indicating that the preparation process has good repeatability and stability and can be used for the laboratory-scale preparation of GlyF-CDs.

[0015] This application also discloses a glycyrrhizin flavonoid carbon nanodot (GlyF-CDs) with stable fluorescence properties and good water dispersibility and biocompatibility. Stability studies show that GlyF-CDs maintain highly stable fluorescence properties within the physiological pH range (5-9), physiological ionic strength (0-1.0 M NaCl), and are stored at 4°C in the dark for 7 days. This demonstrates that GlyF-CDs can adapt to the complex acid-base and ionic environments in vivo, avoiding fluorescence quenching or structural damage during in vivo transport.

[0016] This application also discloses the application of licorice flavonoid carbon nanodots in the preparation of anti-colon cancer drugs. Experimental results show that the growth of tumor volume in nude mice was significantly inhibited after treatment with GlyF-CDs. The GlyF-CDs group showed extensive tumor cell necrosis, accompanied by a small amount of inflammatory cell infiltration and fibrous tissue hyperplasia, which directly demonstrates its killing effect on tumor tissue. Moreover, it has good biosafety within a certain dosage range, making it a candidate drug for the preparation of anti-colon cancer drugs. Attached Figure Description

[0017] Figure 1 Fluorescence emission spectra (excitation wavelength 360 nm) of three batches of GlyF-CDs (n=3): Batch 1 (black), Batch 2 (red), and Batch 3 (blue).

[0018] Figure 2 High-resolution transmission electron microscopy (HRTEM) images of three batches of GlyF-CDs, n=3; Batch 1 (a); Batch 2 (b); Batch 3 (c).

[0019] Figure 3 High-resolution transmission electron microscope image (a); particle size distribution histogram and Gaussian fitting curve (b), n=3.

[0020] Figure 4 The images show the excitation and emission fluorescence spectra of GlyF-CDs.

[0021] Figure 5 The image shows the UV-Vis absorption spectrum of GlyF-CDs.

[0022] Figure 6 Fourier transform infrared spectra of licorice flavonoid precursor (a) and GlyF-CDs (b).

[0023] Figure 7 The X-ray photoelectron spectra of GlyF-CDs are shown in C1s (a), O1s (b), and N1s (c).

[0024] Figure 8 This is the fluorescence decay curve.

[0025] Figure 9 The fluorescence emission spectra and fluorescence intensity stability curves of GlyF-CDs stored in different pH buffer solutions for 7 days are shown (I / I0 is the ratio of relative initial intensity).

[0026] Figure 10 The fluorescence emission spectra and relative fluorescence intensity curves of GlyF-CDs in NaCl solutions of different concentrations (0, 0.1, 0.3, 0.5, 0.7, 0.9, 1.0 M) are shown.

[0027] Figure 11 The fluorescence emission spectrum and fluorescence intensity time stability curve of GlyF-CDs aqueous solution stored in the dark for 7 days (I / I0 is the ratio of the initial intensity on day 1).

[0028] Figure 12 The curves showing the changes in body weight of mice in each group are shown.

[0029] Figure 13 H&E staining images of major organs of mice in the Control group and GlyF-CDs group; (×200).

[0030] Figure 14 The curves showing the changes in subcutaneous tumor volume in each group of mice are shown.

[0031] Figure 15 The gross morphology of subcutaneous xenografts in mice of each group is shown.

[0032] Figure 16 A statistical graph showing the weight of subcutaneous tumors in each group of mice. Figure 17 The tumor inhibition rate of mice in each group is shown below. Note: Compared with the Oxa group, *p<0.05; **p<0.01; ***p<0.001; n=6.

[0033] Figure 18 H&E staining was performed on the xenograft tissues of mice in each group (×100 μ).

[0034] Figure 19 GPX4 immunofluorescence staining was performed on tumor tissues from each group of mice (×400); blue represents DAPI nuclear staining, and red represents the fluorescent signal of the target protein.

[0035] Figure 20 Western blot analysis of ferroptosis-related protein expression in tumor tissues of mice in each group; Note: Compared with the Mod group, *p<0.05, **p<0.01, ***p<0.001, n=3.

[0036] Figure 21 Expression of ferroptosis-related genes mRNA in tumor tissues of mice in each group (qRT-PCR); Note: compared with the Mod group Comparison, *p <0.05, **p <0.01, ***p <0.001, n=3. Detailed Implementation

[0037] It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of this invention can be combined with each other. The technical solutions of this invention will be further described below with reference to the embodiments and accompanying drawings. This invention is not limited to the specific embodiments described below.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0039] In one specific embodiment of this application, a method for preparing licorice flavonoid carbon nanodots includes the following steps: S10. Disperse glycyrrhizin in ultrapure water and stir until homogeneous to form a glycyrrhizin suspension; preferably, the purity of the glycyrrhizin is 80%-99%.

[0040] Glycyrrhiza flavonoids are derived from licorice. To improve resource utilization, this application uses licorice residue after glycyrrhizic acid extraction as raw material and employs an ethanol reflux extraction method to extract licorice flavonoids. Specifically, a certain amount of licorice residue powder is weighed, and a certain volume fraction of ethanol solution is added. The mixture is refluxed at a set temperature for a certain time. After extraction, the mixture is filtered while hot, and the filtrate is collected. The filter residue is then extracted a second time with the same volume fraction of ethanol solution. The two filtrates are combined and concentrated under reduced pressure using a rotary evaporator (55℃, -0.08 MPa) until no alcohol odor remains. The mixture is then vacuum dried to obtain crude licorice flavonoid extract. The optimal extraction conditions for licorice flavonoids are: 60% ethanol, a material-to-liquid ratio of 1:15, 70℃, and 1.5 h. Under these conditions, after three parallel experiments, the average purity of licorice flavonoids was calculated to be 82.61% ± 0.92% (RSD = 1.11%, n = 3), which meets the raw material requirements for the subsequent preparation of licorice flavonoid carbon nanodots.

[0041] Preferably, the concentration of the licorice flavonoid suspension is 0.04-0.12 g / mL; S20. Adjust the pH of the licorice flavonoid suspension to a specified value with hydrochloric acid, react at a specified temperature for a specified time, and after the reaction is completed, allow it to cool naturally to room temperature to obtain a brownish-yellow solution; Furthermore, the pH value is 5-9.

[0042] Furthermore, the specified temperature is 160-180 ℃.

[0043] Furthermore, the specified time is 4-8 hours.

[0044] The specific steps are as follows: Glycyrrhiza flavonoids are weighed and dispersed in ultrapure water. The mixture is stirred at room temperature for 10 min to form a 0.04-0.12 g / mL glycorrhiza flavonoid suspension. The pH of the system is adjusted to 5-9 with 0.1 M HCl solution. The suspension is then transferred to a polytetrafluoroethylene-lined stainless steel reactor and placed in a constant temperature oven at 160-180 ℃ for 4-8 h. After the reaction is complete, the mixture is allowed to cool naturally to room temperature to obtain a brownish-yellow solution.

[0045] S30. The brownish-yellow solution is filtered, centrifuged, and the supernatant is dialyzed and freeze-dried to obtain a solid powder, namely licorice flavonoid carbon nanodots.

[0046] The above steps are as follows: the obtained brownish-yellow solution is filtered through a 0.22μm microporous membrane to remove large particulate impurities, centrifuged at 8000 rpm for 10 min for further purification, the supernatant is dialyzed through a 1000 Da dialysis bag for 24 h (with ultrapure water replaced every 8 h), and after freeze-drying, licorice flavonoid carbon nanodots GlyF-CDs solid powder is obtained and stored at 4℃ in the dark for later use.

[0047] This application selects reaction temperature (A), reaction time (B), glycyrrhizin concentration (C), and system pH (D) as the factors to be investigated in the preparation of GlyF-CDs, with three levels for each. Quantum yield is the core evaluation index, while fluorescence intensity and particle size are auxiliary evaluation indexes (the higher the quantum yield, the greater the fluorescence intensity, and the smaller the particle size, the better the overall evaluation). An L9(3) model is designed. 4 Orthogonal experiments were conducted to determine the influence of various factors on the preparation of GlyF-CDs and the optimal process combination. Experiment 5 (A2B2C3D1, 160℃, 6 h, 0.12 g / mL, pH=5) yielded the highest fluorescence intensity (4450 au) and quantum yield (13.2%) among all experimental groups, and the smallest particle size (3.2 nm), making it the best overall. Combined with range analysis, the optimal preparation process for GlyF-CDs is: reaction temperature 160℃, reaction time 6 h, glycyrrhizin (suspension) concentration 0.12 g / mL, and system pH=5.

[0048] In another specific embodiment of this application, a licorice flavonoid carbon nanodot is prepared by the licorice flavonoid carbon nanodot preparation method described above.

[0049] In another specific embodiment of this application, the application of the above-described licorice flavonoid carbon nanodots in the preparation of drugs for treating colon cancer is described.

[0050] It is worth noting that the process temperature and process time involved in the above embodiments are all temperatures or times used in the experiment. Any reasonable adjustments made by those skilled in the art based on the process temperature and process time provided by the present invention, within the error range, should be included within the protection scope of the present invention.

[0051] The technical solution and effects of the present invention will be further illustrated below through specific embodiments.

[0052] 1. Experimental Materials and Methods 1.1 Experimental reagents (Table 1)

[0053] 1.2 Experimental Apparatus (Table 2)

[0054] 2 Experimental Methods 2.1 Extraction of licorice flavonoids Licorice slices were extracted according to the glycyrrhizic acid extraction method in the 2020 edition of the Chinese Pharmacopoeia. After reflux with ammonia water, the residue was dried at 60℃ and pulverized for later use. A certain amount of licorice residue powder was weighed and added to a 60% (v / v) ethanol solution at a material-to-liquid ratio of 1:15. The mixture was refluxed at 70℃ for 1.5 h. After extraction, the residue was filtered while hot, and the filtrate was collected. The residue was then extracted a second time with the same (v / v) ethanol solution. The two filtrates were combined and concentrated under reduced pressure using a rotary evaporator (55℃, -0.08 MPa) until no alcohol odor remained. After vacuum drying, the licorice flavonoid extract was obtained. After three parallel experiments, the average purity of licorice flavonoids was calculated to be 82.61% ± 0.92% (RSD = 1.11%, n = 3), which meets the raw material requirements for the subsequent preparation of licorice flavonoid carbon nanodots.

[0055] 2.2 GlyF-CDs Sample Preparation According to the factor level combinations in Table 3, the corresponding mass of glycyrrhizin was accurately weighed and dispersed in 10 mL of ultrapure water. The mixture was stirred at room temperature for 10 min to form a homogeneous suspension. The pH of the suspension was adjusted to the specified level using 0.1 M HCl solution, and then transferred to a 25 mL PTFE-lined stainless steel reactor. The reactor was placed in a constant temperature oven and reacted at the specified temperature for the specified time. After the reaction, the mixture was allowed to cool naturally to room temperature. The resulting brownish-yellow solution was filtered through a 0.22 μm microporous membrane to remove large particulate impurities. Further purification was achieved by centrifugation at 8000 rpm for 10 min. The supernatant was dialyzed through a 1000 Da dialysis bag for 24 h (with ultrapure water replaced every 8 h). After freeze-drying, GlyF-CDs solid powder was obtained and stored at 4 °C in the dark for later use. Fluorescence intensity, quantum yield, and particle size were subsequently measured.

[0056] 2.3 Optimization of GlyF-CDs Preparation Process 2.3.1 Orthogonal Experiment Factor Level Design Reaction temperature (A), reaction time (B), glycyrrhizin concentration (C), and system pH (D) were selected as the factors to be investigated in the preparation of GlyF-CDs, with three levels set for each. Fluorescence intensity, quantum yield, and particle size were used as comprehensive evaluation indicators to design L9(3 4 Orthogonal experiments were conducted to determine the influence of each factor on the preparation of GlyF-CDs and the optimal process combination. The factor levels are shown in Table 3.

[0057] Table 3. Factor Level Table for Orthogonal Experiment

[0058] 2.3.2 Orthogonal Experiment 2.3.3 Optimal process repeatability verification The optimal preparation process for GlyF-CDs was determined based on the results of orthogonal experimental range analysis. Three independent parallel experiments were conducted under these conditions, with strict control over the consistency of operations throughout the entire process, including raw material weighing, reaction conditions, purification and separation, and freeze-drying. The fluorescence intensity, quantum yield, and particle size of the three batches of GlyF-CDs were measured, and the average value and relative standard deviation (RSD) of each index were calculated to examine the repeatability and stability of the process. Simultaneously, fluorescence spectrophotometry and high-resolution transmission electron microscopy were used to characterize the fluorescence spectra and morphology / particle size of the three batches of samples, verifying the batch consistency of the products.

[0059] 2.4 Performance Measurement of GlyF-CDs 2.4.1 Determination of fluorescence intensity and quantum yield (QY) The fluorescence quantum yield of GlyF-CDs was determined using a reference method, with quinine sulfate as the standard reference (0.1 MH2SO4 as the solvent, λ). ex The quantum yield at 360 nm is 54%. GlyF-CDs were dispersed in ultrapure water to prepare a series of test solutions ranging from 0.01 to 0.05 mg / mL, ensuring that the absorbance of all solutions at 360 nm was below 0.10 to eliminate the influence of self-absorption.

[0060] In λ ex Under the condition of 360 nm, the fluorescence emission spectra of the GlyF-CDs test solution and the quinine sulfate standard solution were measured respectively (excitation and emission slit widths were both 5 nm, scan speed was 600 nm / min). The integrated area of ​​the fluorescence emission spectrum and the absorbance at the excitation wavelength were recorded, and the quantum yield was calculated according to the following formula:

[0061] Note: In the formula, Φ is the quantum yield; I is the integral area of ​​the fluorescence emission spectrum; A is the absorbance at the excitation wavelength; η is the solvent refractive index (the refractive index of ultrapure water and 0.1 M H2SO4 is 1.33); the subscript x represents the GlyF-CDs sample to be tested, and st represents the quinine sulfate standard reference. Each sample was measured in triplicate, and the average value was taken as the final quantum yield. At the same time, the maximum fluorescence intensity (au) of the fluorescence emission spectrum was recorded.

[0062] 2.4.2 Particle size determination The particle size of GlyF-CDs was characterized by high-resolution transmission electron microscopy (HRTEM). A small amount of GlyF-CDs solid powder was dispersed in ultrapure water and ultrasonically dispersed for 5 min to obtain a uniform suspension. The suspension was then dropped onto the surface of a copper mesh, allowed to air dry naturally, and then placed under HRTEM for morphology observation. Nano Measurer software was used to perform particle size statistical analysis on 100 randomly selected GlyF-CDs particles to calculate the average particle size and particle size distribution range.

[0063] 2.5 Structural and performance characterization of GlyF-CDs 2.5.1 Morphology and Particle Size Distribution Characterization The microstructure, dispersibility, and lattice structure of GlyF-CDs were observed using HRTEM. The particle size of 100 GlyF-CDs particles was statistically analyzed using Nano Measurer software. A particle size distribution histogram was plotted and Gaussian fitting was performed to analyze its size uniformity.

[0064] 2.5.2 Optical performance characterization Fluorescence spectroscopy: GlyF-CDs were prepared into an aqueous solution of 0.05 mg / mL, and their excitation and emission spectra were measured using a fluorescence spectrophotometer (excitation wavelength scanning range 300~400 nm, emission wavelength scanning range 380~600 nm, slit width 5 nm) to determine the maximum excitation and maximum emission wavelengths. Ultraviolet-visible absorption spectroscopy: Using ultrapure water as a blank control, the ultraviolet absorption spectrum of 0.05 mg / mL GlyF-CDs aqueous solution was measured in the wavelength range of 200~500 nm using an ultraviolet-visible spectrophotometer, and its optical absorption characteristics were analyzed.

[0065] 2.6.3 Characterization of Surface Chemical Composition Fourier transform infrared spectroscopy (FT-IR): KBr pellet method was used, in the range of 4000~400 cm⁻¹. -1 The FT-IR spectra of licorice flavonoid raw material and GlyF-CDs were measured in the wavenumber range to analyze the types of surface functional groups and bonding characteristics of GlyF-CDs. X-ray photoelectron spectroscopy (XPS): Using Al Kα rays as the excitation source, high-resolution XPS spectra of C1s, O1s, and N1s of GlyF-CDs were measured. Peak fitting was performed on the spectra to analyze the surface elemental composition, elemental valence states, and bonding modes.

[0066] 2.5.4 Fluorescence kinetic characterization The fluorescence decay curve (λ) of a 0.05 mg / mL GlyF-CDs aqueous solution was determined using time-resolved fluorescence spectroscopy. ex =360 nm, λem =460 nm) The decay curve data were fitted using a double exponential decay model to calculate the fluorescence lifetime components and their relative proportions, determine the average fluorescence lifetime, and analyze the luminescence mechanism of GlyF-CDs.

[0067] 2.6 Stability Study of GlyF-CDs 2.6.1 pH stability GlyF-CDs were dispersed in buffer solutions with pH values ​​of 3, 5, 7, 9, and 11 to prepare solutions with a concentration of 0.05 mg / mL. The solutions were stored at room temperature in the dark for 7 days, and their fluorescence emission spectra were measured on days 1, 3, 5, and 7. The maximum fluorescence intensity was recorded, and the relative fluorescence intensity (I / I0, where I0 is the initial fluorescence intensity) was calculated to investigate the effect of different pH environments on the fluorescence performance of GlyF-CDs.

[0068] 2.6.2 Salt stability GlyF-CDs were dispersed in NaCl solutions of different concentrations (0, 0.1, 0.3, 0.5, 0.7, 0.9, 1.0 M) to prepare solutions with a concentration of 0.05 mg / mL. After being placed at room temperature in the dark, their fluorescence emission spectra were measured immediately, the maximum fluorescence intensity was recorded, and the relative fluorescence intensity (I / I0, where I0 is the fluorescence intensity of the 0 M NaCl solution) was calculated to investigate the effect of ionic strength on the fluorescence performance of GlyF-CDs.

[0069] 2.6.3 Long-term storage stability GlyF-CDs were prepared into an aqueous solution of 0.05 mg / mL, sealed, and stored at 4°C in the dark for 7 days. The fluorescence emission spectra were measured on days 1 to 7, the maximum fluorescence intensity was recorded, and the relative fluorescence intensity (I / I0, where I0 is the initial fluorescence intensity on day 1) was calculated to investigate the effect of long-term storage on the fluorescence performance of GlyF-CDs.

[0070] 3 Results 3.1 Results of Preparation Process Optimization L9(3 4 The results of the orthogonal experiments are shown in Table 4. Quantum yield was used as the core evaluation index, while fluorescence intensity and particle size were used as auxiliary evaluation indexes (higher quantum yield, greater fluorescence intensity, and smaller particle size resulted in better overall evaluation). As shown in Table 4, the GlyF-CDs obtained in experiment 5 (A2B2C3D1, 160℃, 6 h, 0.12 g / mL, pH=5) had the highest fluorescence intensity (4450 au) and quantum yield (13.2%) among all experimental groups, and the smallest particle size (3.2 nm), resulting in the best overall evaluation.

[0071] Table 4. Results of the orthogonal experiment

[0072] 3.1.1 Results of range analysis Range analysis was performed on the quantum yield results of the orthogonal experiments to examine the influence of each factor on the preparation of GlyF-CDs. The results are shown in Table 5. The larger the range R value, the more significant the influence of the factor on the experimental results. The table shows that the influence of each factor on the quantum yield of GlyF-CDs is ranked as follows: A. Reaction temperature > B. Reaction time > C. Glycyrrhizin concentration > D. System pH. Based on the factor levels and experimental results, the optimal preparation process for GlyF-CDs was determined to be: A2B2C3D1, i.e., reaction temperature 160℃, reaction time 6 h, glycyrrhizin concentration 0.12 g / mL, and system pH=5.

[0073] Table 5 Range Analysis Table

[0074] Note: QY represents quantum yield; the larger the value of R, the more significant the effect.

[0075] 3.1.2 Results of Optimal Process Verification Test Three batches of parallel replicate experiments were conducted under the optimal preparation process (160℃, 6 h, glycyrrhizin concentration 0.12 g / mL, pH=5), and the results are shown in Table 6. The average fluorescence intensity of the three batches of GlyF-CDs was 4450 au, the average quantum yield was 13.2%, and the average particle size was 3.2 nm. The RSDs of each indicator were 1.6%, 0.8%, and 3.1%, respectively, all <5%, indicating that the preparation process has good reproducibility and stability and can be used for the laboratory-scale preparation of GlyF-CDs.

[0076] Table 6 Results of Process Validation Tests

[0077] Note: RSD is the relative standard deviation; n=3.

[0078] 3.2 Reproducibility verification of GlyF-CDs preparation process To further verify the reliability of the optimal preparation process and the batch-to-batch consistency of the product, three batches of GlyF-CDs were characterized by fluorescence spectroscopy and HRTEM, respectively. The results are shown in [Figure number missing]. Figure 1 , Figure 2 .

[0079] Depend on Figure 1 It can be seen that the fluorescence emission spectra of the three batches of GlyF-CDs are highly overlapping, all showing symmetrical single-peak emission characteristics, with the maximum emission wavelength located in the range of 459~461 nm, and the RSD of fluorescence intensity being 1.6%, indicating that there is no significant difference in the optical performance of the three batches of products.

[0080] Depend on Figure 2 It can be seen that all three batches of GlyF-CDs exhibited good monodispersity, with no obvious agglomeration. The particles were approximately spherical or ellipsoidal with clear edges, and the average particle sizes were 3.1 nm, 3.3 nm, and 3.2 nm, respectively, with a particle size RSD of 3.1%.

[0081] The above results confirm that the solvothermal preparation process of GlyF-CDs established in this study has good repeatability and stability. The optical properties, morphology and particle size of the obtained products are highly consistent, which can provide uniform and stable experimental raw materials for subsequent pharmacodynamic studies of colorectal cancer.

[0082] 3.3 Morphology and particle size distribution characterization results of GlyF-CDs HRTEM images and particle size distribution results of GlyF-CDs are shown in [link to image]. Figure 3 .Depend on Figure 3 As can be seen from the image, GlyF-CDs are approximately spherical or ellipsoidal in shape, with clear particle edges, uniform contrast, and no obvious agglomeration, exhibiting excellent monodispersity. Clear lattice fringes are observed in the image, with a crystal interplanar spacing of approximately 0.21 nm, indicating that glycyrrhizin undergoes controlled carbonization during solvothermal processes, forming sp... 2 It has a hybrid graphitized carbon core structure and good crystallinity.

[0083] Depend on Figure 3 As shown in b, the particle size distribution of 100 randomly selected GlyF-CDs particles from the HRTEM images conformed to a normal distribution. Gaussian fitting yielded an average particle size of 3.2 ± 0.4 nm, with the majority of particles ranging from 2.6 to 3.8 nm. This indicates that the GlyF-CDs prepared using this process exhibit good size uniformity. The small particle size structure facilitates the transmembrane transport, tissue penetration, and in vivo metabolism of GlyF-CDs, laying a structural foundation for subsequent in vitro and in vivo experiments on their anti-colon cancer effects.

[0084] 3.4 Optical performance characterization results of GlyF-CDs 3.4.1 Fluorescence Spectral Characteristics The excitation and emission spectra of GlyF-CDs aqueous solution are shown in [reference needed]. Figure 4 As shown in the figure, the excitation spectrum of GlyF-CDs exhibits a distinct single-peak characteristic, with the maximum excitation wavelength (λ) being... ex The absorption peak at λmax is 361 nm, which is attributed to the n-π* electronic transition of the C=O bond on the carbon core surface; at λ... ex Under the excitation condition of 360 nm, the maximum emission wavelength (λ) of GlyF-CDs is... emThe fluorescence intensity (max) is 460 nm, exhibiting bright blue fluorescence. There is a significant Stokes shift between the excitation and emission spectra, which can effectively avoid the influence of self-absorption effect on fluorescence performance and ensure the accuracy of fluorescence detection.

[0085] 3.4.2 Ultraviolet-Visible Absorption Spectral Characteristics The UV-Vis absorption spectrum of GlyF-CDs aqueous solution is shown below. Figure 5 It exhibits typical carbon nanodot characteristic absorption peak shapes, which can be divided into three characteristic absorption regions: ① A strong absorption peak appears at 252 nm, corresponding to aromatic sp. 2 ① The π-π electronic transitions in the carbon domain confirm that GlyF-CDs form a graphitized carbon core structure, consistent with the lattice structure results characterized by HRTEM; ② A distinct shoulder peak appears at 311 nm, which is attributed to the n-π electronic transitions of oxygen-containing functional groups such as C=O and C–O on the surface, indicating that the surface of GlyF-CDs is rich in polar oxygen-containing functional groups such as hydroxyl, carbonyl, and carboxyl groups, giving it good water dispersibility and biocompatibility; ③ A gradually decaying tail absorption is observed in the range of 320~450 nm and extends into the visible light region, corresponding to the light absorption of the surface state energy levels of GlyF-CDs.

[0086] GlyF-CDs aqueous solution appears pale yellow and transparent under fluorescent light, and emits bright blue fluorescence under 365 nm ultraviolet light. Its appearance characteristics are highly consistent with its ultraviolet absorption and fluorescence spectral characteristics, further verifying its excellent optical performance.

[0087] 3.5 Surface chemical composition characterization results of GlyF-CDs 3.5.1 Fourier Transform Infrared Spectroscopy (FT-IR) Characterization The FT-IR spectra of glycyrrhizin flavonoid precursors and GlyF-CDs are shown in the figure. Figure 6 Comparative analysis reveals that the molecular structure of glycyrrhizin undergoes significant changes during solvothermal carbonization, and abundant polar functional groups form on the surface of GlyF-CDs, with specific characteristics as follows. Glycyrrhizin at 3377 cm⁻¹... -1 The broadened OH / NH stretching vibration peak at 3012 cm⁻¹ is retained in GlyF-CDs, indicating that the surface of GlyF-CDs is rich in hydroxyl functional groups; glycyrrhizin at 3012 cm⁻¹... -1 The olefin =CH stretching vibration peak and 1746 cm⁻¹ at the position. -1 The C=O stretching vibration peak at 2894 cm⁻¹ completely disappears in GlyF-CDs, indicating that the unsaturated olefin structure and ester group undergo breakage and decomposition during solvothermal processes, completing the carbonization reaction; GlyF-CDs at 2894 cm⁻¹... -1 A new CH stretching vibration peak appears at 1592 cm⁻¹, attributed to the stretching vibration of the residual methylene group; glycyrrhizin peaks at 1592 cm⁻¹.-1 The aromatic C=C skeleton vibration peak at this location shifts to 1575 cm⁻¹ in GlyF-CDs. -1 Furthermore, the relative strength increased significantly, confirming the formation of sp during the solvothermal process. 2 Hybridized graphitized conjugated carbon core structure; GlyF-CDs at 1314 cm⁻¹ -1 The presence of CO stretching vibration peaks indicates that oxygen-containing functional groups such as ether bonds and phenolic hydroxyl groups still exist on its surface.

[0088] The aforementioned surface polar oxygen-containing functional groups not only endow GlyF-CDs with excellent water solubility and colloidal stability, but also serve as surface passivation sites, effectively suppressing nonradiative recombination processes and improving the fluorescence quantum yield of GlyF-CDs.

[0089] 3.5.2 X-ray photoelectron spectroscopy (XPS) characterization The high-resolution XPS spectra of C1s, O1s, and N1s of GlyF-CDs are shown in [reference needed]. Figure 7 The results showed that the surface of GlyF-CDs mainly contains three elements: C, O, and N, and there are multiple bonding modes.

[0090] C1s spectrum ( Figure 7 a): After peak fitting, the convolution can be deconvolved into four characteristic peaks. The peak at 284.6 eV belongs to C–C / C=C(sp). 2 Hybridized carbon (C₁₂O) accounts for 37.2%; C₁₂O at 286.2 eV, C₂=O at 287.8 eV, and O₁₂C₂=O at 288.9 eV together account for 62.8%, confirming that the surface of GlyF-CDs is rich in oxygen-containing functional groups such as hydroxyl, carbonyl, and carboxyl groups, consistent with the FT-IR characterization results. O1s spectrum ( Figure 7 b): Peak fitting revealed two main characteristic peaks, with C=O double bonds accounting for 62.4% and C–O single bonds accounting for 37.6%, further verifying the presence and relative content of oxygen-containing functional groups on the surface of GlyF-CDs. N1s spectrum ( Figure 7 c): Two characteristic peaks can be fitted at 400.8 eV and 403.4 eV, indicating that the natural nitrogen-containing components in glycyrrhizin were successfully doped into the carbon framework during solvothermal carbonization. The introduction of nitrogen can form an n-type doping effect and surface defect states in the carbon framework, which helps to regulate the band structure of GlyF-CDs and optimize their optical properties.

[0091] 3.6 Fluorescence kinetics characterization results of GlyF-CDs The fluorescence decay curve of GlyF-CDs at an excitation wavelength of 360 nm is shown in the figure. Figure 8The fluorescence decay curves of GlyF-CDs exhibit typical multi-exponential decay characteristics, indicating that the luminescence process involves multiple excited-state relaxation pathways. This characteristic is consistent with the structural properties of carbon nanodots, which simultaneously possess intrinsic carbon core states and surface defect states. A double-exponential decay model was used to fit the fluorescence decay curve data, yielding two fluorescence lifetime components: a short-lived component of 4.2 ns (65% relative), attributed to the intrinsic fluorescence of the carbon core sp² hybrid graphitized structure; and a long-lived component of 15.8 ns (35% relative), originating from surface trap states formed by oxygen-containing functional groups (C=O, COC, etc.) on the GlyF-CDs surface. The calculated average fluorescence lifetime of GlyF-CDs is 8.5 ns, which falls within the typical fluorescence lifetime range (4–15 ns) of carbon nanodots, indicating stable fluorescence performance and a luminescence mechanism consistent with typical carbon nanodot characteristics.

[0092] 3.7 Stability test results of GlyF-CDs 3.7.1 pH stability The changes in fluorescence performance of GlyF-CDs after 7 days of storage in different pH buffer solutions are shown in the figure. Figure 9 As shown in the figure, within the pH range of 3 to 11, the maximum emission wavelength of GlyF-CDs remained at 460±3 nm without any obvious red or blue shift, indicating that the chemical structure of its surface luminescent centers remained stable under acidic or alkaline conditions. Within the pH range of 5 to 9, the relative fluorescence intensity retention rate of GlyF-CDs was above 95%, demonstrating excellent pH tolerance.

[0093] The excellent pH stability of GlyF-CDs stems from the abundance of oxygen-containing functional groups on their surface. Within the pH range of 5–9, functional groups such as carboxyl and hydroxyl groups exist in a dissociated state, forming a stable hydration layer and electrostatic repulsion on the particle surface. This effectively maintains the dispersibility of GlyF-CDs and avoids fluorescence quenching caused by aggregation. This characteristic allows GlyF-CDs to adapt to the acid-base environment in vivo (human physiological pH 7.35–7.45, tumor microenvironment pH 6.5–6.8), providing a reliable guarantee for subsequent in vivo experiments against colon cancer.

[0094] 3.7.2 Salt stability The fluorescence performance changes of GlyF-CDs in NaCl solutions of different concentrations and pH values ​​are shown in the figure. Figure 10As shown in the figure, within the NaCl concentration range of 0–1.0 M, the maximum emission wavelength of GlyF-CDs remained consistently around 460 nm without significant shift, indicating that the electronic structure of its surface luminescent centers is insensitive to changes in ion intensity. As the NaCl concentration increased from 0 to 1.0 M, the fluorescence intensity of GlyF-CDs showed a trend of first slightly increasing and then slightly decreasing, reaching its maximum value under 0.5 M NaCl conditions, and the relative fluorescence intensity remained consistently between 99% and 102%.

[0095] Moderate ionic strength can compress the electric double layer on the surface of GlyF-CDs particles, promoting their uniform dispersion in aqueous solution and reducing fluorescence quenching caused by particle aggregation. Therefore, the fluorescence intensity is slightly enhanced in low-concentration NaCl solutions. High-concentration NaCl slightly disrupts the hydration layer on the particle surface, leading to a slight decrease in fluorescence intensity, but the overall fluorescence intensity remains stable. These results indicate that GlyF-CDs exhibit stable fluorescence performance within the physiological ionic strength range and can adapt well to the ionic environment of body fluids in vivo.

[0096] 3.7.3 Long-term storage stability The fluorescence performance changes of GlyF-CDs aqueous solution after 7 days of storage at 4℃ in the dark are shown in the figure. Figure 11 As shown in the figure, during the 7-day storage period, the maximum emission wavelength of GlyF-CDs remained within the range of 460±2 nm, and the peak shape of the fluorescence emission spectrum did not change significantly. Based on the initial fluorescence intensity on day 1, the relative fluorescence intensity of GlyF-CDs remained between 99% and 102%, with an average value of 100.3% and an RSD of only 1.0%, demonstrating excellent long-term storage stability.

[0097] The high storage stability of GlyF-CDs stems from two structural advantages: first, the stable hydration layer formed by abundant oxygen-containing functional groups on the surface effectively prevents particle aggregation and sedimentation, maintaining its dispersibility; second, the rigid structure of the graphitized carbon core effectively inhibits photochemical degradation reactions, ensuring stable optical performance. The slight increase in fluorescence intensity during storage may be related to further evaporation of residual solvents on the surface and the slow rearrangement of surface functional groups, reducing defect states.

[0098] 4. Pharmacodynamic studies of GlyF-CDs 4.1 Materials and Methods 4.1.1 Experimental Apparatus (Table 7)

[0099] 1.1.2 Experimental reagents (Table 8)

[0100] 2. Experimental Methods 2.1 Constructing a mouse model of SW480 colon cancer xenograft Sixty male SPF-grade BALB / c nude mice, 3-4 weeks old and weighing 16-20g, were used. They were purchased from the Experimental Animal Center of Ningxia Medical University. Animal experiments were approved by the Ethics Committee of the Experimental Animal Center of Ningxia Medical University. After passing animal quarantine, the mice were acclimatized for one week in a standard SPF environment with free access to food and water. Human colon cancer cell line SW480 (purchased from Wuhan Pronosai Life Science Technology Co., Ltd.) was cultured and passaged using conventional cell culture methods. Cells in the logarithmic growth phase were used for model establishment. The cell count was 5 × 10⁶. 6 Cells / 100μL were resuspended in PBS, dispensed into syringes, and injected subcutaneously into the right axilla of nude mice. After inoculation, the mice were fed as usual, and their living conditions were observed. Tumor formation was monitored daily.

[0101] After the tumor has grown, use calipers to measure the major axis (A) and minor axis (B) of the tumor, and calculate the value using the formula V = A × B. 2 / 2, Calculate the volume of the transplanted tumor, which reaches approximately 50 mm. 3 That is, the first day of the experiment.

[0102] The optimal preparation process for GlyF-CDs was as follows: A2B2C3D1, i.e., reaction temperature 160℃, reaction time 6 h, glycyrrhizin concentration 0.12 g / mL, and system pH=5. GlyF-CDs were prepared for later use. The mice were randomly divided into 8 groups (n=8 per group): control group, model group, positive control group (Oxaliplatin), high- and low-dose GlyF-CDs groups, high- and low-dose glycyrrhizin GlyF groups, and Con+GlyF-CDs group. Based on the preliminary experimental results, combined with the bioavailability (42%) and maximum tolerated dose (60 mg / kg) of gavage in nude mice, the following dose levels were set: low-dose GlyF 30 mg / kg, high-dose GlyF 50 mg / kg; low-dose GlyF-CDs 10 mg / kg, high-dose GlyF 20 mg / kg.

[0103] 2.2 Bioactivity Study and Safety Evaluation in Tumor-Bearing Mice Tumor-bearing mice were intraperitoneally injected with saline (Model group, 0.1 mL / 10 g), GlyF-CDs (low-dose group 10 mg / kg, high-dose group 20 mg / kg), and GlyF (low-dose group 30 mg / kg, high-dose group 50 mg / kg), respectively. The oxaliplatin group received intraperitoneal injection of oxaliplatin at a dose of 5 mg / kg, twice weekly (days 1, 4, 7, and 10), with saline as the solvent. Normal mice were injected with saline (Control group, 0.1 mL / 10 g) and GlyF-CDs (Con+GlyF-CDs group, 5 mg / kg). Administered daily at fixed times, with dosage calculated based on body weight, for 14 consecutive days via gavage. The mice's behavior and tumor growth were closely monitored. Subcutaneous tumor diameter was measured every 1-2 days using calipers.

[0104] After the administration was completed, all nude mice were euthanized, subcutaneous tumor tissue was removed and weighed, and whole blood was collected, centrifuged after standing. Serum was collected, tumor tissue was photographed, measured, and weighed, and the tumor inhibition rate of mice in each group was calculated. Tumor inhibition rate (%) = [average tumor mass in the model group (g) - average tumor mass in the drug-treated group (g)] / average tumor mass in the model group (g) × 100%. Partial tumor tissue and major organs were fixed for subsequent pathological experiments, while the remainder was rapidly stored at -80℃ to facilitate subsequent detection of related proteins and mRNA expression in tumor tissue. Subsequent experimental groups were all represented by the high-dose drug group.

[0105] Table 9 Dosing regimens for mice in each group

[0106] 2.3 Paraffin sections Tumor tissue blocks fixed in the Con, Mod, GlyF, and GlyF-CDs groups were trimmed to appropriate sizes and placed in clearly marked embedding cassettes. These cassettes were then subjected to gradient dehydration and paraffin infiltration sequentially in an automated dehydrator. After paraffin infiltration, the tissue was embedded into paraffin blocks using an embedding machine. Once the paraffin blocks had completely solidified, they were serially sectioned using a paraffin microtome with a section thickness set to 5 μm. The sectioned tissue slides were flattened in warm water, transferred to glass slides, and then fixed using a slide warmer. The dried slides were then sealed and stored at 4°C for later use.

[0107] 2.4 Histological HE staining and pathological observation 2.4.1 HE staining of transplanted tumor tissue Paraffin sections of tumor tissue from each group were routinely stained with hematoxylin and eosin (HE), dewaxed with xylene, hydrated in graded ethanol, stained with hematoxylin for 5 min, rinsed with tap water to regain blue color, stained with eosin for 5 min, dehydrated again in graded ethanol, cleared with xylene, and mounted with neutral resin. The pathological morphology of the tumor tissue was observed under a light microscope and images were acquired.

[0108] 2.4.2 HE staining of major organs (biosafety assessment) Heart, liver, spleen, lung, and kidney tissues were collected from mice in the Con and Con+GlyF-CDs groups and processed using the same HE staining procedure described above. The pathological structures of each organ were observed under a light microscope to assess abnormal changes such as inflammation, degeneration, and necrosis, and to evaluate the biosafety of GlyF-CDs.

[0109] 2.4.3 Immunofluorescence staining of transplanted tumor tissue After immersing the slides in 95℃ pH 6.0 citrate antigen retrieval solution for 10 min, they were allowed to cool naturally. They were then washed three times (5 min each time) with PBS (containing 0.05% Tween-20), and incubated with 0.1% Triton X-100 at room temperature for 15 min to allow permeation. After washing with PBS, they were blocked with 5% BSA at 37℃ for 1 h. The blocking solution was discarded, and rabbit anti-mouse GPX4 polyclonal antibody diluted 1:200 (approximately 50 μL per slide, diluted with 5% BSA) was added. The slides were incubated overnight in a humidified chamber at 4℃ (the negative control was replaced with 5% BSA instead of the primary antibody). The next day, the slides were warmed to room temperature for 30 min, washed three times with PBS, and then incubated with 1:500 diluted Alexa Fluor 488-labeled secondary antibody at room temperature in the dark for 1 h. After washing three times with PBS in the dark, DAPI staining solution was added and incubated at room temperature in the dark for 5 min. After washing with PBS, the liquid was blotted dry, and anti-fluorescence quenching mounting medium was added for mounting. The slides were then air-dried in the dark for 24 h. The slides were then examined under a laser confocal microscope (DAPI excitation wavelength 405 nm). Five random × 400x field-of-view images were acquired using Alexa Fluor 488 excitation wavelength (nm). The average fluorescence intensity of GPX4 protein was quantitatively analyzed using ImageJ software. During the operation, it is necessary to pay attention to rapid tissue processing, fixation and dehydration to maintain low temperature to avoid GPX4 protein degradation, strictly control the antigen retrieval temperature and time, ensure the humidity of the humidified chamber during incubation, keep the fluorescent reagent away from light throughout the process, and wash thoroughly to reduce non-specific staining interference.

[0110] 2.5 Western blot analysis of expression levels of tumor-related proteins Tumor tissue was minced (3 mm × 3 mm), and 0.5–1 mL of pre-chilled RIPA lysis buffer (containing protease inhibitor) was added per 100 mg of tissue. After adding sterile steel balls, the mixture was cryogenically ground at 60 Hz for 90 s, centrifuged at 12000 rpm for 5 min at 4°C, and the supernatant was collected. Protein concentration was quantified using the BCA method. Protein samples were mixed with 5× loading buffer at a 4:1 ratio, denatured in a boiling water bath for 5 min, separated by SDS-PAGE gel electrophoresis at 120 V constant voltage, and transferred to a methanol-activated PVDF membrane at a constant current of 200 mA. The membrane was blocked with 5% skim milk powder at room temperature for 1 h, incubated overnight at 4°C with primary antibody, washed three times with PBST (10 min each time), incubated with HRP-labeled secondary antibody at room temperature for 1 h, and washed three more times with PBST. Chemiluminescence reagent was added, and images were acquired using a fully automated chemiluminescence analyzer. The grayscale values ​​of the bands were quantified using ImageJ software.

[0111] 2.6 qRT-PCR detection of tumor tissue-related mRNA expression levels Take 10-20 mg of cryopreserved tumor tissue, add 300 μL of lysis buffer RL, freeze-mill, centrifuge at 12000 rpm for 5 min, collect the supernatant, add 0.5 volume of anhydrous ethanol, mix well, and purify by adsorption column CR3. Then, treat with protein removal buffer RD and washing buffer RW sequentially (each washing step repeated once), elute with RNase-free ddH2O to obtain total RNA, and verify A using a nucleic acid protein analyzer. 260 / A 280 purity.

[0112] Take ≤5 μg of total RNA, place it on ice, add 1 µL of Oligo(dT) and nuclease-free water to a total volume of 12 µL, incubate in a PCR instrument at 65°C for 5 min, then cool on ice. Add 5×Reaction Buffer, RNase inhibitor, dNTP Mix and reverse transcriptase to a total volume of 20 µL, mix gently, and incubate at 42°C for 60 min and 70°C for 5 min to terminate the reaction and synthesize cDNA. It can be used directly for experiments or stored in a -80°C freezer.

[0113] Using β-actin as an internal control, the target genes included GPX4, ACSL4, TFR1, and NOX1 (primer sequences are shown in Table 10). A qPCR reaction solution (containing 2×SYBR Master Mix, cDNA, and upstream and downstream primers) was prepared in a 20 μL system and amplified using a real-time quantitative PCR instrument: pre-denaturation at 95℃ for 5 min, 40 cycles (95℃ for 10 s, 60℃ for 30 s), followed by melting curve analysis. Two... -ΔΔCt The method calculates the relative expression level of the target gene.

[0114] Table 10 Primer Sequences

[0115] 2.7 Statistical Analysis All experiments were performed in triplicate. Graphs were generated using GraphPad Prism 9.5.1 software. Experimental data between groups were compared using one-way ANOVA. A p-value < 0.05 was considered statistically significant (*p < 0.05, *p < 0.01, ***p < 0.001).

[0116] 3 Results 3.1 In vivo biosafety evaluation of GlyF-CDs The growth status of mice in each group was assessed by monitoring changes in their body weight. Figure 12 The results of weight changes showed that the weight of mice in the Control group showed a steady and continuous increase throughout the experimental period. The weight of mice in the Control+GlyF-CDs group increased at a slightly slower rate in the first 7 days, but then increased rapidly as the experiment progressed. By day 13, there was no significant difference in weight between the Control group and the Control group. This suggests that GlyF-CDs have good biocompatibility and did not have any adverse effects on the normal growth and metabolism of mice.

[0117] Mice in the Model group showed a slight decrease in body weight on day 4. At this stage, the subcutaneous transplanted tumor volume had not yet significantly increased, presumably due to disordered energy metabolism and nutrient consumption under tumor-bearing conditions. Later, as the mice adapted to the tumor-bearing state, they consumed energy to cope with the condition, resulting in a slow recovery in body weight, but the overall rate of increase was significantly lower than that in the Control group. Among the drug administration groups, the Oxaliplatin group showed a significantly different body weight change compared to other groups. From day 4 to 7, the body weight showed a marked decrease, although it recovered slightly later, but by the end of the experiment, it had not returned to the initial level and was significantly lower than that in the Control group (P<0.01). Combined with behavioral observations, mice injected with Oxaliplatin exhibited decreased activity and reduced food intake, suggesting that the commonly used chemotherapy drug Oxaliplatin has significant in vivo toxic side effects and can produce strong stress stimulation in mice, leading to weight loss. The overall body weight of mice in the low- and high-dose GlyF groups and the high- and low-dose GlyF-CDs groups showed a slow recovery trend, with a growth rate higher than that of the Mod and Oxaliplatin groups. Furthermore, the high-dose group showed slightly better weight gain than the low-dose group, suggesting that neither drug class produced significant systemic toxicity in mice during anti-tumor treatment and had minimal impact on normal growth. The weight gain trend of mice in the high-dose GlyF-CDs group was similar to that of the Control group, and by day 13, their body weight was significantly higher than other treatment groups, further confirming that GlyF-CDs maintains excellent biocompatibility at high doses and has no significant in vivo toxicity.

[0118] HE staining was performed on the heart, liver, spleen, lung, and kidney tissues of mice in the Control group and the Control+ GlyF-CDs group to observe changes in organ pathological structure. The results are shown in [Figure 1]. Figure 13 In the control group mice, the myocardial fibers of the heart were neatly arranged and the striations were clear. The liver lobule structure was intact, the central vein and hepatic cords were arranged regularly, the red pulp and white pulp of the spleen were clearly demarcated, the lymph node structure was intact, the alveolar cavities of the lung tissue were of uniform size, and there was no proliferation of alveolar epithelial cells. The glomeruli of the kidneys were of normal morphology, the renal tubular epithelial cells were neatly arranged, and there was no vacuolar degeneration or cast formation.

[0119] The pathological structures of major organs in mice in the Control+GlyF-CDs group were not significantly different from those in the Control group. No abnormal pathological changes were observed in myocardium, hepatocytes, alveolar epithelial cells, etc., indicating that GlyF-CDs did not cause significant toxic damage to important organs of mice after in vivo administration, further verifying the good in vivo biocompatibility and safety of GlyF-CDs.

[0120] 3.2 Inhibitory effect of GlyF-CDs on tumors in vivo like Figure 14 The results of tumor volume changes show that the tumor volume of the Mod group mice increased rapidly and exponentially during the experimental period, reflecting the malignant proliferative characteristics of colon cancer xenografts in vivo.

[0121] Tumor growth was inhibited to varying degrees in all treatment groups, and all showed highly significant differences compared to the model group (P<0.01), exhibiting a clear dose-dependent effect. The tumor growth rate in both the low- and high-dose GlyF groups was lower than that in the model group, suggesting that glycyrrhizin has a certain in vivo inhibitory effect on colorectal cancer xenografts, but the effect is relatively mild. The oxaliplatin group showed significantly better tumor inhibition than the low- and high-dose GlyF groups and the low-dose GlyF-CDs group, demonstrating the potent antitumor effect of chemotherapy drugs. The high-dose GlyF-CDs group showed the most significant tumor growth inhibition effect, with tumor volume at all time points lower than that in the oxaliplatin group (P<0.05), suggesting that high-dose GlyF-CDs has a potent and sustained inhibitory effect on the growth of colorectal cancer xenografts.

[0122] On day 13 of the experiment, all tumor-bearing mice were sacrificed, and the tumor tissue was dissected, photographed, and weighed. Figure 15 The tumor inhibition rate of each group was calculated, and the in vivo antitumor effect of each drug administration group was evaluated. The results of tumor weight and tumor inhibition rate are shown in the table below. Figure 16 , Figure 17 The results of tumor weight and tumor inhibition rate showed that the tumor weight in each treatment group was lower than that in the Mod group (P<0.01), and the tumor inhibition rate increased with increasing dosage, showing a dose-dependent relationship.

[0123] The tumor inhibition rates of the low- and high-dose GlyF groups were 19.67% and 33.61%, respectively, showing a certain anti-tumor effect, but the inhibition rate was relatively low. The tumor inhibition rate of the low-dose GlyF-CDs group was 45.08%, significantly higher than that of the GlyF group, but slightly lower than that of the oxaliplatin group, suggesting that the anti-tumor activity of GlyF-CDs was significantly better than that of glycyrrhizin. The tumor inhibition rate of the oxaliplatin group was 49.18%, reflecting the strong anti-tumor effect of the chemotherapy drug, while the tumor inhibition rate of the high-dose GlyF-CDs group was significantly higher than that of the oxaliplatin group (P<0.05). These results indicate that GlyF-CDs have a significant in vivo inhibitory effect on subcutaneous xenografts of colon cancer.

[0124] 3.3 GlyF-CDs Observation on the Histopathological Morphology of Transplanted Tumors in Tumor-Bearing Mice like Figure 18 Under a light microscope, the model group showed a large number of tumor cells (black arrows), densely packed with varying sizes and irregular shapes, exhibiting significant atypia, large and deeply stained nuclei, and a high nucleoplasm-to-nucleus ratio. Only a few scattered nuclear pyknosis phenomena were observed, with no obvious necrotic areas. The overall structure of the tumor tissue remained intact, reflecting the malignant proliferative characteristics of colon cancer xenografts. In the glycyrrhizin group, the tumor tissue showed a small number of mitotic figures (black arrows), moderate-scale tumor cell necrosis (red arrows), nuclear pyknosis, fragmentation, or dissolution, and numerous necrotic cell fragments. No obvious inflammatory cell infiltration was observed. In the Oxa group, tumor cell necrosis (red arrows) was observed, with nuclear pyknosis, fragmentation, or dissolution, presenting as unstructured eosinophilic material, and numerous necrotic cell fragments were visible. Focal stromal proliferation of a small number of fibroblasts was observed (green arrows), accompanied by a small amount of lymphocyte infiltration (blue arrows). In the GlyF-CDs group, extensive tumor cell necrosis (red arrows) was observed, with nuclear pyknosis, fragmentation, or dissolution. The tumor presents as unstructured eosinophilic material with numerous necrotic cell fragments and occasional hemorrhage (yellow arrows). Small-scale proliferation of fibroblasts and fine collagen fibers is visible in the stroma (green arrows), accompanied by a small amount of lymphocyte infiltration (blue arrows). HE staining results visually confirm that GlyF-CDs can cause extensive necrosis of tumor tissue and inhibit tumor growth in vivo.

[0125] 3.4 Immunofluorescence staining to observe the location and expression of ferroptosis-associated protein GPX4 The expression and distribution of the target protein GPX4 in tumor tissues of each group were detected by immunofluorescence staining. The results are as follows: Figure 19As shown in the figure (scale bar: 100 μm), the Mod group exhibited abundant red fluorescence signals, indicating widespread expression of GPX4 in tumor tissues, distributed in the cytoplasm and cell membrane. The Oxa group showed a significantly weakened red fluorescence signal intensity, indicating a marked decrease in protein expression levels. The GlyF group showed a decrease in red fluorescence signal intensity compared to the model group, but remained higher than the Oxa group. The GlyF-CDs group exhibited the lowest red fluorescence signal intensity, with significantly downregulated protein expression levels and sparser distribution. These results suggest that GlyF-CDs can significantly inhibit the expression of ferroptosis-related proteins in xenograft tumor tissues.

[0126] 3.5 Effect of Western blot detection on tumor tissue-related protein expression Western blot analysis was performed to detect the expression levels of key regulatory proteins of ferroptosis, GPX4, ACSL4, TFR1, and NOX1, using GAPDH as an internal control. The results are as follows: Figure 20 As shown in the figure, compared with the Mod group, the expression levels of GPX4 protein in the GlyF group, Oxa group, and GlyF-CDs group were all downregulated (*p<0.05, **p<0.01, ***p<0.001), with the most significant downregulation in the GlyF-CDs group (***p<0.001). GPX4 is a core inhibitor of ferroptosis, and its downregulation is an important marker of ferroptosis. Compared with the Mod group, the expression levels of ACSL4, TFR1, and NOX1 proteins in the GlyF group, Oxa group, and GlyF-CDs group were all significantly upregulated (*p<0.05, **p<0.01, ***p<0.001), with the largest upregulation in the GlyF-CDs group (**p<0.01, ***p<0.001). ACSL4 drives ferroptosis by promoting lipid peroxidation, TFR1 increases intracellular iron overload by regulating iron uptake, and NOX1 exacerbates oxidative stress by promoting reactive oxygen species (ROS) generation. The upregulation of these three factors jointly promotes the process of ferroptosis.

[0127] 3.6 qRT-PCR detection of ferroptosis-related gene mRNA expression Using β-actin as an internal control, the mRNA expression levels of key regulatory genes of ferroptosis, GPX4, ACSL4, TFR1, and NOX1, were detected. The results are as follows: Figure 21As shown in the figure, compared with the Mod group, the expression levels of GPX4 gene in the GlyF group, Oxa group, and GlyF-CDs group were significantly downregulated (*p<0.05, **p<0.01, ***p<0.001), with the GlyF-CDs group showing the most significant downregulation (***p<0.001). Compared with the Mod group, the expression levels of ACSL4, TFR1, and NOX1 mRNA in the GlyF group, Oxa group, and GlyF-CDs group were significantly upregulated (*p<0.05, **p<0.01, ***p<0.001), with the GlyF-CDs group showing the largest upregulation (**p<0.01, ***p<0.001), consistent with the protein expression trend. This further confirms that GlyF-CDs can regulate the expression of ferroptosis-related molecules at the transcriptional and translational levels.

[0128] In summary, this application successfully prepared glycyrrhizin-based carbon nanodots (GlyF-CDs) using glycyrrhizin as the carbon source via a solvothermal method, optimized the preparation conditions, and systematically characterized the morphology, structure, and optical properties of the obtained nanomaterials. Regarding the preparation method, a solvothermal method was used to prepare GlyF-CDs. This method has advantages such as relatively mild reaction conditions, simple operation, and good product dispersibility, and is widely used in the preparation of carbon nanodots. Experimental results show that factors such as reaction temperature, reaction time, raw material concentration, and system pH all have a certain influence on the formation and properties of carbon nanodots. With increasing reaction temperature, the carbonization reaction gradually intensifies, which is beneficial to the formation of carbon core structures. However, excessively high temperatures may lead to particle aggregation or increased structural defects, thus affecting their fluorescence performance. This application determined that 160℃ is a suitable reaction temperature, under which carbon nanodots with relatively uniform particle size distribution and stable fluorescence performance can be obtained. Reaction time also plays an important role in the formation of carbon nanodots. In the initial stage of the reaction, precursor decomposition and carbonization reactions mainly occur in the system. As the reaction time increases, carbon cores gradually form and tend to stabilize. However, if the reaction time is too long, the carbon nuclei may further aggregate or undergo structural changes, leading to a decrease in fluorescence performance. Therefore, after comprehensively considering the product stability and fluorescence properties, the reaction time was determined to be 6 h. The concentration of raw materials and the pH of the system also have a certain influence on the degree of carbonization and the formation of surface functional groups in the reaction system. In this study, the optimal conditions were determined through orthogonal experiments to be a glycyrrhizin flavonoid concentration of 0.12 g / mL, a pH of 5, a temperature of 160℃, and a reaction time of 6 h.

[0129] Morphological characterization results showed that the obtained GlyF-CDs exhibited a uniformly dispersed spherical structure with an average particle size of approximately 3.2 ± 0.4 nm, indicating that the prepared material belongs to the typical carbon nanodot scale range. The nanoscale particles possess a large specific surface area and good dispersibility, providing a fundamental condition for their application in biological systems. HRTEM results showed no obvious aggregation, indicating that the prepared carbon nanodots have good dispersion performance. FT-IR and XPS analyses revealed that the surface of GlyF-CDs contains oxygen-containing functional groups such as hydroxyl and carbonyl groups, along with nitrogen. These polar groups not only improve the water solubility of the carbon nanodots but may also participate in subsequent biological interactions to some extent. Furthermore, the presence of surface functional groups provides possibilities for further functionalization modifications.

[0130] Optical performance characterization showed that GlyF-CDs emitted blue fluorescence at 460 nm under 361 nm excitation, exhibiting a significant Stokes shift, which effectively avoided interference from self-absorption effects on fluorescence detection. The average fluorescence lifetime was 8.5 ns, within the typical fluorescence lifetime range of carbon nanodots, indicating stable fluorescence performance. The luminescence mechanism is a combination of intrinsic carbon nucleus eigenstate luminescence and surface defect state luminescence. Stability testing results showed that GlyF-CDs maintained highly stable fluorescence performance within the physiological pH range (5–9), physiological ionic strength (0–1.0 M NaCl), and for 7 days at 4°C in the dark. This demonstrates their ability to adapt to the complex acid-base and ionic environments in vivo, preventing fluorescence quenching or structural damage during in vivo transport, providing a reliable material guarantee for subsequent cell and animal experiments.

[0131] Biosafety evaluation is a crucial prerequisite for nanomedicine development. Experimental results showed that GlyF-CDs treatment significantly inhibited tumor growth in nude mice, with the high-dose group exhibiting a more pronounced inhibitory effect. This suggests that GlyF-CDs possesses certain antitumor activity in vivo, exhibiting a dose-dependent effect. The weight gain trend in the Control+GlyF-CDs group was largely consistent with that of the normal group, and HE staining of major organs showed no significant pathological damage, indicating that this material has good biosafety within a certain dosage range.

[0132] Overall, this study successfully constructed a carbon nanodot system using glycyrrhizin as the carbon source. This material not only exhibits good structural stability and dispersibility but also demonstrates certain advantages in optical properties, providing a foundation for subsequent pharmacodynamic studies.

[0133] In vivo tumor suppression experiments showed that the tumor volume and weight in the high-dose GlyF-CDs group were significantly lower than those in the model group, and the tumor inhibition rate was significantly higher than that in the glycyrrhizin group, and superior to that in the positive control drug oxaliplatin group, indicating that the in vivo anti-colon cancer activity of GlyF-CDs was significantly better than that of free glycyrrhizin. HE staining of tumor tissues showed that the tumor cells in the model group were densely packed, exhibited significant atypia, and had no obvious necrosis areas. In contrast, the GlyF-CDs group showed extensive tumor cell necrosis, accompanied by a small amount of inflammatory cell infiltration and fibrous tissue proliferation, directly demonstrating its killing effect on tumor tissue.

[0134] The above embodiments merely illustrate several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A method for preparing licorice flavonoid carbon nanodots, characterized in that, Includes the following steps: S10. Disperse glycyrrhizin in ultrapure water and stir until homogeneous to form a glycyrrhizin suspension; S20. Adjust the pH of the licorice flavonoid suspension to a specified value with hydrochloric acid, react at a specified temperature for a specified time, and after the reaction is completed, allow it to cool naturally to room temperature to obtain a brownish-yellow solution; S30. Filter and centrifuge the brownish-yellow solution, and take... After dialyzing of the supernatant, it was freeze-dried to obtain a solid powder, namely licorice flavonoid carbon nanodots.

2. The method for preparing licorice flavonoid carbon nanodots as described in claim 1, characterized in that, The purity of the licorice flavonoids is 80%-99%.

3. The method for preparing licorice flavonoid carbon nanodots as described in claim 1, characterized in that, The concentration of the licorice flavonoid suspension is 0.04-0.12 g / mL.

4. The method for preparing licorice flavonoid carbon nanodots as described in claim 1, characterized in that, The pH value is 5-9.

5. The method for preparing licorice flavonoid carbon nanodots as described in claim 1, characterized in that, The specified temperature is 160-180 ℃.

6. The method for preparing licorice flavonoid carbon nanodots as described in claim 1, characterized in that, The specified time is 4-8 hours.

7. A type of licorice flavonoid carbon nanodots, characterized in that, The licorice flavonoid carbon nanodots were prepared using the method described in any one of claims 1 to 6.

8. The use of the licorice flavonoid carbon nanodots as described in claim 7 in the preparation of drugs for treating colon cancer.

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