Amino acid carbon dots, methods of making the same, and applications thereof

Abstract

The application relates to the technical field of carbon dot preparation, and provides a preparation method and application of amino acid carbon dots, wherein the preparation method of the amino acid carbon dots comprises the following steps: grinding amino acids to obtain primary-treated amino acids; and heating the primary-treated amino acids to obtain amino acid carbon dots. The amino acid carbon dots are prepared by using a pyrolysis method, the amino acid carbon dots are directly heated, further purified by using methods such as grinding, dissolving, centrifugal separation, filtering and dialysis, and finally high-purity amino acid carbon dots are obtained. The preparation method is simple and efficient, the high-pressure environment necessary for preparing the carbon dots by using a hydrothermal method is avoided, safety is better, the requirement for production equipment is reduced, and the method is more conducive to popularization and large-scale production.

Description

Technical Field

[0001] This invention relates to the field of carbon dot preparation technology, and more particularly to amino acid carbon dots, their preparation methods, and their applications. Background Technology

[0002] Carbon dots (CDs) are a novel type of zero-dimensional carbon nanomaterial with excellent fluorescence properties, good biocompatibility, and low toxicity. Furthermore, due to their small size, large specific surface area, and ease of surface functionalization, carbon dots can be rapidly absorbed and distributed by cells after entering the human body, enabling precise targeted therapy. In addition, the metabolic pathways of carbon dots in vivo are relatively well-defined, minimizing long-term bioaccumulation, which is another significant advantage in biomedical applications. Therefore, carbon dots have enormous potential for applications in human biology; for example, they can be used in multiple fields such as bioimaging, drug delivery, disease diagnosis, and treatment.

[0003] Amino acid carbon dots (AACDs), as a derivative of carbon dots, possess unique advantages in biological applications due to the introduction of amino acid molecules on their surface. Simultaneously, amino acids themselves are the basic building blocks of proteins, exhibiting good biocompatibility and low immunogenicity. Therefore, the safety and biocompatibility of amino acid carbon dots in vivo are further enhanced.

[0004] The main methods for preparing carbon dots include hydrothermal and pyrolysis methods. Currently, amino acid carbon dots are mostly prepared using the hydrothermal method. The hydrothermal method for preparing amino acid carbon dots involves reacting amino acids and a carbon source under mild conditions through the action of chemical reagents. Specifically, the amino acid is dissolved in an aqueous solution with a specific pH, and then heated in a closed container such as a hydrothermal reactor to obtain the amino acid carbon dot product. This method produces amino acid carbon dots with good uniformity, but the high-pressure environment generated by water evaporation during the reaction poses a certain risk and is not conducive to large-scale production. Furthermore, some amino acids have poor solubility and cannot dissolve in water, thus making it impossible to prepare amino acid carbon dots using the hydrothermal method.

[0005] Therefore, providing a method for preparing amino acid carbon dots is of great significance. Summary of the Invention

[0006] The technical problem solved by this invention is to provide a method for preparing amino acid carbon dots. The preparation method provided by this application can prepare amino acid carbon dots with smaller particle size and better uniformity. Moreover, the method is simple and can achieve the preparation of amino acid carbon dots without a high-pressure environment.

[0007] This application provides a method for preparing amino acid carbon dots, comprising the following steps:

[0008] The amino acids were ground to obtain pre-treated amino acids;

[0009] The pre-treated amino acids were heated to obtain amino acid carbon dots.

[0010] In some specific embodiments, the grinding process further includes:

[0011] The amino acids are dried for 6 to 10 hours.

[0012] In some specific embodiments, the grinding time is 10 to 30 minutes.

[0013] In some specific embodiments, the heating temperature is 150~250℃, and the heating time is 3~10h.

[0014] In some specific embodiments, the heating temperature is 160~240℃, and the heating time is 4~7h.

[0015] In some specific embodiments, the heating process further includes:

[0016] The heated carbon dot mixture was subjected to secondary grinding, dissolution, centrifugation, filtration, dialysis, and freeze-drying in sequence.

[0017] In some specific embodiments, the secondary grinding time is 0.5~1 hour;

[0018] And / or, the dissolution specifically includes:

[0019] The amino acid carbon dot mixture after secondary grinding was ultrasonically dissolved for 30-50 minutes to obtain the first supernatant.

[0020] The centrifugation specifically refers to:

[0021] The first supernatant is centrifuged to obtain a second supernatant; the centrifugation time is 20-40 min and the centrifugation speed is 1000-1500 rpm.

[0022] The filtering specifically refers to:

[0023] The filter was sequentially filtered using a Buchner funnel and a microporous membrane with a particle size of 0.22 μm, and the filtration was performed 2 to 3 times.

[0024] Alternatively, a filter head with a pore size of 0.22 μm can be used for single-pass filtration;

[0025] The dialysis membrane used in the dialysis is a dialysis membrane with a density of 500~1000D, and the dialysis time is 12~24h.

[0026] In some specific embodiments, the particle size of the amino acid carbon dots is 2.0~3.5 nm.

[0027] In some specific embodiments, the amino acid includes methionine, selenomethionine, valine, proline, glutamine, or arginine.

[0028] This application also provides the application of the amino acid carbon dots prepared by the above preparation method or the amino acid carbon dots described in the above method in the preparation of drugs to relieve oxidative stress, drugs to delay aging, drugs to promote osteogenic formation, or antibacterial drugs.

[0029] This application also provides amino acid carbon dots prepared by the preparation method described above.

[0030] This application provides a method for preparing amino acid carbon dots. First, amino acids are ground to obtain pre-treated amino acids. Then, the pre-treated amino acids are heated to obtain amino acid carbon dots. In the preparation process of amino acid carbon dots, heating is performed directly after grinding the amino acids to obtain them. This preparation method does not require a high-pressure environment, thus improving safety and reducing the requirements for production equipment. Furthermore, this application allows for adjustments to the heating temperature and time to obtain amino acid carbon dots with smaller particle size and better uniformity. In addition, the amino acid carbon dots prepared by this application exhibit excellent biocompatibility and can be used as biological probes. Attached Figure Description

[0031] Figure 1 The particle size distribution and corresponding transmission electron microscope images (scale bar 20 nm) of valine carbon dots (Val-CD), methionine carbon dots (Met-CD), and selenomethionine carbon dots (Se-Met-CD) prepared for embodiments of the present invention are shown in Figure (A), Figure (B), and Figure (C).

[0032] Figure 2 Fourier transform infrared (FT-IR) spectra of Val-CD, Met-CD, and Se-Met-CD prepared for embodiments of the present invention.

[0033] Figure 3 FT-IR spectra of valine (Val), methionine (Met), and selenomethionine (Se-Met) used in the preparation of amino acid carbon dots in the embodiments of the present invention;

[0034] Figure 4 X-ray diffraction patterns of Val-CD, Met-CD, and Se-Met-CD prepared according to embodiments of the present invention;

[0035] Figure 5 The XRD patterns of Val, Met, and Se-Met used to prepare the amino acid carbon dots in the embodiments of the present invention are shown in Figure (A), which is the XRD pattern of Val, Figure (B) is the XRD pattern of Met, and Figure (C) is the XRD pattern of Se-Met.

[0036] Figure 6 The X-ray photoelectron spectrum of Val-CD prepared in this embodiment of the invention is shown in Figure (A), which is the full XPS spectrum of Val-CD; Figure (B) is the high-resolution C 1s XPS spectrum of Val-CD; Figure (C) is the high-resolution N 1s XPS spectrum of Val-CD; and Figure (D) is the high-resolution O 1s XPS spectrum of Val-CD.

[0037] Figure 7 The X-ray photoelectron spectrum of the Met-CD prepared in this embodiment of the invention is shown in Figure (A), which is the full XPS spectrum of the Met-CD; Figure (B) is the high-resolution S 2p XPS spectrum of the Met-CD; Figure (C) is the high-resolution C 1s XPS spectrum of the Met-CD; Figure (D) is the high-resolution N 1s XPS spectrum of the Met-CD; and Figure (E) is the high-resolution O 1s XPS spectrum of the Met-CD.

[0038] Figure 8 The X-ray photoelectron spectra of the Se-Met-CD prepared in this embodiment of the invention are shown in Figure (A), which is the full XPS spectrum of the Se-Met-CD; Figure (B) is the high-resolution XPS Se 3d spectrum of the Se-Met-CD; Figure (C) is the high-resolution XPS C 1s spectrum of the Se-Met-CD; Figure (D) is the high-resolution XPS N 1s spectrum of the Se-Met-CD; and Figure (E) is the high-resolution XPS O 1s spectrum of the Se-Met-CD.

[0039] Figure 9 The UV-Vis absorption spectra of the amino acid carbon dots (CDs) aqueous solution prepared in the embodiments of the present invention are shown in Figure (A), which is the UV-Vis absorption spectrum of Val-CD, Figure (B) is the UV-Vis absorption spectrum of Met-CD, and Figure (C) is the UV-Vis absorption spectrum of Se-Met-CD.

[0040] Figure 10 The photoluminescence spectra of the CDs aqueous solution prepared in the embodiments of the present invention are shown in Figure (A), which is the photoluminescence spectrum of the Val-CD aqueous solution, Figure (B) is the photoluminescence spectrum of the Met-CD aqueous solution, and Figure (C) is the photoluminescence spectrum of the Se-Met-CD aqueous solution.

[0041] Figure 11The excitation and emission peaks of Val-CD, Met-CD and Se-Met-CD prepared in the embodiments of the present invention are shown in Figure (A) as the excitation and emission peaks of Val-CD, Figure (B) as the excitation and emission peaks of Met-CD, and Figure (C) as the excitation and emission peaks of Se-Met-CD.

[0042] Figure 12 The following are bar charts showing the cell viability results detected by the CCK-8 assay after treatment with different concentrations (0 to 100 μg / mL) of CDs according to the present invention; Figure (A) is a bar chart of cell viability results for Val-CD, Figure (B) is a bar chart of cell viability results for Met-CD, and Figure (C) is a bar chart of cell viability results for Se-Met-CD.

[0043] Figure 13 These are representative confocal microscopy images (the images are superimposed images of fluorescence channels and bright field channels) of NPCs taking up 100.0 μg / mL of three CDs in the embodiments of the present invention.

[0044] Figure 14 This invention provides an embodiment of the ImageJ software for... Figure 13 The bar charts show the quantitative analysis of integrated fluorescence intensity of Val-CD (A), Met-CD (B), and Se-Met-CD (C). Figure (A) is a bar chart of integrated fluorescence intensity of Val-CD, Figure (B) is a bar chart of integrated fluorescence intensity of Met-CD, and Figure (C) is a bar chart of integrated fluorescence intensity of Se-Met-CD.

[0045] Figure 15 These are bright-field and confocal microscopy images of cellular uptake of Val-CD, Met-CD, and Se-Met-CD in embodiments of the present invention;

[0046] Figure 16 Images of Calcein AM / PI staining in cells pretreated with CDs under H2O2 stimulation; live cells show green fluorescence (Calcein AM), while dead cells show red fluorescence (PI).

[0047] Figure 17 To detect the ROS level in NPCs after co-culturing H2O2 with each carbon point using fluorescent probes (the image is an overlay of the fluorescence channel and the bright field channel).

[0048] Figure 18 To use ImageJ software for Figure 17 Quantitative analysis of ROS levels was performed; blue represents Val-CD, brown represents Met-CD, and green represents Se-Met-CD;

[0049] Figure 19 The images show DR images of the rat caudal vertebrae 4 weeks after treatment. The images are representative DR images of all groups at the 4-week time point. The red box indicates the Co7 / 8 intervertebral disc space.

[0050] Figure 20 The figure shows the disc height index (DHI) at week 4. The image shows the DHI (%) of all treatment groups relative to the normal group after normalization to 1 (the DHI value of the normal group at week 4 postoperatively).

[0051] Figure 21 The images show DR images of the rat caudal spine 8 weeks after treatment. The images are representative DR images of all groups at the 8th week time point. The red box indicates the Co7 / 8 intervertebral disc space.

[0052] Figure 22 The image shows the DHI at week 8, with the DHI value of the normal group at week 8 post-surgery being 100%, and the DHI (%) of all treatment groups relative to the normal group after normalization.

[0053] Figure 23 T2-weighted MRI images and corresponding pseudocolor images at week 4. The images are representative MRI scans of the Co7 / 8 intervertebral discs in all groups at week 4 postoperatively. The Co7 / 8 intervertebral disc space is marked in red. Pseudocolor was applied to the MRI scans to enhance visualization.

[0054] Figure 24 Pfirrmann quantitative scores were obtained for all groups to be evaluated based on T2-weighted MRI scans at the week 4 endpoint.

[0055] Figure 25 T2-weighted MRI images and corresponding pseudocolor images at week 8 postoperatively. The images are representative MRI scans of the Co7 / 8 intervertebral discs in all groups at week 8 postoperatively. The red box indicates the Co7 / 8 intervertebral disc space. Pseudocolor was applied to the MRI scans to enhance visualization.

[0056] Figure 26 Pfirrmann quantitative scores were obtained for all groups to be evaluated based on T2-weighted MRI scans at the week 8 endpoint.

[0057] Figure 27 H&E and Safranin O-Fix Green staining images of Co7 / 8 intervertebral disc sections from all groups 4 weeks post-operation;

[0058] Figure 28 To determine the histological quantitative score of the intervertebral disc at week 4, histological grading was performed on stained sections of all experimental groups. The lower the score, the healthier the intervertebral disc structure.

[0059] Figure 29 H&E and Safranin O-Fix Green staining images of Co7 / 8 intervertebral disc sections from all groups 8 weeks post-operation;

[0060] Figure 30 To determine the histological quantitative score of the intervertebral disc at week 8, histological grading was performed on stained sections of all experimental groups. The lower the score, the healthier the intervertebral disc structure.

[0061] Figure 31 Acan immunohistochemical staining and Col-II immunohistochemical staining of Co7 / 8 intervertebral disc sections from all groups 8 weeks post-operation;

[0062] Figure 32 For Pro-CD particle size analysis, (A) Representative image of Pro-CD transmission electron microscopy, (B) Pro-CD particle size distribution;

[0063] Figure 33 FTIR spectra of Pro CD and Pro precursor;

[0064] Figure 34 XRD patterns of Pro CD and Pro precursor;

[0065] Figure 35 The following are XPS spectra of Pro-CD: (A) XPS full spectrum scan of Pro-CD; (B) XPS energy spectrum of Pro-CD C 1s; (C) XPS energy spectrum of Pro-CD O 1s; (D) XPS energy spectrum of Pro-CD N 1s.

[0066] Figure 36 The optical properties of Pro-CD; (A) Photoluminescence spectrum of Pro-CD; (B) Excitation and generation peaks of Pro-CD; (C) UV-Vis absorption spectrum of Pro-CD;

[0067] Figure 37 The antioxidant capacity of Pro-CD and Pro; (A) ABTS test results of Pro-CD and Pro; (B) DPPH scavenging capacity test results of Pro-CD and Pro;

[0068] Figure 38 (A) Representative images of SA-β-Gal in each group; (B) Quantitative analysis of SA-β-Gal;

[0069] Figure 39Immunofluorescence images for Pro-CD to slow down cell senescence; (A) Immunofluorescence images of γH2A.X, MMP-13, Col II and Acan in each group; (B) Quantitative analysis of γH2A.X fluorescence intensity in each group; (C) Quantitative analysis of MMP-13 fluorescence intensity in each group; (D) Quantitative analysis of Col II fluorescence intensity in each group; (E) Quantitative analysis of Acan fluorescence intensity in each group;

[0070] Figure 40 DR assessment of IVDD progress at various time points; (A) DR images at 1, 4 and 8 weeks; (B) DHI heat maps at 1, 4 and 8 weeks for each group;

[0071] Figure 41 MRI assessment of IVDD progression at various time points; (A) MRI images and pseudocolor images of each group at 1, 4 and 8 weeks; (B) Pfirrman grade thermograms of each group at 1, 4 and 8 weeks.

[0072] Figure 42 Representative images of each group stained with H&E and Safranin O / Fast Green at weeks 1, 4 and 8;

[0073] Figure 43 (A) Histological quantitative scores of each group at 1, 4 and 8 weeks; (B) Statistical analysis of the differences in histological quantitative scores of each group at 1, 4 and 8 weeks.

[0074] Figure 44 Immunohistochemical staining of Col II and Acan in each group at 4 and 8 weeks;

[0075] Figure 45 Particle size distribution and TEM image of Gln-CD (scale bar: 20 nm);

[0076] Figure 46 The image shows the FTIR spectrum of the Gln-CD.

[0077] Figure 47 XPS spectral image of Gln-CD;

[0078] Figure 48 To promote the proliferation of BMSCs treated with different concentrations of Gln-CD (A), Pro-CD (B), and Gln (C);

[0079] Figure 49 To perform ARS staining on BMSCs without drugs, with or without Gln or Gln-CD treatment;

[0080] Figure 50 To perform ALP staining on BMSCs without drugs, with or without Gln or Gln-CD treatment;

[0081] Figure 51 To perform qPCR analysis of osteogenic-related genes (positive) in BMSCs under drug-free, Gln, or Gln-CD treatment;

[0082] Figure 52 To perform qPCR analysis of osteogenic-related genes in BMSCs (negative) under drug-free, Gln, or Gln-CD treatment;

[0083] Figure 53 To perform Western blot analysis of osteogenic-related genes in BMSCs under drug-free, Gln, or Gln-CD treatment;

[0084] Figure 54 To perform semi-quantitative analysis of osteogenic-related genes in BMSCs using Western blot under drug-free, Gln, or Gln-CD treatment;

[0085] Figure 55 Representative immunofluorescence images of Runx2 and Ocn in BMSCs under drug-free, Gln, or Gln-CD treatment;

[0086] Figure 56 Semi-quantitative analysis of representative immunofluorescence images of Runx2 and Ocn in BMSCs under drug-free, Gln, or Gln-CD treatment;

[0087] Figure 57 Representative immunofluorescence images of Col1a1 in BMSCs on days 3, 7, and 14 under drug-free, Gln, or Gln-CD treatment;

[0088] Figure 58 Semi-quantitative analysis of representative immunofluorescence images of Col1a1 in BMSCs on days 3, 7, and 14 under drug-free, Gln, or Gln-CD treatment;

[0089] Figure 59 The experimental procedure for a rat tibial osteomyelitis model;

[0090] Figure 60 Six independent replicate experiments were conducted to obtain three-dimensional CT images of the tibia of rats in different groups at 0, 4 and 8 weeks.

[0091] Figure 61For (A) bone volume (BV) analysis, (B) bone volume / tissue volume (BV / TV) analysis, (C) trabecular thickness (Tb.Th) analysis, and (D) trabecular number (Tb.N) analysis, all statistical data are presented as mean ± standard deviation (SD; n = 6; NS, no significant difference; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0092] Figure 62 Masson staining was performed on tissue sections at 4 and 8 weeks post-treatment (bone tissue stained blue, collagen stained red); collagen is indicated by yellow arrows. Scale bars: 1000 μm and 100 μm.

[0093] Figure 63 For semi-quantitative analysis of Masson staining of tissue sections at 4 and 8 weeks post-treatment, all statistical data are presented as mean ± standard deviation (SD; n = 3; NS, no significant difference; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0094] Figure 64 Immunohistochemical staining of BMP2 (A) and ATF4 (B) in tissue sections 8 weeks after treatment. Scale bar: 1000 μm and 100 μm.

[0095] Figure 65 To perform semi-quantitative analysis of immunohistochemical staining of BMP2 (A) and ATF4 (B) in tissue sections at 8 weeks post-treatment, all statistical data are presented as mean ± standard deviation (SD; n = 3; NS, no significant difference; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0096] Figure 66 The particle size distribution of Lys-CD and the corresponding TEM image (scale bar: 20 nm).

[0097] Figure 67 XPS spectral image of Lys-CD;

[0098] Figure 68 Particle size distribution and corresponding TEM images of L-Arg-CD (A) and D-Arg-CD (B) (scale bar: 20 nm).

[0099] Figure 69 XPS spectral images of L-Arg-CD (A) and D-Arg-CD (B);

[0100] Figure 70 The minimum inhibitory concentrations of bacteria after treatment with different concentrations (0 to 500 μg / mL) of L-Lys-CD and L-Arg-CD were determined. All statistical data are expressed as mean ± standard deviation.

[0101] Figure 71 (A) The minimum inhibitory concentrations of D-Lys-CD and D-Arg-CD against Staphylococcus aureus were detected; (B) The minimum inhibitory concentrations of D-Lys-CD and D-Arg-CD against Escherichia coli were detected; (C) The minimum inhibitory concentrations of D-Lys and D-Arg were detected.

[0102] Figure 72 These are live and dead bacteria stained after treatment with different concentrations of L-Arg-CD;

[0103] Figure 73 The image is a field emission scanning electron microscope image of Staphylococcus aureus after co-incubation with antibacterial CD.

[0104] Figure 74 Field emission scanning electron microscope images of Escherichia coli after co-incubation with antibacterial CD;

[0105] Figure 75 The cell membrane permeability of Staphylococcus aureus and Escherichia coli was measured.

[0106] Figure 76 H&E staining of tissue sections was performed at 4 and 8 weeks post-treatment. Scale bar: 1000 μm and 100 μm.

[0107] Figure 77 To perform semi-quantitative analysis of H&E staining of tissue sections at 4 and 8 weeks post-treatment, all statistical data are presented as mean ± standard deviation (SD; n = 3; NS, no significant difference; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0108] Figure 78 Gram staining of tissue sections was performed at 4 and 8 weeks post-treatment. Scale bar: 1000 μm and 100 μm.

[0109] Figure 79 To perform semi-quantitative analysis of Gram staining of tissue sections at 4 and 8 weeks post-treatment, all statistical data are presented as mean ± standard deviation (SD; n = 3; NS, no significant difference; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0110] Figure 80Gram staining of tissue sections (longitudinal sections) was performed 8 weeks after treatment. Scale bar: 1000 μm and 100 μm.

[0111] Figure 81 To perform semi-quantitative analysis of Gram staining of tissue sections (longitudinal sections) 8 weeks after treatment, all statistical data are presented as mean ± standard deviation (SD; n = 3; NS, no significant difference; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0112] Figure 82 Image showing the CFU count of Staphylococcus aureus after homogenization of infected tissue;

[0113] Figure 83 For the quantitative analysis of Staphylococcus aureus CFU count after homogenization of infected tissue, all statistical data are expressed as mean ± standard deviation (SD; n = 3; NS, no significant difference; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Detailed Implementation

[0114] To further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and not for limiting the scope of the claims of the present invention.

[0115] In view of the high risk associated with preparing amino acid carbon dots under high pressure in existing technologies, this application provides a method for preparing amino acid carbon dots that uses direct heating to prepare amino acid carbon dots with smaller particle size and better uniformity, simplifying the preparation method. Specifically, this invention discloses a method for preparing amino acid carbon dots, including the following steps:

[0116] The amino acids were ground to obtain pre-treated amino acids;

[0117] The pre-treated amino acids were heated to obtain amino acid carbon dots.

[0118] In the method for preparing amino acid carbon dots provided in this application, in order to ensure the smooth progress of the reaction, the amino acids are preferentially dried for 6-10 hours. Specifically, the drying is carried out in a vacuum drying oven for 8-9 hours.

[0119] After drying, the carbon dots are ground to obtain pre-treated amino acids. The grinding can be carried out in an agate mortar for 10-30 minutes, more specifically, for 15-20 minutes. The grinding can also be carried out in a ball mill.

[0120] This application then heats the pre-treated amino acids at a temperature of 150-250°C for 3-10 hours, specifically at 160-240°C for 4-7 hours, and more specifically at 180-220°C for 5-6 hours. At higher temperatures, the amino acids will be over-carbonized, forming an insoluble brown to black powder; at lower temperatures, the reaction may not occur or may be incomplete, resulting in no product being obtained after final freeze-drying.

[0121] The preparation method provided in this application is applicable to all amino acids. In specific embodiments, different amino acid carbon dots were prepared using methionine (Met), selenomethionine (Se-Met), valine (Val), proline (Pro), glutamine (Gln), or arginine (Arg) as examples. The heating temperature and heating time differ for different amino acids. Specifically, the heating temperature for methionine was 180℃ for 7 hours, the heating temperature for arginine was 240℃ for 3 hours, the heating temperature for proline was 240℃ for 5 hours, the heating temperature for valine was 220℃ for 5 hours, and the heating temperature for selenomethionine was 160℃ for 3 hours. The above heating can be carried out in an oven or by other heating methods such as a muffle furnace, tubular furnace, or hot plate.

[0122] Following the aforementioned heating, this application preferably further purifies the obtained amino acid carbon dots by sequentially performing secondary grinding, dissolution, centrifugation, filtration, dialysis, and freeze-drying to separate the insoluble portion and large carbonized particles from the amino acid carbon dot mixture. Specifically, the secondary grinding involves:

[0123] The heated amino acid carbon dot mixture is placed in an agate mortar treated with liquid nitrogen and ground at a low temperature for 0.5 to 1 hour. The grinding can also be performed in a low-temperature ball mill.

[0124] The dissolution process specifically employs ultrasonic dissolution, which involves using an ultrasonic instrument to dissolve the ground amino acid carbon dot mixture at low temperature. After ultrasonication for 30 minutes, the undissolved solid at the bottom layer is removed to obtain the supernatant.

[0125] The centrifugation specifically involves centrifuging the supernatant obtained above to obtain a second supernatant. The centrifugation time is 20-40 minutes, and the centrifugation speed is 1000-1500 rpm. Specifically, the centrifugation time is 30-35 minutes, and the centrifugation speed is 1200-1400 rpm. This centrifugation process aims to separate impurities from amino acid carbon dots in the amino acid carbon dot mixture within the supernatant.

[0126] Furthermore, the second supernatant is filtered to remove large particles from the amino acid carbon dots. The filtration process specifically involves:

[0127] The filter was sequentially filtered using a Buchner funnel and a microporous membrane with a particle size of 0.22 μm, and the filtration was performed 2 to 3 times.

[0128] Alternatively, a filter head with a pore size of 0.22 μm can be used for single-pass filtration.

[0129] Subsequently, the filtered amino acid carbon dots are dialyzed to separate small impurities and amino acid monomers. Specifically, the supernatant obtained from filtration is dialyzed using a 500-1000D dialysis membrane for 12-24 hours, with water changed every 2 hours. Furthermore, the dialysis can also be performed using ultrafiltration tubing.

[0130] Following the above process, the dialyzed amino acid carbon dots are finally freeze-dried to separate the residual water from the amino acid carbon dots, obtaining high-purity amino acid carbon dots. The freeze-drying process involves first freeze-drying the carbon dots into a solid state, and then placing it in a vacuum freeze dryer for further freeze-drying. In some specific embodiments, rotary evaporation can be used as an alternative to or supplementary method for separating the carbon dots from the water.

[0131] The amino acid carbon dots prepared in this application have a particle size of 2.0~3.5nm and high uniformity.

[0132] Furthermore, this application also provides amino acid carbon dots prepared by the above preparation method.

[0133] Due to the differences in amino acid raw materials, the prepared amino acid carbon dots are different. For example, methionine is prepared into methionine carbon dots, selenomethionine into selenomethionine carbon dots, valine into valine carbon dots, proline into proline carbon dots, glutamine into glutamine carbon dots, and arginine into arginine carbon dots.

[0134] This application also provides the application of the above-mentioned amino acid carbon dots in the preparation of drugs to relieve oxidative stress, drugs to delay aging, drugs to promote osteogenic formation, or in the preparation of antibacterial drugs.

[0135] In some specific embodiments, methionine carbon dots and selenomethionine carbon dots are used in the preparation of drugs to alleviate oxidative stress; specifically, the preparation of drugs to alleviate oxidative stress is for the treatment of intervertebral disc degeneration. In some specific embodiments, proline carbon dots are used in the preparation of drugs to delay aging. In some specific embodiments, glutamine carbon dots are used in the preparation of drugs to promote osteogenic growth. In some specific embodiments, lysine carbon dots and arginine carbon dots are used in antibacterial and anti-inflammatory drugs.

[0136] This application provides a method for preparing amino acid carbon dots. The method involves direct heating to form carbon dots from amino acid monomers under high-temperature conditions. The carbon dots are then purified through grinding, dissolution, centrifugation, and filtration to obtain high-purity amino acid carbon dots. This method is simple, efficient, and avoids the high-pressure environment required for hydrothermal carbon dot preparation, thus improving safety. The reduced requirements for production equipment also facilitate wider adoption and large-scale production. Furthermore, by adjusting the heating time and temperature, different types of amino acid carbon dots can be prepared, allowing them to exert their biological functions related to amino acid structure. These carbon dots exhibit excellent biocompatibility and can be used as biological probes.

[0137] To further understand the present invention, the preparation method of amino acid carbon dots provided by the present invention will be described in detail below with reference to the embodiments. The scope of protection of the present invention is not limited by the following embodiments.

[0138] Example 1: Preparation of amino acid carbon dots

[0139] 1) Dry the amino acid raw material in a vacuum drying oven for 8 hours, take out the dried amino acid raw material, weigh the required weight of amino acids for the experiment and put them into an agate mortar, grind for 15~20 minutes.

[0140] 2) Place the ground amino acids into a crucible and heat it in an oven. Keep track of the time while heating. Immediately after heating, remove the synthesized carbon dots and cool them to terminate the reaction and obtain a mixture of carbon dots.

[0141] The reaction temperature and reaction time used to synthesize different carbon dots are different. The reaction temperature is 150℃~240℃ and the heating time is 3h~10h. The reaction temperature and reaction time of different amino acids are shown in Table 1.

[0142] Table 1. Reaction temperature and reaction time data for different amino acids.

[0143]

[0144] 3) Separate the insoluble portion and large carbonized particles from the carbon dot mixture obtained in step 2) using methods such as grinding, dissolving, ultrasonication, and filtration; the specific steps are as follows:

[0145] Grinding and dissolving with double-distilled water: Take the carbon dot mixture obtained in step 2) out of the crucible and put it into an agate mortar treated with liquid nitrogen. Grind the carbon dot mixture finely at low temperature (about 30 minutes). After grinding into small particles, add double-distilled water to dissolve and continue grinding.

[0146] Ultrasonic dissolution: The ground amino acid carbon dots were dissolved by ultrasonication at low temperature. After sonication for 30 minutes, the undissolved solids in the lower layer were removed, and the supernatant was transferred into a 50ml centrifuge tube.

[0147] Centrifugation to separate carbon dots and impurities: The supernatant containing amino acid carbon dots was centrifuged for 30 minutes at a speed of 12,000 rpm, and the supernatant was collected after centrifugation.

[0148] Filtration: The supernatant above is filtered three times using a Buchner funnel and a microporous membrane (0.22 μm), or filtered once using a filter head with a pore size of 0.22 μm.

[0149] Carbon dot dialysis: Take out the supernatant after filtration, and perform dialysis on the liquid after filtration. Select a 500~1000D dialysis membrane, and perform dialysis for 24 hours, changing the water every 2 hours.

[0150] Carbon dot freeze-drying: The dialyzed carbon dot solution is placed in a refrigerator and freeze-dried into a solid. Then, it is placed in a vacuum freeze dryer to separate the carbon dots from the water by vacuum freeze-drying, thus obtaining high-purity powdered carbon dots.

[0151] Different amino acid carbon dots were prepared according to the above scheme. The reaction temperature and time for different amino acids are shown in Table 1. Other preparation conditions remained unchanged as described above.

[0152] Example 2 Biological applications of selenomethionine, methionine, and valine

[0153] The technical effects of selenomethionine carbon dots (Se-Met-CD), methionine carbon dots (Met-CD), and valine carbon dots (Val-CD) are explained, and their biological applications are explored.

[0154] The structure of amino acid CDs was characterized by observing their morphology, size and dispersion using transmission electron microscopy (TEM).

[0155] Its crystal structure and degree of graphitization were analyzed using X-ray diffraction (XRD).

[0156] The chemical functional groups present on its surface were identified using Fourier transform-infrared (FT-IR) spectroscopy.

[0157] The composition, relative abundance, and chemical valence state of the surface elements were determined using X-ray photoelectron spectroscopy (XPS).

[0158] First, the morphology and particle size of the amino acid carbon dots were characterized. The particle size of the carbon dots was obtained by transmission electron microscopy. The morphologies of the three amino acid carbon dots (CDs) are as follows: Figure 1 As shown, from Figure 1 (A) The average particle size of Val-CD measured in the figure is 2.44 nm. Figure 1 (B) The Met-CD measured in the figure is 2.94 nm. Figure 1 (C) shows that the Se-Met-CD is 3.08 nm. The similar morphology and size of the above amino acid carbon dots may be due to the consistency of the synthesis method and the structural similarity between the precursor amino acids. Compared with the amino acid carbon dots prepared by the hydrothermal method, the amino acid carbon dots prepared by the heating method of this application have smaller particle size and better uniformity.

[0159] To investigate the structural transformations during synthesis, the chemical structures of amino acid CDs and their precursors were analyzed using FT-IR. The FT-IR spectra of amino acid CDs are shown below. Figure 2 As shown, the FT-IR spectra of its precursor amino acids are as follows: Figure 3 As shown, by Figure 2 and Figure 3 It can be seen that the FT-IR spectrum of amino acid CDs is at 3425 cm⁻¹. -1 An absorption peak is observed at 2968 cm⁻¹, attributed to the stretching vibrations of the NH and OH bonds. -1 The absorption peak at 1587 cm⁻¹ corresponds to the stretching vibration of the CH bond, while the peak at 1587 cm⁻¹ corresponds to the stretching vibration of the CH bond. -1 The peak at 1510 cm⁻¹ is attributed to the stretching vibration of the C=O bond in the carboxyl group. -1 and 1395cm -1 The peaks at the positions are attributed to the bending vibrations of NH and CH bonds, respectively. The infrared absorption peaks of amino acid CDs and their corresponding precursors are basically in the same position, but the peak intensity of the pyrolysis product is weakened, indicating that some chemical bonds of the amino acid precursor have been broken and the number of functional groups has been reduced. These changes confirm that the amino acid has been successfully converted into carbon dots of carbon-based nanostructures after heating.

[0160] To confirm the formation of the graphitized carbon framework, a characteristic feature of CDs, further XRD analysis was performed, such as... Figure 4As shown, all three amino acid CDs exhibit a broad and weak diffraction peak between 20° and 30° (2θ), corresponding to the (002) crystal plane of graphite carbon, indicating that the CDs have been successfully synthesized. The peak of Val-CD appears at 24.7°, while the peaks of Met-CD and Se-Met-CD appear at 19.3° and 19.6°, respectively. The similarity in the diffraction peak positions of Met-CD and Se-Met-CD can be attributed to the high structural similarity of their precursor amino acids, which differ by only one atom. For comparison, XRD analysis was also performed on the precursor amino acids, such as... Figure 5 As shown, most of the peaks in the amino acid raw material have disappeared in the XRD results of CDs, which once again proves the successful synthesis of CDs.

[0161] To further investigate the surface elemental composition and chemical bonding state of CDs, XPS analysis was employed. Full-spectrum surveys confirmed that the chemical composition of each CD highly corresponded to the structure of its respective precursor amino acid, while high-resolution spectroscopy revealed the specific chemical states of key elements. Figure 6 As shown, no S or Se signal was detected for Val-CD, confirming the absence of heteroatom contamination in the sample. Its high-resolution C 1s spectrum can be decomposed into four peaks, corresponding to 284.8 eV (CC / C=C), 286.2 eV (CH), 286.9 eV (CO), and C=O (288.3 eV), respectively. The O 1s spectrum shows absorption peaks at 531.4 eV (C=O), 532.7 eV (CO), and 534.2 eV (adsorbed oxygen), with the relatively high CO ratio indicating that COC bridging bonds were formed through condensation reactions during carbonization. Analysis of the N 1s spectrum revealed multiple nitrogen configurations, including pyridine nitrogen, pyrrole nitrogen, and graphitic nitrogen, indicating that nitrogen atoms are embedded in the carbon skeleton in a pyrrole-like configuration.

[0162] like Figure 7 As shown, for Met-CD, its S 2p spectrum exhibits absorption peaks at 163.6 eV and 164.8 eV, which is attributed to the S 2p in the typical thioether structure. 3 / 2 The C 1s spectrum shows absorption peaks at 284.8 eV (CC / C=C), 286.5 eV (CN / CO), and 288.2 eV (C=O), consistent with those observed in Se-Met-CD. This result confirms that the S atoms from the Met precursor are covalently bonded to the carbon framework and have not undergone oxidation.

[0163] As shown in Figure 8, for Se-Met-CD, its high-resolution Se 3d spectrum shows a double peak at 54.2 eV, which is a characteristic peak of the Se-C bond. No signal was detected in the 58–60 eV range, ruling out the presence of oxidized Se and confirming that Se is stably incorporated into the structure in an organically bound state. Deconstruction of the C 1s spectrum revealed absorption peaks at 284.8 eV (CC / C=C), 286.1 eV (CN / CO / C-Se), and 288.3 eV (C=O), indicating that CDs have a partially graphitized carbon core and retain amino and carboxyl groups. The N 1s spectrum shows a double peak at 399.8 eV and 401.1 eV, corresponding to neutral amines (-NH2) and protonated amines (-NH3), respectively. + Functional groups. In summary, these XPS results indicate that the chemical structure of CDs is directly determined by their amino acid precursors.

[0164] The XPS results above collectively verify that the carbon dots of amino acids synthesized by the heating method can retain the special elements within the amino acids, thereby enabling them to perform their specific functions.

[0165] The optical properties of carbon dots (CDs) are crucial for their biomedical applications. Therefore, the amino acid carbon dots were characterized using UV-Vis absorption spectroscopy and photoluminescence (PL) spectroscopy.

[0166] like Figure 9 As shown, no obvious absorption peaks were observed in the UV-Vis spectra of Val-CD and Met-CD. In contrast, the UV-Vis spectrum of the Se-Met-CD aqueous solution showed a significant absorption peak at 320 nm, which may be attributed to n→π* transitions involving surface or molecular states (from carboxyl groups and / or CN / or C-Se). This absorption peak showed a slight blue shift compared to previously reported CDs derived from selenocysteine ​​(which had a similar absorption peak at about 340 nm). This difference in the position of the absorption peak can be attributed to the inherent structural differences between the two precursors and variations in synthetic conditions.

[0167] Furthermore, the wavelength-dependent photoluminescence excitation characteristic of CDs was investigated. For example... Figure 10 As shown, similar to other CDs, these three CDs exhibit typical excitation wavelength-dependent photoluminescence characteristics, meaning their fluorescence emission spectra shift with changes in the excitation wavelength; for example, when the excitation wavelength of Val-CD is changed from 300 nm to 460 nm, its emission peak red-shifts from 415 nm to 525 nm. This is a common optical phenomenon in CDs, further confirming the successful synthesis of these three CDs. Figure 10As shown, Val-CD exhibits the strongest photoluminescence intensity under 360 nm excitation, while Met-CD and Se-Met-CD both reach their strongest photoluminescence intensity under 390 nm excitation. Based on UV-Vis and fluorescence spectroscopy calculations, the QY values ​​for Se-Met-CD, Met-CD, and Val-CD are 5.4%, 7.1%, and 12.1%, respectively.

[0168] To accurately characterize the optical parameters of amino acid CDs, such as Figure 11 As shown, the excitation and emission spectra of each CD were measured. Val-CD has a distinct excitation peak at 360 nm, with a corresponding emission peak at 436 nm. In contrast, the optimal excitation wavelength for both Met-CD and Se-Met-CD is 390 nm, and their maximum emission peaks are both at 470 nm. The high similarity in optical behavior between Met-CD and Se-Met-CD may be due to the structural similarity of their precursor amino acids.

[0169] The biocompatibility of the synthesized amino acid CDs with NPCs was tested using the CCK-8 assay, such as... Figure 12 As shown, after co-incubation with CDs at concentrations ranging from 0 to 100 μg / mL for 24 hours, no significant decrease in cell viability was observed, and the survival rate was not statistically different from the control group (P>0.05). This result confirms that these three amino acid CDs all have good biocompatibility.

[0170] To assess the cellular uptake of amino acids by CDs and their potential bioprobe utility, intracellular fluorescence of (nucleus pulposus) NPCs after co-culturing with 100.0 μg / mL CDs was observed using laser confocal microscopy (CLSM). Figure 13 As shown, CLSM observed a stable and strong intracellular fluorescence signal, confirming that the cells successfully took up CDs.

[0171] Then, the integrated fluorescence intensity of the confocal image was quantized using ImageJ software, such as... Figure 14 As shown, the results indicate that all three CDs were effectively internalized by cells within 3 hours, with intracellular fluorescence intensity peaking at 9 hours, indicating that CDs were mainly localized in the cytoplasm. After 12 hours, the fluorescence intensity decreased slightly. Meanwhile, Se-Met-CD showed weaker fluorescence, attributed to its lower QY. The average fluorescence intensity of Met-CD was similar to that of Val-CD, possibly because the 488 nm laser line used for excitation was closer to the optimal excitation range of Met-CD. Complete imaging data are shown below. Figure 15 As shown.

[0172] To visually demonstrate the protective effect of CDs against oxidative stress-induced cell death, Calcein-AM / PI live / dead cell staining was performed. NPCs were treated with DMEM / F12 medium containing Se-Met-CD (10.0 μg / mL), Met-CD (60.0 μg / mL), or Val-CD (100.0 μg / mL), and simultaneously exposed to H2O2 at a final concentration of 300.0 μM. After 8 hours of incubation, the treatment medium was removed, and cell death rate was quantified. Cell staining results are shown in [image / image / details]. Figure 16 As shown in the figure, the Se-Met-CD treatment group at a concentration of 10.0 μg / mL exhibited the strongest protective effect, with a significantly reduced cell death rate of only 5.6% compared to the H2O2-only treatment group (mortality rate of 18.8%). Met-CD at 60.0 μg / mL also provided significant protection, with a mortality rate of 8.3%. In contrast, the effect of Val-CD was less pronounced, with a mortality rate of 15.1%. These findings are consistent with the results of CCK-8 activity assays, jointly highlighting the powerful cytoprotective role of Se-Met-CD against oxidative stress.

[0173] To determine whether the observed cell protection was attributable to ROS clearance, ROS levels within NPCs were measured. To analyze intracellular ROS levels, NPCs were treated with DMEM / F12 medium containing Se-Met-CD (10.0 μg / mL), Met-CD (60.0 μg / mL), Val-CD (100.0 μg / mL), or corresponding concentrations of amino acid precursors, and then co-incubated with H2O2. Intracellular ROS concentrations were then visualized using an intracellular ROS detection probe and CLSM. Figure 17 The image is an overlay of bright field and fluorescence channels, where green fluorescence represents the intracellular ROS level. As shown in the figure, after stimulation with H2O2, the intracellular fluorescence signal representing the ROS level was significantly enhanced, while the group treated with CDs showed varying degrees of fluorescence signal attenuation.

[0174] Then, the fluorescence intensity was quantified using ImageJ software. For example... Figure 18As shown, compared with the group treated with H2O2 alone, treatment with 100.0 μg / mL Val-CD, 60.0 μg / mL Met-CD, and 10.0 μg / mL Se-Met-CD reduced intracellular ROS levels to 33.8%, 25.1%, and 17.7%, respectively (all differences were statistically significant, P < 0.0001). In contrast, treatment with the three precursor amino acids did not lead to any significant reduction in ROS levels. These findings confirm that amino acid-derived CDs, especially Se-Met-CD, can effectively scavenge intracellular ROS under oxidative stress conditions.

[0175] Then, using a rat model of intervertebral disc degeneration, the study investigated the role of CDs in alleviating oxidative stress by scavenging reactive oxygen species. The treatment method involved physically mixing Met-CD and Se-Met-CD in a hydrogel carrier, which was then injected into the rat intervertebral discs. Disc degeneration was induced by needle puncture at the Co7 / 8 intervertebral disc. At the experimental endpoints of weeks 4 and 8, the degree of intervertebral disc narrowing and local bone destruction was assessed using DR (disc resection) to analyze the severity of disc degeneration. At week 4, as... Figure 19 As shown, compared with the normal group, the intervertebral space in the blank control group showed a certain degree of narrowing, while the narrowing in the hydrogel group, Met-CD / Gel group, and Se-Met-CD / Gel group was milder. Figure 20 As shown, the assessment of DHI% revealed that the DHI of the puncture-induced control group was 62.9% of that of the normal group, while the DHI% of the hydrogel group, Met-CD / Gel group and Se-Met-CD / Gel group remained at 68.7%, 67.7% and 75.5%, respectively.

[0176] By the end of the eighth week, as Figure 21 As shown, compared with the normal group, the control group exhibited more significant signs of intervertebral space narrowing and bone damage, accompanied by a significant decrease in DHI. Figure 22 As shown, the DHI% value of the blank control group was 44.6%, while the DHI% values ​​of the hydrogel group, Met-CD / Gel group, and Se-Met-CD / Gel group were 60.9%, 65.0%, and 71.2%, respectively. The hydrogel group preserved significantly more intervertebral disc height than the blank control group, and the DHI values ​​of both the Se-Met-CD / Gel group and the Met-CD / Gel group were significantly higher than those of the control group. However, no statistically significant difference in DHI was observed among the Se-Met-CD / Gel group, the Met-CD / Gel group, and the hydrogel group.

[0177] To assess the health of the intervertebral disc, T2-weighted MRI is used to evaluate the hydration and degeneration of the nucleus pulposus. Figure 23 As shown, at week 4, normal intervertebral discs exhibited high T2 signal intensity, while this signal was almost absent in the control group induced by disc degeneration. Hydrogel treatment alone preserved a weak T2 signal, while Se-Met-CD / hydrogel treatment preserved a significantly stronger signal intensity. We applied pseudocolor enhancement technology to better visualize the differences between groups. Figure 24 As shown, quantitative grading using the Pfirrmann grading system revealed that the mean gradations for the blank control group, hydrogel group, Met-CD / Gel group, and Se-Met-CD / Gel group were 4.3, 3.3, 2.7, and 2.3, respectively.

[0178] In week 8, the performance of each group was similar to that in week 4. Figure 25 As shown, normal intervertebral discs still exhibit high-intensity T2 signal, which was completely invisible in the blank control group. A weak T2 signal was observed in the hydrogel group. Both the Met-CD / Gel and Se-Met-CD / Gel groups showed strong signals, but the T2 signal in the Se-Met-CD / Gel group was more regular, exhibiting a complete circle. The Pfirrmann grading results are presented in… Figure 26 In the control group, hydrogel group, Met-CD / Gel group, and Se-Met-CD / Gel group, the mean Pfirrmann scores were 4.7, 3.7, 3.3, and 2.3, respectively. These results indicate that the treatment effect was significantly improved in the Se-Met-CD / Gel group.

[0179] Further histological analysis was performed to examine tissue morphology and ECM composition. Intervertebral disc sections were prepared at 4 and 8 weeks post-injection and stained with H&E and SOFG. Figure 27 As shown, at week 4, normal intervertebral discs exhibited a regular structure and clear nucleus pulposus boundaries, while in the control group, the nucleus pulposus tissue almost completely disappeared. The hydrogel group retained more proteoglycans, but its nucleus pulposus structure remained disordered. The Met-CD / Gel group maintained intervertebral disc height and partially restored the nucleus pulposus structure, while the Se-Met-CD / Gel group showed the most complete morphology and clear nucleus pulposus boundaries.

[0180] The degree of degeneration was quantified using a standardized histological scoring system for rat intervertebral disc degeneration. This scoring system assesses the morphology of the annulus fibrosus, the clarity of the boundary between the annulus fibrosus and the nucleus pulposus, the number of cells in the nucleus pulposus, and the condition of the nucleus pulposus matrix. Higher scores indicate more severe intervertebral disc degeneration. Figure 28As shown, in week 4, the mean score of the blank control group was 10.3, while the scores of the hydrogel group, Met-CD / Gel group and Se-Met-CD / Gel group were 8.7, 5.3 and 3.0, respectively.

[0181] The biopsy taken at 8 weeks post-surgery is as follows: Figure 29 As shown, the intervertebral discs in the normal group had regular structures and clear nucleus pulposus boundaries, while the intervertebral discs in the blank control group showed significant loss of height and almost complete disappearance of nucleus pulposus tissue. The hydrogel group retained a certain intervertebral disc height, but the nucleus pulposus structure was still disordered. The Met-CD / Gel group maintained the intervertebral disc height and partially restored the nucleus pulposus structure, while the Se-Met-CD / Gel group showed the most complete morphology and clear nucleus pulposus boundaries.

[0182] Histological scores at 8 weeks post-surgery: Figure 30 As shown, the histological score of the blank control group increased to 11.3, while the scores of the hydrogel group, Met-CD / Gel group, and Se-Met-CD / Gel group were 8.7, 5.7, and 4.0, respectively, indicating that the degeneration was continuing to progress. At the 4-week and 8-week postoperative time points, the scores of both CDs treatment groups were significantly lower than those of the blank control group, and Se-Met-CD showed the most significant relief effect on intervertebral disc degeneration.

[0183] To further investigate the anabolic state of the intervertebral disc at the protein level, IHC of Acan and Col II was performed on intervertebral disc sections. The results are as follows: Figure 31 As shown, the nucleus pulposus of the normal group exhibited strong and widespread positive staining for Acan and Col II, confirming high baseline expression levels of these matrix proteins. In stark contrast, the staining area and intensity of Acan and Col II were significantly reduced in the blank control group, indicating severe impairment of matrix synthesis. Although hydrogel therapy alone provided some relief, severe deficiency of Acan and Col II persisted in the hydrogel group, indicating that it was essentially insufficient to prevent matrix degradation. While the Met-CD / Gel group achieved partial recovery of Acan and Col II expression, the effect was significantly more pronounced in the Se-Met-CD / Gel group. Notably, the Se-Met-CD / Gel group demonstrated the strongest protective ability; by strongly upregulating the synthesis of its main structural proteins, Se-Met-CD / hydrogel effectively maintained ECM homeostasis, thereby delaying the overall progression of intervertebral disc degeneration.

[0184] Example 3: Characterization of proline carbon dots and its biological applications in delaying aging

[0185] The morphology of Pro CD prepared by pyrolysis was observed by transmission electron microscopy, such as... Figure 32As shown, the particles exhibit a uniform circular structure with a uniform particle size distribution and an average particle size of 2.17 ± 0.16 nm. The lattice spacing of 0.32 nm corresponds to the (002) plane of graphite carbon, thus verifying that the Pro precursor successfully formed crystalline carbon dots.

[0186] To further elucidate the functional group transitions and the evolution of characterization structures, such as Figure 33 As shown, FTIR spectral analysis was performed on Pro and the generated Pro-CD. The results show that the FTIR spectrum is at 3450 cm⁻¹. -1 A broad absorption band is observed at 2800–2900 cm⁻¹, which can be attributed to the overlap of OH and NH stretching vibrations, indicating that the hydrophilic surface groups and nitrogen-containing functional groups in the proline precursor are retained in Pro-CD. -1 The absorption peaks within this range correspond to the CH stretching vibrations of aliphatic hydrocarbons, reflecting the saturated carbon skeleton structure of the pyrrolidine ring. In Pro, the peak at 1650 cm⁻¹... -1 A distinct C=O stretching band exists at this point, which is a characteristic peak of a free carboxyl group. This peak disappears after carbonization, indicating that the C=O group underwent a condensation reaction with available NH or OH in the system. Simultaneously, at 1630–1660 cm⁻¹... -1 and 1500~1550 cm -1 New absorption bands appear in the region, attributed to vibrations of the amide I and amide II bands, respectively. These features are attributable to the C=O bonds in the newly formed amide and ester bonds during carbonization, as well as the C=C stretching vibrations in the conjugated aromatic domains. (1510 cm⁻¹) -1 and 1330 cm -1 The peaks at these locations correspond to the NH bending vibration and the C−N stretching vibration, respectively.

[0187] XRD analysis was performed to confirm the formation of the graphitized carbon framework, which is a characteristic feature of carbon dots. The results are as follows: Figure 34 The results showed a broad diffraction peak in the range of 20-30° at the 2θ angle, which is a characteristic peak of the graphitized carbon (002) crystal plane. This confirms that both graphitized microregions and functionalized surfaces are present in the Pro-CD.

[0188] X-ray photoelectron spectroscopy (XPS) was used to further analyze the chemical state and bonding configuration of Pro-CD. The results are as follows: Figure 35As shown, the elemental composition of Pro-CD is 73.07 at%, 12.36 at%, and 14.57 at%, which is highly consistent with the theoretical stoichiometry of Pro, indicating that nitrogen was effectively retained during carbonization. Peak fitting of the C 1s spectrum identified four characteristic components, which is consistent with the partial retention of carboxyl functional groups. The O 1s spectrum shows a peak corresponding to the O=CO group, indicating that some groups underwent dehydrogenation to form ether bonds. The N 1s spectrum exhibits two main forms: the main peak is a pyrrolidine-type secondary amine (-NH-, binding energy 399.6 eV), reflecting the preservation of the Pro cyclic structure; the secondary peak is a protonated amine (-NH2). + The binding energy (401.2 eV) indicates that some N−H participated in the condensation reaction, thus forming a newly formed conjugated network.

[0189] Pro-CD exhibits excitation wavelength-dependent photoluminescence, meaning its fluorescence emission spectrum shifts with changes in excitation wavelength, a typical characteristic of carbon dots. The results for this carbon dot experiment are as follows: Figure 36 As shown, when the excitation wavelength increases from 340 nm to 480 nm, the emission peak gradually redshifts from 440 nm to 540 nm. Further analysis indicates that the maximum excitation intensity occurs at 424 nm, the emission peak is located at approximately 512 nm, and the Stokes shift is approximately 88 nm. Subsequently, it was characterized using a UV-Vis spectrophotometer, and the results showed that a characteristic absorption band exists in the 290–320 nm range. This is attributed to the n→π* transition of the C=O group and the π−π transition of the conjugated C=C structure, which is consistent with the optical characteristics of carbon-based nanomaterials.

[0190] Given the sp² hybridized carbon core in the carbon dots and the resulting π-π conjugated structure, this structure enhances the electron-donating ability of the material. This study used ABTS and DPPH kits to further quantify the antioxidant capacity of Pro-CD and the Pro precursor. Results are as follows... Figure 37 As shown, Pro-CD exhibits stronger antioxidant capacity compared to the amino acid precursor. ABT test results showed that the scavenging rate of the Pro precursor was less than 5% at all concentrations, at 2000 μg / mL. -1 At a concentration of 2000 μg / mL, Pro-CD exhibited an ABTS scavenging rate 7.2 times that of the precursor. DPPH scavenging experiments further confirmed Pro-CD's superior health and wellness properties. -1 At the specified concentration, the DPPH scavenging rate was 13.8%, while that of the Pro precursor was only 1.1%, indicating almost no detectable activity.

[0191] Oxidative stress-induced cellular senescence triggers the senescence-associated secretory phenotype (SASP), negatively impacting cellular physiological function. To assess whether Pro-CD could alleviate oxidative damage-induced senescence, the overall cellular senescence status of different groups was first evaluated using the SA-β-Gal cellular senescence kit. Results are as follows: Figure 38 The results showed that oxidative damage increased the proportion of senescent cells in NPCs, while the addition of Pro-CD significantly reduced the proportion of positive cells. These results preliminarily suggest that Pro-CD alleviates cellular senescence caused by oxidative damage.

[0192] Increased cellular senescence is accompanied by increased DNA double-strand damage, which exacerbates genomic instability and DNA damage accumulation. Using γH2AX, we investigated whether Pro-CD could alleviate the increased DNA double-strand damage caused by cellular senescence. Pro-CD reduced DNA double-strand breaks to 82.81% of the control level. Furthermore, senescent NPCs reduce the secretion of Col II and Acan, substances crucial for maintaining the elasticity and hydration function of normal NPs. Senescent NPCs also secrete MMP13, promoting ECM degradation. To verify whether Pro-CD can maintain the normal secretory function of NPCs and ensure they are not affected by oxidative damage and senescence, immunofluorescence was used to further verify the levels of Col II, Acan, and MMP13 in NPCs. The results are as follows: Figure 39 As shown, Pro-CD significantly alleviated the downregulation of Col II and Acan expression caused by cellular senescence and further alleviated the abnormal degradation of ECM by reducing MMP13 expression. This result indicates that Pro-CD alleviates abnormal cellular secretory function caused by cellular senescence, can maintain the normal secretory function of NPCs, and thus maintain the stability of ECM components.

[0193] To verify its biological efficacy in delaying aging, a rat intervertebral disc degeneration model was used as a template. At 1, 4 and 8 weeks postoperatively, the IVD height and local bone structure of rats in each group were dynamically monitored using DR, and the degree of degeneration was quantified by calculating the intervertebral disc height index (DHI).

[0194] like Figure 40As shown, in the first week post-operation, the DHI in the control group decreased to 79.97% of the normal group, indicating that the puncture injury successfully induced early IVDD. The DHI in the GEL group was 82.48% of the normal group, showing a slight improvement compared to the control group, but the difference was not significant; while the DHI in the GEL / Pro-CD group remained at 96.34% of the normal group, demonstrating a good early protective effect. By the fourth week, the IVD height in each group further decreased: the DHI in the control group was only 61.10% of the normal group, the GEL group was 69.74%, and the GEL / Pro-CD group maintained a higher level, at 83.47% of the normal group. These results indicate that GEL hydrogel can delay the loss of IVD height to a certain extent, which may be attributed to the physical support provided by its high water content and its inherent ROS scavenging ability. The GEL / Pro-CD group showed a more significant height preservation effect, the advantage of which lies in the fact that Pro-CD, on the basis of GEL's ROS scavenging function, further exerts anti-cellular oxidative damage and anti-aging effects, thereby more effectively protecting NPC function and maintaining the structural integrity of the intervertebral disc.

[0195] like Figure 41 As shown, to further assess the progression of IVDD, this study used MRI T2-weighted sequences to dynamically monitor the water content of NP tissue in each group of rats, and semi-quantitatively assessed the degree of degeneration using the Pfirrmann grading system. MRI results showed that the acupuncture model successfully induced progressive IVDD. At week 1 post-surgery, a small amount of T2 high signal was still visible in the NP area of ​​the control group, indicating some water retention; by week 4, the water signal in the NP of the control group had almost completely disappeared, indicating rapid degeneration progression. At week 4, a small amount of residual water signal was still observed in the NP of the GEL-only treatment group, suggesting that the hydrogel could delay water loss to some extent, possibly related to the physical support provided by its high water content and its inherent ROS scavenging ability. Notably, the GEL / Pro-CD combined treatment group showed superior protective effects: good NP morphology and water retention were observed at week 1 post-surgery, and a relatively ideal NP water content was maintained at week 8, significantly better than the control group and the GEL-only treatment group at the same time point.

[0196] Quantitative Pfirrmann grading analysis further validated the above observations. At week 4, the mean Pfirrmann grades for the control group, GEL group, and GEL / Pro-CD group were 4.33, 4.00, and 3.00, respectively; at week 8, the mean grades for the three groups were 5.00, 4.00, and 3.33, respectively. These results indicate that the GEL / Pro-CD composite hydrogel can effectively delay the decline in NP hydration function during IVDD and maintain intervertebral disc height to a certain extent, demonstrating superior therapeutic potential compared to single hydrogels.

[0197] like Figure 42 As shown, to further characterize the microstructural changes within the IVD, this study used H&E staining and Safranin O / Fixed Green staining to perform histological evaluation of the surgical segment of the intervertebral disc, observing the overall tissue structure and the distribution of proteoglycans in the extracellular matrix. In the normal group, the IVD tissue structure was orderly, with clear NP boundaries, intact morphology, and a regular arrangement of the annulus fibrosus lamellar structure. Safranin O / Fixed Green staining showed abundant proteoglycans within the NP matrix. In contrast, the saline-treated group exhibited progressive structural destruction: at week 1 post-surgery, the boundary between the NP and the annulus fibrosus (AF) began to blur; by week 4, the NP region significantly shrank, and the annulus fibrosus lamellar structure became disordered; by week 8, the NP tissue was almost unrecognizable, the boundary between the annulus fibrosus and the nucleus pulposus completely disappeared, and the Safranin O staining signal was significantly weakened, indicating a large loss of proteoglycans. In the simple GEL hydrogel group, some NP structures were still observed to be preserved at week 1, and the NP morphology was still recognizable; however, by weeks 4 and 8, the NP tissue was no longer clearly distinguishable, the boundary between the AF and NP was blurred, and the Safranin O staining signal was significantly weakened. This result indicates that the physical support and inherent antioxidant activity of hydrogels alone are insufficient to provide long-term, sustained protection against IVDD. In stark contrast, the EGEL / Pro-CD composite hydrogel group exhibited excellent structure maintenance throughout the observation period: the NP morphology was intact and the boundaries were clear at week 1; a relatively complete NP structure was still visible at week 4; and at week 8, it maintained a clear NP morphology and well-defined AF boundaries, with good safranin O staining signal retention. These findings suggest that this composite hydrogel can effectively maintain the inherent microstructure of IVD and slow down the progression of IVDD.

[0198] like Figure 43 As shown, quantitative histological scoring further validated the above observations. The saline treatment group showed a progressive deterioration trend, with mean histological scores of 10.33, 11.00, and 11.67 at 1, 4, and 8 weeks, respectively. The GEL-only treatment group showed scores of 8.67, 9.33, and 10.00 at each time point, indicating some early protective effect, but this was difficult to maintain. The GEL / Pro-CD composite hydrogel group showed significant improvement in scores at the corresponding time points, at 5.00, 5.33, and 5.67, significantly better than the saline group and the hydrogel-only group at each time point. These results collectively confirm that the GEL / Pro-CD composite hydrogel can effectively reduce intervertebral disc degeneration and maintain the integrity of the tissue microstructure.

[0199] like Figure 44As shown, the protective effect was further confirmed by immunohistochemical staining of Col II and Acan in the NP region. In normal IVD tissue, the NP region showed strong positive staining for Col II and Acan, with brownish-yellow granules evenly distributed in the extracellular matrix, consistent with the structural characteristics of healthy NP tissue. In contrast, the saline-treated group showed almost complete loss of NP tissue, with extremely weak and sparse positive signals for Col II and Acan, only seen around a few remaining cells, consistent with the pathological characteristics of late-stage intervertebral disc degeneration.

[0200] In the GEL hydrogel-only group, some Col II and Acan positive areas were still observed at 4 weeks post-surgery, indicating that the hydrogel could provide some protection to the extracellular matrix in the short term. However, by week 8, the positive signal significantly weakened, failing to effectively halt subsequent matrix degradation and highlighting the limitations of single antioxidant therapy in maintaining matrix homeostasis in the long term. The GEL / Pro-CD composite hydrogel group, on the other hand, showed excellent matrix retention throughout the observation period: the intensity and distribution of Col II and Acan positive signals were significantly better than in the hydrogel-only group, maintaining relatively abundant matrix expression even at week 8. These results demonstrate that the GEL / Pro-CD composite hydrogel can effectively slow the loss of key matrix components in NP tissue, promote ECM remodeling, and restore matrix metabolic homeostasis, thus providing strong support for the structural and functional maintenance of IVD.

[0201] Comprehensive imaging and histological analysis results indicate that the GEL / Pro-CD composite hydrogel provides superior protection against IVDD compared to GEL hydrogel alone. This combined treatment strategy effectively maintains the hydration status of IVD, delays the significant loss of IVD levels, and restores matrix metabolic homeostasis by promoting the expression of key ECM components, thereby enhancing the balance between anabolism and catabolism. These in vivo observations are highly consistent with in vitro cell experiments, further confirming the important role of Pro-CD in alleviating NPC aging, inhibiting oxidative stress damage, and maintaining cell function.

[0202] Example 4: Characterization of glutamine carbon dots and biological verification of its osteogenic function

[0203] Glu-CD was prepared using the aforementioned method. The morphology of the glut-CD prepared by the pyrolysis method was observed using transmission electron microscopy, and the results are as follows. Figure 45 As shown, it exhibits a uniform circular structure with a uniform particle size distribution and an average particle size of 3 nm.

[0204] FT-IR test results are as follows Figure 46 As shown. In Gln-CD, most vibrational modes are preserved, indicating that the structure and function of the precursor are largely retained. 3332 cm-1 and 3192 cm -1 The peak values ​​are attributed to the tensile vibrations of OH and NH, respectively. 2959 cm⁻¹ -1 and 1420 cm -1 The vibrations correspond to asymmetric CH stretching and CH bending vibrations, respectively. However, at wavenumbers below 1680 cm⁻¹... -1 In this region, the peaks broaden significantly and their number decreases. (1040 cm⁻¹) -1 Up to 1400 cm -1 The vibrations between the atoms are related to the stretching of CN and CO in the aromatic heterocycles, further supporting the formation of heterocyclic polycyclic structures during the preparation of Gln-CD. L-Arg-CD, D-Arg-CD, and Pro-CD exhibit similar properties. CD and its corresponding precursors show infrared absorption peaks at almost the same positions, but the absorption intensity of the pyrolysis products is weaker, indicating that the original amino acids undergo bond breaking and the functional groups are reduced. These changes confirm the phenomenon of amino acids being transformed into carbon-based nanostructures.

[0205] XPS analysis results are as follows: Figure 47 As shown, Gln-CD possesses abundant surface functional groups, such as C–N / C–O bonds in C1, protonated amino groups in N1s, and carboxyl / hydroxyl / adsorbed oxygen groups in O1s. Furthermore, the basic amino groups help neutralize the local acidic microenvironment, stabilize osteoblast activity, and serve as chelating sites for calcium ions, initiating mineral deposition.

[0206] The proliferation of BMSCs was detected using CCK-8 assay. Results are as follows: Figure 48 As shown, Gln-CD most significantly promoted BMSC proliferation in a concentration-dependent biphasic mode: at low concentrations (250–1000 μg / mL), cell viability increased with culture time (by 40%). However, at high concentrations (>1000 μg / mL), proliferation was inhibited due to Gln-CD aggregation inducing excessive activation of mTORC1 and inhibition of mTORC2. In contrast, although free Gln showed some proliferative effects, its efficacy was inferior to Gln-CD at equivalent low concentrations. Pro-CD did not show significant biological effects.

[0207] Alizarin Red S staining results are as follows: Figure 49As shown, Gln-CD significantly regulated the metabolic activity of BMSCs. In the three groups, the control group showed only scattered light red mineralized nodules, indicating weak mineralization under basal culture conditions. The Gln group showed a significant increase in mineralized nodules, but its proliferation-promoting effect was not as strong as Gln-CD. In contrast, the Gln-CD group exhibited a wide and intense red mineralization zone, with a significantly increased number of mineralized nodules. Consistent with the Alizarin Red S results, alkaline phosphatase (ALP) staining was as follows... Figure 50 As shown, the Gln-CD group most strongly activated osteogenic differentiation of BMSCs. The control group only showed pale blue granular deposition. The free Gln group showed increased purplish-blue granules and plaque aggregation, but the staining remained scattered. However, the Gln-CD group showed dense, deep blue-purple precipitation throughout the field, with a tightly connected intercellular mineralization network, indicating excellent osteogenic potential.

[0208] The mechanism of action of Gln-CD was further explored. Analysis of the expression profiles of key bone formation genes using qPCR yielded the following results: Figure 51 The results showed that Gln-CD had a statistically significant enhancing effect on all five genes (Runx2, Ocn, Col1, Alp, and Bmp2). Its relative expression level was not only higher than that of the control group, but also significantly higher than that of the free Gln group.

[0209] In addition, the effects of Gln-CD on eight bone metabolism-related genes / proteins (Wnt3a, Axin2, Slc1a5, Slc7as, Yap1, Ctgf, Gls, and Glul) were systematically evaluated. Results are as follows: Figure 52 As shown, none of these targets were upregulated in the Gln-CD group.

[0210] To further verify whether osteogenic formation is mediated by BMP2, we performed Western blot analysis at the protein level, and the results are as follows: Figure 53 and Figure 54 As shown in the figure, this study clearly demonstrates that Gln-CD activates the BMP2 / SMAD / Runx2 signaling axis through metabolic and transcriptional pathways, driving bone formation and differentiation. Gln-CD first induces cellular metabolic stress, prompting the phosphorylation of the eukaryotic initiation factor 2α subunit (eIF2α) in stressed cells by protein kinases. This process significantly increases the expression of activated transcription factor 4 (ATF4), which travels to the BMP2 promoter region, driving BMP2 protein expression. Activation of the BMP2 ligand binds to the receptor on the BMP2 cell membrane, triggering the specific phosphorylation of SMAD1 / 5 / 8 phosphorylated R-SMADs to form a complex, and SMAD4 translocates to the nucleus.

[0211] Within the nucleus, the SMAD complex binds to the promoter of osteogenic genes via Runx2, promoting increased transcriptional expression. This cascade ultimately leads to Col1a1, Alp, and Ocn, resulting in mineral deposition. Immunofluorescence staining results are as follows... Figure 55 and 56 As shown, the expression levels of Runx2 and Ocn in BMSCs treated with Gln-CD were significantly increased compared to the other two groups. The expression levels of Col1a1 in BMSCs at different treatment times were also observed, and the results are as follows: Figure 57 and 58 As shown, it can be seen that Gln-CD can promote osteogenic differentiation in the early stage of cell incubation.

[0212] This study systematically evaluated the synergistic therapeutic effect of composite CD hydrogel (Gel / L-Lys-Gln-CD) on infectious bone defects in rats through multi-timepoint and multi-index in vivo experiments. The experimental design is as follows: Figure 59 As shown, a rat model of tibial osteomyelitis was successfully established and rats underwent group intervention.

[0213] To evaluate the in vivo bone regeneration properties of the composite CD hydrogel, such as Figure 60 The system shown evaluates the repair status of rat tibial defects using in vivo micro-CT scanning and skeletal morphology measurements. The reconstructed 3D CT images clearly demonstrate the bone repair process in different groups at weeks 0, 4, and 8. Regular ring-shaped bone defects appeared in all groups post-surgery. At 4 weeks post-treatment, the defect area remained clearly visible in the blank control group (control group), with only minimal marginal mineralization. The L-Lys-CD group was similar to the control group. The Gel / L-Lys-CD group showed relatively obvious bridging new bone formation. In contrast, the defect area in the Gel / L-Lys-Gln-CD group was mainly filled with high-density mineralized new bone.

[0214] By week eight, both groups showed significant bone repair. The control group, which received no effective treatment, exhibited extensive bone destruction accompanied by abscess formation. Despite antibacterial intervention in the L-Lys-CD group, complete bacterial eradication was not achieved, and osteomyelitis symptoms persisted. In contrast, the Gel / L-Lys-Lys-Gln-CD and Gel / L-Lys-Gln-CD groups, which utilized esterase-responsive hydrogels for CD delivery, achieved complete bacterial clearance due to the sustained-release properties of the hydrogels. Notably, the Gel / L-Lys-Gln-CD group showed almost complete repair of bone defects, with new bone density comparable to surrounding healthy bone tissue, restoration of cortical bone continuity, and good trabecular structure. The repair effect in this group was significantly superior to all other groups.

[0215] like Figure 61The bone morphology measurements further quantitatively confirmed these results. Regarding bone volume fraction (BV / TV), the composite hydrogel group maintained the highest values ​​at both weeks 4 and 8, significantly higher than the blank control group and the single-function hydrogel group, demonstrating its effective ability to promote mineralized bone matrix deposition. In terms of trabecular thickness (Tb.Th), the composite hydrogel group increased to 0.44 mm after 8 weeks, more than doubling the value at week 4, significantly exceeding the levels of other groups at the same time point. This indicates that the composite material not only increased bone quality but also promoted the maturation and compaction of new bone structures. The composite group also showed a continuously increasing trend in trabecular number (Tb.N), significantly higher than the control group and the L-Lys-CD group, proving that it can generate more new trabeculae by efficiently activating the bone formation process.

[0216] These results collectively indicate that the composite hydrogel loaded with bifunctional CDs exhibits significant osteogenic capacity in vivo, and its repair effect is significantly better than that of single-functional material groups and free drug groups.

[0217] To further investigate the in vivo bone formation properties and molecular mechanisms of the composite CD hydrogel, we systematically analyzed collagen deposition and key bone protein expression during rat bone defect repair using histological staining and immunohistochemistry (IHC) techniques.

[0218] Masson staining results at different time points (4 weeks and 8 weeks) Figure 62 This visually demonstrates the deposition of collagen fibers in each group. For example... Figure 63 As shown, for Figure 62 The results were semi-quantitatively analyzed. At 4 weeks post-surgery, the defect area in the control group was still primarily filled with fibrous tissue, showing sparse collagen deposition. The L-Lys-CD group and the Gel / L-Lys-CD group exhibited moderate collagen formation, but with disordered fibrous structure. In contrast, the gel / L-Lys-Gln-CD group displayed abundant, dense, and orderly arranged red collagen fibers. By 8 weeks, the collagen deposition area and density in the composite hydrogel group further increased, almost filling the entire defect area, with a morphology similar to normal bone tissue, significantly superior to all other groups.

[0219] Semi-quantitative analysis of collagen deposition quantitatively confirmed these observations: the relative collagen content in the composite hydrogel group was significantly higher at both 4 and 8 weeks, and the difference at 8 weeks compared with other groups was extremely significant. This indicates that collagen synthesis and secretion in the extracellular matrix are continuously and efficiently promoted, laying a solid structural foundation for subsequent mineralization.

[0220] like Figure 64 and 65As shown, to further elucidate the molecular mechanism, immunohistochemical and semi-quantitative analyses were performed on the key bone-derived regulatory protein BMP2 and its upstream transcription factor Atf4. BMP2 IHC showed the strongest and most widely distributed positive signal (brownish-yellow) in the bone defect area and surrounding bone matrix of the composite hydrogel group, indicating the highest expression level. Semi-quantitative analysis confirmed that its relative expression was significantly higher than other groups at both 4 and 8 weeks. Similarly, ATF4 IHC and its semi-quantitative analysis showed a consistent trend, with the composite hydrogel group exhibiting significantly superior ATF4 positivity and expression intensity.

[0221] These results collectively demonstrate that, at both the tissue morphology and molecular expression levels, the composite hydrogel loaded with bifunctional CDs (Gel / L-Lys-Gln-CD) strongly promotes osteogenic differentiation by significantly upregulating the Atf4-Bmp2 signaling axis. This effectively promotes collagen synthesis and orderly deposition—a major component of the bone matrix—ultimately accelerating the repair and maturation of bone defects.

[0222] Example 5: Carbon dot characterization of lysine and arginine and their antibacterial biological applications

[0223] Lys-CD was synthesized using the aforementioned method, and its TEM images and particle size statistics are as follows: Figure 66 As shown, its XPS analysis results are as follows: Figure 67 As shown.

[0224] Arg-CD was synthesized using the aforementioned method. Based on the difference in chirality of the amino acid raw materials, L-Arg-CD and D-Arg-CD were synthesized respectively. Their TEM images and particle size distribution results are shown below. Figure 68 As shown. Its XPS analysis results are as follows. Figure 69 As shown.

[0225] To analyze their antibacterial activity and in vitro mechanism, the minimum inhibitory concentration (MIC) values ​​of various antibacterial CDs against different strains (including Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus) were determined using the standard dilution method. The MIC values ​​for L-Lys-CD, D-Lys-CD, L-Arg-CD, and D-Arg-CD against Staphylococcus aureus were 200 μg / mL, 600 μg / mL, 300 μg / mL, and 600 μg / mL, respectively, while the MIC values ​​against Escherichia coli were 400 μg / mL, 600 μg / mL, 450 μg / mL, and 600 μg / mL, respectively. L-Lys and L-Arg did not show significant antibacterial activity, as shown in the results below. Figure 70 and 71 As shown in the figure. These results indicate that the antibacterial activity of L-type CD is significantly better than that of D-type CD.

[0226] The in vitro antibacterial activity and potential antibacterial mechanism of CD were further investigated. To assess changes in membrane integrity, Staphylococcus aureus was stained for live and dead cells using SYTO 9 / iodophor malondiamide. The results showed that the intensity of red PI fluorescence increased over time, indicating progressive membrane damage, as shown in the figure. Figure 72 As shown.

[0227] After two hours of L-Lys-CD treatment, no viable bacteria were detected. Further morphological examination using field emission scanning electron microscopy (FE-SEM) yielded the following results: Figure 73 and 74 As shown, the membranes of untreated *E. coli* and *Staphylococcus aureus* were intact and smooth. In contrast, after incubation with L-Lys-CD or L-Arg-CD, CDs were observed to aggregate significantly on the bacterial surface, accompanied by membrane shrinkage and rupture. The bacterial surface carried a negative charge.

[0228] To quantify the degree of membrane damage, an NPN probe was used to assess changes in membrane permeability. For example... Figure 75 As shown, the permeability of both bacterial strains treated with L-Lys-CD increased more than that of the L-Arg-CD group, with Staphylococcus aureus increasing by 45.8% and Escherichia coli by 27.4%. These results support the mechanism by which the highly positively charged L-Lys-CD exacerbates the destruction of the membrane phospholipid layer through strong electrostatic adsorption. Notably, Staphylococcus aureus lacks an outer membrane, and its permeability changes are more pronounced, indicating that its monolayer peptidoglycan structure is more susceptible to physical damage.

[0229] like Figure 76 and Figure 77 As shown, histopathological analysis revealed significant differences in the gradient of bone tissue repair and inflammatory status between the two groups at 4 and 8 weeks post-treatment. The control group consistently exhibited extensive inflammatory cell infiltration, tissue necrosis, and fibrosis. The free Lys-CD group (L-Lys-CD) showed some anti-inflammatory effect at 4 weeks, but inflammation control was incomplete at 8 weeks, with residual local abscess formation. The Lys-CD hydrogel group (Gel / L-Lys-CD) benefited from the sustained release effect, exhibiting superior anti-inflammatory efficacy compared to the free group, but new bone formation remained limited. In contrast, the composite CD hydrogel group (Gel / L-Lys-Gln-CD) essentially eliminated inflammation within 4 weeks, and abundant mature trabecular bone formation was observed at 8 weeks, with almost complete repair of the bone defect area. Semi-quantitative scoring confirmed that its histological repair score was significantly better than all other groups.

[0230] like Figures 78-81As shown, to accurately assess antibacterial efficacy, residual bacterial load was quantified by Gram staining. Gram staining revealed that the number of bacterial-positive areas gradually decreased over time in all treatment groups; however, the composite hydrogel group showed the lowest residual bacteria at both 4 and 8 weeks, indicating the best antibacterial effect.

[0231] like Figure 82 and Figure 83 As shown, the bacterial culture results also provided conclusive evidence: after 8 weeks of treatment, the colony-forming unit (CFU) count in the composite hydrogel group was significantly lower than that in all other groups, approaching a sterile state, while obvious colonies were still observed in the other groups.

[0232] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

[0233] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing amino acid carbon dots, comprising the following steps: The amino acids were ground to obtain pre-treated amino acids; The pre-treated amino acids were heated to obtain amino acid carbon dots.

2. The preparation method according to claim 1, characterized in that, The grinding process also includes: The amino acids are dried for 6 to 10 hours.

3. The preparation method according to claim 1, characterized in that, The grinding time is 10-30 min; and / or the heating temperature is 150-250℃, and the heating time is 3-10 h.

4. The preparation method according to claim 1 or 3, characterized in that, The heating temperature is 160~240℃, and the heating time is 4~7h.

5. The preparation method according to claim 1, characterized in that, The heating process also includes: The heated carbon dot mixture was subjected to secondary grinding, dissolution, centrifugation, filtration, dialysis, and freeze-drying in sequence.

6. The preparation method according to claim 5, characterized in that, The secondary grinding time is 0.5~1 hour; And / or, the dissolution specifically includes: The amino acid carbon dot mixture after secondary grinding was ultrasonically dissolved for 30-50 minutes to obtain the first supernatant. The centrifugation specifically refers to: The first supernatant is centrifuged to obtain a second supernatant; the centrifugation time is 20-40 min and the centrifugation speed is 1000-1500 rpm. The filtering specifically refers to: The filter was sequentially filtered using a Buchner funnel and a microporous membrane with a particle size of 0.22 μm, and the filtration was performed 2 to 3 times. Alternatively, a filter head with a pore size of 0.22 μm can be used for single-pass filtration; The dialysis membrane used in the dialysis is a dialysis membrane with a density of 500~1000D, and the dialysis time is 12~24h.

7. The preparation method according to any one of claims 1 to 6, characterized in that, The particle size of the amino acid carbon dots is 2.0~3.5 nm.

8. The preparation method according to claim 7, characterized in that, The amino acids include methionine, selenomethionine, proline, glutamine, or arginine.

9. The amino acid carbon dots prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the amino acid carbon dots prepared by the preparation method according to any one of claims 1 to 8 or the amino acid carbon dots according to claim 9 in the preparation of drugs to relieve oxidative stress, drugs to delay aging, drugs to promote osteogenic formation, or drugs to prepare antibacterial drugs.

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