A novel North American senna glycoside carbon dot, its preparation method, and its application in the preparation of regenerative materials for inflammatory bone defects.

Abstract

This invention discloses a novel Neo-Neo-Symplocosone carbon dot, its preparation method, and its application in the preparation of regenerative materials for inflammatory bone defects. The preparation method involves preparing a Neo-Neo-Symplocosone solution followed by a hydrothermal reaction. After cooling, the reaction solution is subjected to precipitation removal, filtration, dialysis, and freeze-drying. The concentration of Neo-Neo-Symplocosone in the solution is 5-20 mg / mL. The hydrothermal reaction conditions are 190-200℃ for 9.5-11.5 h. By converting Neo, which has poor water solubility and low bioavailability, into nanoscale carbon dots, the abundant functional groups on its surface significantly improve dispersion stability, providing a technical basis for delivery systems. Under LPS-simulated inflammatory conditions, it promotes osteogenic differentiation of hPDLSCs and simultaneously inhibits the release of pro-inflammatory factors, achieving a synergistic regulation of "osteoproliferative-anti-inflammatory".

Description

Technical Field

[0001] This invention relates to a novel North American sage glycoside carbon dot, its preparation method, and its application in the preparation of regenerative materials for inflammatory bone defects, belonging to the field of pharmaceutical technology. Background Technology

[0002] Periodontitis is a chronic infectious disease caused by plaque biofilm, and the resulting irreversible alveolar bone resorption is the main cause of tooth loss in adults. While conventional clinical treatments can control the progression of inflammation, they are insufficient to achieve complete regeneration of periodontal tissues. Human periodontal ligament stem cells (hPDLSCs), considered ideal seed cells for periodontal regeneration, face significant challenges in the inflammatory microenvironment of periodontitis. Plaque microbial residue and immune microenvironment dysregulation significantly inhibit their osteogenic differentiation capacity, becoming a core bottleneck hindering bone tissue repair.

[0003] Currently, technologies for promoting bone regeneration mainly include growth factor induction and gene therapy. While recombinant human bone morphogenetic protein-2 (BMP-2) and other growth factors can effectively induce osteogenic differentiation of stem cells, they suffer from drawbacks such as high production costs, short in vivo half-life, and a tendency to induce ectopic osteogenic formation. Gene therapy, on the other hand, faces challenges related to safety and ethical controversies. Natural small molecule compounds, due to their wide availability, low cost, and multi-target regulation, have been considered ideal tools for regulating stem cell fate in recent years. Neoeriocitrin (Neo), a flavonoid mainly found in citrus fruits and *Drynaria fortunei*, has been shown to have the potential to promote osteogenic differentiation and reduce inflammation. However, Neo suffers from the inherent drawbacks of flavonoids, such as poor water solubility and low bioavailability, making it difficult to deliver effectively and exert sustained effects in the complex inflammatory microenvironment, severely limiting its clinical application.

[0004] Carbon dots (CDs) are a class of fluorescent carbon nanomaterials with a size of less than 10 nm, exhibiting excellent water solubility, biocompatibility, and ease of surface functionalization. Studies have shown that converting natural active molecules into carbon dots can not only improve their water solubility and bioavailability but also endow them with new biological activities. For example, carbon dots derived from Dendrobium officinale showed significantly better therapeutic effects than their precursors in a colitis model; metformin-derived carbon dots not only retained their anti-inflammatory properties but also significantly promoted osteogenic differentiation of bone marrow mesenchymal stem cells in the inflammatory microenvironment. These studies demonstrate that nanoconversion can endow precursor molecules with superior biological activity. Currently, no research has been conducted to convert Neo-CDs into carbon dots to overcome their solubility and bioavailability deficiencies, nor have Neo-CD-based adaptors been developed to address the problem of suppressed osteogenic function of hPDLSCs in the inflammatory microenvironment. Summary of the Invention

[0005] The purpose of this invention is to provide a novel North American senna glycoside carbon dot, its preparation method, and its application in the preparation of regenerative materials for inflammatory bone defects. By converting Neo, which has poor water solubility and low bioavailability, into nanoscale carbon dots, the rich functional groups on its surface significantly improve dispersion stability, providing a technical basis for delivery systems. Under LPS-simulated inflammatory conditions, it promotes osteogenic differentiation of hPDLSCs and simultaneously inhibits the release of pro-inflammatory factors, achieving a synergistic regulation of "promoting osteogenic differentiation and inhibiting inflammation".

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A method for preparing new North American senna carbon dots involves preparing a solution of new North American senna and then carrying out a hydrothermal reaction. After cooling the reaction solution, the solution is subjected to precipitation removal, filtration, and dialysis. The concentration of new North American senna in the solution is 5-20 mg / mL. The hydrothermal reaction conditions are: 190-200℃, 9.5-11.5 h.

[0008] Preferably, the conditions for removing sediment are: 12000-18000 rpm, centrifugation for 8-15 min.

[0009] Preferably, the filtration conditions are: using a filter membrane with a pore size of 0.2-0.5μm.

[0010] Preferably, the molecular weight cutoff for dialysis is 500-2000 Da, and the dialysis time is 20-30 hours.

[0011] A new North American sage carbon dot prepared by any of the above methods, the obtained new North American sage carbon dots (Neo-CDs) have an average particle size of 2.10 nm, exhibit a flat disc-shaped morphology, are rich in carboxyl functional groups on the surface, have a negative zeta potential, and exhibit wavelength-dependent fluorescence.

[0012] The application of the new North American sage carbon dots prepared by any of the above methods in the preparation of regenerative materials for inflammatory bone defects, wherein the material is a gelatin methacryloyl (GelMA) hydrogel loaded with Neo-CDs.

[0013] The beneficial effects of this invention are as follows: 1. Precise design of process parameters: By controlling the hydrothermal reaction temperature and time, the efficient carbonization of the Neo precursor is ensured in a coordinated manner, avoiding excessive decomposition of the flavonoid structure; dialysis purification can effectively retain small molecule impurities and improve biocompatibility; 2. Overcoming the inherent defects of Neo: Neo, which has poor water solubility and low bioavailability, is transformed into nanoscale carbon dots. The abundant carboxyl groups on its surface significantly improve dispersion stability, providing a technological basis for delivery systems; 3. Adaptability to the inflammatory microenvironment: Neo-CDs promote osteogenic differentiation of hPDLSCs under LPS-simulated inflammatory conditions (upregulate the expression of ALP, Runx2, BMP2, and OPN, and promote the formation of mineralized nodules), while simultaneously inhibiting the release of pro-inflammatory factors (IL-6, TNF-α, and IL-1β), achieving a synergistic regulation of "promoting osteogenic differentiation and anti-inflammatory effects". 4. Enhanced In vivo regenerative performance: In a rat model of inflammatory bone defect in the mandible, implantation of Neo-CDs-loaded GelMA hydrogel (LPS+GelMA / Neo-CDs group) effectively inhibited local inflammatory response and significantly promoted new bone formation. Micro-CT quantitative analysis showed that bone mineral density (BMD), bone volume (BV), and bone volume fraction (BV / TV) were significantly better than those in the Neo-loaded hydrogel group (LPS+GelMA / Neo group) and the blank hydrogel group (LPS+GelMA group). Histological staining results further confirmed that the bone defect area in the LPS+GelMA / Neo-CDs group was filled with a large amount of new bone, BMP2 expression was upregulated, and osteoclast activity was inhibited. 5. Good biocompatibility: In vitro and in vivo safety assessments showed that Neo-CDs and their composite hydrogels have good cell compatibility and in vivo biocompatibility. No obvious pathological damage was found in major organs, and no abnormalities were found in blood routine and blood biochemical indicators. Attached Figure Description

[0014] Figure 1 Characterization images of the newly prepared Neo-North American sage glycoside carbon dots (Neo-CDs). In the images, A shows scanning electron microscopy (SEM) images and high-resolution transmission electron microscopy (HRTEM) lattice fringes; B shows atomic force microscopy (AFM) images and height profiles; C shows the Raman spectra and peak fitting of Neo and Neo-CDs; D shows the UV-Vis absorption spectra of Neo-CDs; E shows the fluorescence emission spectra of Neo-CDs at different excitation wavelengths; F shows the Fourier transform infrared (FTIR) spectra of Neo and Neo-CDs; G shows the full-spectrum X-ray photoelectron spectroscopy (XPS) of Neo and Neo-CDs; H shows the high-resolution C 1s XPS spectrum of Neo-CDs; I shows the high-resolution O 1s XPS spectrum of Neo-CDs; and J shows the Zeta potential diagram of Neo and Neo-CDs. Figure 2This image shows the effect of Neo-CDs in inhibiting inflammation and promoting osteogenic differentiation of hPDLSCs in an in vitro inflammatory microenvironment. In the image, A represents the proliferative activity of hPDLSCs after different treatments at days 1, 3, 5, and 7, detected by CCK-8 assay; BD represents the mRNA expression of pro-inflammatory factors IL-1β, IL-6, and TNF-α, detected by qRT-PCR; EH represents the mRNA expression of osteogenic markers ALP, SPP1, Runx2, and BMP2, detected by qRT-PCR; IM represents the protein expression and quantification analysis of the corresponding osteogenic markers, detected by Western blot; NO represents ALP staining and quantification on day 7; and PQ represents Alizarin Red S (ARS) mineralization nodules staining and quantification on day 21. Figure 3 Figure 1 shows the construction of Neo-CDs composite hydrogels and their in vivo anti-inflammatory effects. A shows photographs of GelMA, GelMA / Neo, and GelMA / Neo-CDs hydrogels before and after photocrosslinking; B shows scanning electron microscopy (SEM) images of the three hydrogels; C shows the in vitro degradation curves of the three hydrogels over 28 days; D shows the cumulative release curves of Neo and Neo-CDs from the hydrogels; E shows the quantitative analysis of pore size distribution; FH shows the quantitative analysis results of IL-1β, IL-6, and TNF-α detected by immunohistochemical (IHC) staining; I shows a representative IHC stained image with a scale bar of 200 μm (100 μm in the magnified image). Labels in the figures: D represents dentin, C represents cementum, AB represents alveolar bone, and NAB represents newly formed alveolar bone. Figure 4 This image represents the in vivo evaluation of the effect of Neo-CDs composite hydrogel on promoting periodontal bone regeneration in an inflammatory microenvironment. A is a Micro-CT 3D reconstructed image; BF represents quantitative analysis of bone mineral density (BMD), bone volume (BV), bone volume fraction (BV / TV), trabecular bone thickness (Tb.Th), and bone surface area to volume ratio (BS / BV); G represents histological images stained with HE, Masson's trichrome, and toluidine blue, with a scale bar of 200 μm (100 μm in magnified images); H represents a representative image of BMP2 stained with IHC, with a scale bar of 200 μm (100 μm in magnified images); I represents an image stained with tartrate-resistant acid phosphatase (TRAP), with a scale bar of 100 μm. Labels in the image: D represents dentin, C represents cementum, AB represents alveolar bone, and NAB represents newly formed alveolar bone. Figure 5 These are auxiliary data diagrams for the present invention. A shows live / dead cell staining to assess the cell compatibility of the hydrogel, with a scale bar of 100 μm; B shows HE staining images of major organs (heart, liver, spleen, lung, kidney, and brain), with a scale bar of 200 μm. Figures 1-5In the range, ***p < 0.001, **p < 0.01, and *p < 0.05. Detailed Implementation

[0015] Example 1: Preparation and characterization of Neo-CDs (new North American sage glycoside carbon dots).

[0016] 200 mg of New North American sennae (Chengdu Pusi Biotechnology Co., Ltd.) was dissolved in 20 mL of deionized water to prepare a solution with a concentration of 10 mg / mL. The solution was transferred to a polytetrafluoroethylene-lined hydrothermal reactor and reacted at 200 °C for 10 hours. After the reaction, the solution was cooled to room temperature, centrifuged at 15,000 rpm for 10 minutes, and the supernatant was filtered through a 0.22 μm filter membrane and dialyzed against deionized water for 24 hours using a dialysis bag with a molecular weight cutoff (MWCO) of 1000 Da. The purified product was lyophilized and stored at 4 °C for later use.

[0017] The obtained Neo-CDs were characterized, and the results are as follows: Figure 1 As shown. Transmission electron microscopy (JEOL, Japan) and atomic force microscopy (Bruker, Germany) revealed that Neo-CDs have an average particle size of 2.10 nm, a thickness of less than 1.3 nm, and exhibit a flat, disc-like morphology. Figure 1 (Middle AB region). Raman spectroscopy (WITec, Germany) shows Neo-CDs at 1377 cm⁻¹. -1 and 1587cm -1 D-band and G-band appear at this location, with an ID / IG value of 1.6 ( Figure 1 The C region). UV-Vis absorption spectroscopy (Shimadzu, Japan) shows that Neo-CDs have a characteristic absorption peak at 285 nm. Figure 1 (Middle D region). Fluorescence spectroscopy (Hong Kong, China) shows that it is wavelength-dependent, emitting the strongest fluorescence at 463 nm under 410 nm excitation. Figure 1 (Mid-E region). Fourier transform infrared spectroscopy (ThermoFisher Scientific, USA) shows Neo-CDs at 1575 cm⁻¹. -1 and 1394cm -1 Characteristic peaks appear at ( Figure 1 The F region). X-ray photoelectron spectroscopy (Thermo Fisher Scientific, USA) confirmed the presence of C, O, and Na elements. Figure 1 The GI region). Zeta potential (Malvern, UK) measurement result was negative. Figure 1 The presence of the J region indicates that it has good colloidal stability.

[0018] Example 2: Functional regulation of hPDLSCs by Neo-CDs in an in vitro inflammatory microenvironment.

[0019] hPDLSCs were cultured in α-MEM medium (Gibco, USA) containing 10% FBS (Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA) at 37°C and 5% CO2. Cells from passages 3-6 were used in the experiments. hPDLSCs were seeded in 96-well plates (2 × 10⁶ cells / wells). 3 Cells / well were treated with LPS (10 μg / mL, Merck, USA) and Neo or Neo-CDs (3 μg / mL) for 1, 3, 5, and 7 days. 10 μL of CCK-8 reagent (APExBIO, USA) was added to each well, and the cells were incubated at 37°C for 2 hours. The absorbance at 450 nm was then measured (Molecular Devices, USA). Results are as follows: Figure 2 As shown in region A. The mRNA expression of IL-1β, IL-6, TNF-α, and osteogenic markers ALP, SPP1, Runx2, and BMP2 was detected by qRT-PCR. The protein expression of ALP, OPN, Runx2, and BMP2 was detected by Western blot. The results are as follows. Figure 2 The BM region is shown. ALP staining was performed 7 days after culture. Figure 2 (NO region), ARS staining was performed 21 days later. Figure 2 (Middle PQ region). The results showed that Neo-CDs could significantly inhibit inflammation and promote osteogenic differentiation, with better effects than Neo precursors.

[0020] Example 3: Preparation of Neo-CDs composite hydrogel and evaluation of its in vivo bone regeneration effect.

[0021] Dissolve 0.05 g LAP in 20 mL PBS and heat to 40-50 °C for 15 minutes to prepare a 0.25% photoinitiator solution. Dissolve 10 g GelMA (Engineering for Life, China) in the above solution and heat at 60-70 °C without heat for 20-30 minutes. After filtration, add Neo or Neo-CDs to a final concentration of 60 μg / mL, and crosslink under blue light to obtain GelMA / Neo and GelMA / Neo-CDs hydrogels. The hydrogel morphology, changes in residual mass, release behavior, and cell compatibility test results are as follows: Figure 3 Middle BE region and Figure 5 As shown in region A.

[0022] Six-week-old male SD rats were injected every other day with 0.1 mL of PBS containing 2 mg / mL LPS into the buccal gingival sulcus of the first and second mandibular molars for one week. They were randomly divided into six groups (n=4): ① Control group: no surgical treatment, serving as a blank control; ② Blank group: mandibular bone defects were prepared without LPS pretreatment or implantation of any hydrogel; ③ LPS group: mandibular bone defects were prepared and LPS was injected, but no hydrogel was implanted; ④ LPS+GelMA group: mandibular bone defects were prepared, LPS was injected, and blank GelMA hydrogel was implanted; ⑤ LPS+GelMA / Neo group: mandibular bone defects were prepared, LPS was injected, and Neo-loaded GelMA hydrogel was implanted; ⑥ LPS+GelMA / Neo-CDs group: mandibular bone defects were prepared, LPS was injected, and Neo-loaded GelMA hydrogel was implanted. All rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (0.3 mL / 100 g body weight). A 2 cm incision was made along the lower border of the mandible to expose the alveolar bone. Under saline irrigation, a buccal bone defect was prepared using a ball bur, measuring 5 mm mesiodistal × 1 mm buccal-lingual × 2 mm vertically. In the hydrogel group, 10 μL of the corresponding formulation was injected and cross-linked and cured using blue light. No material was implanted in the Blank and LPS groups. Penicillin was administered for three consecutive days post-surgery to prevent infection. Blood samples, mandible samples, and major organs were collected four weeks post-surgery for testing.

[0023] Immunohistochemical staining was used to detect IL-1β, IL-6, and TNF-α. The results are as follows: Figure 3 The FI region is shown. Micro-CT analysis of bone parameters yields the following results. Figure 4 The AF region is shown in the image. Histological staining (HE, Masson's, toluidine blue, TRAP) and BMP2IHC staining results are shown below. Figure 4 The GI region is shown. HE staining results of major organs are shown below. Figure 5 As shown in section B, the results of routine blood tests are shown in Table 1, and the results of blood biochemistry tests are shown in Table 2.

[0024] Table 1 Blood routine indicators of rats in each group

[0025] index Control Blank LPS LPS+GelMA LPS+GelMA / Neo LPS+GelMA / Neo-CDs <![CDATA[White blood cell count (10 9 / L)]]> 8.488 ±1.220 8.385 ±1.964 8.435 ±1.930 8.780 ±2.125 8.510 ±2.592 8.535 ± 2.207 <![CDATA[Red blood cell count (10 12 / L)]]> 6.915 ±0.121 6.968 ±0.405 6.788 ±0.211 6.880 ±0.408 6.728 ±0.418 6.868 ± 0.543 Hemoglobin (g / L) 138.250 ±6.076 135.800 ±6.843 137.500 ±8.103 139.750 ±8.421 137.750 ±10.782 138.250 ±2.500 Mean corpuscular volume (fL) 59.750 ±2.965 59.525 ±2.831 59.200 ±1.966 59.650 ±1.411 60.625 ±1.410 59.800 ±2.857 <![CDATA[Platelet count (10 9 / L)]]> 648.250 ±37.464 654.250 ±41.987 664.500 ±44.852 674.000 ±38.358 671.750 ±50.109 669.250 ±51.713 <![CDATA[Number of lymphocytes (10 9 / L)]]> 7.548 ±1.337 7.715 ±1.665 7.763 ±1.968 8.128 ±2.205 7.180 ±1.615 7.793 ±1.939 <![CDATA[Intermediate cell count (10 9 / L)]]> 0.163 ±0.046 0.170 ±0.088 0.158 ±0.048 0.165 ±0.042 0.140 ±0.109 0.160 ±0.091 <![CDATA[Granulocyte count (10 9 / L)]]> 0.605 ±0.362 0.595 ±0.197 0.720 ±0.507 0.715 ±0.183 0.648 ±0.269 0.670 ±0.214 Hematocrit (%) 40.475 ±1.601 40.750 ±2.385 39.575±1.338 42.000 ±1.846 40.625 ±2.089 41.275 ±1.394 Red blood cell distribution width coefficient of variation (%) 12.375 ±1.031 12.350 ±0.420 12.425 ±0.556 12.250 ±1.115 12.525 ±0.634 12.325 ±0.556 Mean corpuscular hemoglobin content (pg) 20.175 ±1.115 19.875 ±0.988 19.975 ±0.793 20.400 ±0.898 19.925 ±0.750 20.100 ±1.433

[0026] Table 2. Blood biochemical indicators of rats in each group

[0027] index Control Blank LPS LPS+GelMA LPS+GelMA / Neo LPS+GelMA / Neo-CDs Alanine aminotransferase (U / L) 36.000 ± 3.304 34.925 ± 3.702 37.950 ± 4.549 32.875 ± 6.234 34.925 ± 4.686 33.150 ± 4.230 Aspartate aminotransferase (U / L) 105.250 ±16.001 105.550 ±9.755 107.150 ±19.022 106.675 ±11.818 108.525 ±10.759 107.000 ±6.943 Total bilirubin (μmol / L) 1.195 ± 0.289 1.335 ± 0.393 1.228 ± 0.362 1.378 ± 0.124 1.325 ± 0.348 1.283 ± 0.332 Albumin (g / L) 31.500 ± 0.779 31.925 ± 1.150 30.700 ± 1.944 31.225 ± 1.001 31.475 ± 1.115 31.950 ± 0.777 Creatinine (μmol / L) 21.275 ± 3.397 21.900 ± 4.152 20.725 ± 1.021 21.350 ± 1.182 21.975 ± 2.579 21.050 ± 1.250 Urea (mmol / L) 4.615 ± 0.674 4.500 ± 0.811 4.760 ± 0.478 4.510 ± 0.694 4.785 ± 0.259 4.538 ± 0.637 Uric acid (μmol / L) 111.575 ±15.651 114.325 ±18.332 116.675 ±15.203 113.650 ±23.132 108.850 ±20.107 113.900 ±20.124 Glucose (mmol / L) 9.293 ± 1.752 9.375 ± 2.477 9.368 ± 2.599 9.170 ± 1.115 9.208 ± 2.065 9.278 ± 1.933

[0028] The results showed that GelMA / Neo-CDs hydrogels could effectively inhibit local inflammation, significantly promote new bone formation, and have good biocompatibility.

Claims

1. A method for preparing novel North American senna glycoside carbon dots, characterized in that, The reaction is carried out by preparing a solution of neo-North American senna extract and then performing a hydrothermal reaction. After the reaction solution is cooled, it is subjected to precipitation removal, filtration, dialysis, and freeze-drying. The concentration of neo-North American senna extract in the solution is 5-20 mg / mL. The hydrothermal reaction conditions are: 190-200℃, 9.5-11.5h.

2. The method for preparing the new North American sage glycoside carbon dots according to claim 1, characterized in that, The conditions for removing sediment are: centrifugation at 12000-18000 rpm for 8-15 minutes.

3. The method for preparing the new North American sage glycoside carbon dots according to claim 1, characterized in that, The filtration conditions are: use a filter membrane with a pore size of 0.2-0.5μm.

4. The method for preparing the new North American sage glycoside carbon dots according to claim 1, characterized in that, The molecular weight cutoff for dialysis is 500-2000 Da, and the dialysis time is 20-30 hours.

5. A novel North American senna glycoside carbon dot, characterized in that, Prepared by the method described in any one of claims 1-4.

6. The application of the new North American sage carbon dots prepared by the method according to any one of claims 1-4 in the preparation of regenerative materials for inflammatory bone defects.

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