Application of system carrying loxanthin in treatment of periodontitis and its medicine and equipment

Abstract

A system for carrying breviscapine, comprising extracellular vesicles and breviscapine, wherein the breviscapine is carried in the extracellular vesicles. The system provided by the present application can target enrichment in the damaged periodontal microvascular area. The vasodilation activity of breviscapine and the proangiogenic factors (such as VEGF, miR-126) carried by the Schwann cell extracellular vesicles play a synergistic role, effectively repairing the capillary basement membrane thickening and endothelial dysfunction caused by diabetes, significantly reversing the degenerative vascular changes of periodontal tissue, restoring local blood supply, and reversing vascular degeneration, providing the necessary nutrients and oxygen supply basis for periodontal tissue regeneration. The drug or medical device prepared by taking the system of the present application as an active ingredient can be effectively applied to the field of periodontal regeneration.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and tissue regeneration technology, specifically to an exovesicle carrying natural plant compounds for targeted delivery, and its application in the preparation of drugs or medical devices for treating diabetic periodontitis. Background Technology

[0002] Diabetes mellitus is a significant systemic risk factor for the development and progression of periodontitis. Diabetic periodontitis clinically manifests as an intensified inflammatory response, rapid alveolar bone resorption, and impaired tissue repair capacity. Its core pathological mechanisms, in addition to local flora imbalance and immune disturbances, also involve diabetic-induced microvascular complications, manifested as impaired endothelial cell function in periodontal tissues, thickening of the basement membrane, and reduced blood perfusion, ultimately leading to tissue malnutrition and an imbalance in the regenerative microenvironment. While current clinical treatments such as scaling and root planing and systemic antibiotics can temporarily control infection, they are insufficient to effectively reverse the microcirculatory disturbances and insufficient tissue regeneration caused by diabetes.

[0003] Breviscapine is a natural flavonoid active ingredient extracted from the chrysanthemum plant (Erigeron breviscapus), belonging to the Asteraceae family. As a component, researchers have attempted to use it in the treatment of diabetic complications, but its clinical application is limited. Due to its highly polar molecular structure and poor water solubility, breviscapine exhibits low bioavailability, a short biological half-life, and lacks the ability to target and accumulate at periodontal sites, making it difficult to achieve a sustained and effective therapeutic concentration locally. Therefore, it has not been observed to be used as an active ingredient in the treatment of diabetic periodontitis.

[0004] In existing technologies, the delivery of scutellarin largely relies on synthetic nanocarriers such as liposomes or PLGA, which generally have limitations such as immunogenicity risks and limited targeting. On the other hand, current clinical treatment strategies for diabetic periodontitis often focus on single antibacterial or anti-inflammatory approaches, neglecting the synergistic repair of the multi-dimensional microenvironment encompassing the neuro-immune-vascular-bone regions. Therefore, developing a synergistic treatment system that combines excellent biocompatibility and high targeting, and can achieve multi-effect linkage against multiple pathological damages in diabetic periodontitis, has significant clinical value. Summary of the Invention

[0005] One object of the present invention is to provide a system for delivering scutellarin to periodontal tissues and increasing the retention time of scutellarin in periodontal tissues.

[0006] Another objective of this invention is to provide a system carrying scutellarin, which utilizes its low immunogenicity and multi-target action characteristics to achieve clinical application in diabetic periodontitis, promote alveolar bone regeneration, and reverse diabetic vascular degenerative changes.

[0007] Another object of the present invention is to provide the application of a system carrying scutellarin in the preparation of a medicament for treating periodontitis.

[0008] Another object of the present invention is to provide an application of a system carrying scutellarin in the preparation of medical devices for treating periodontitis.

[0009] A system for loading scutellarin includes extracellular vesicles and scutellarin, with the scutellarin loaded within the extracellular vesicles. For example, the drug loading capacity reaches 35 wt%.

[0010] Schwann cells (SCs) are the main supporting cells of the peripheral nervous system. The Schwann extracellular vesicles (SC-EVs) they secrete not only serve as drug delivery carriers, but also possess biological functions such as nerve repair, anti-inflammation, angiogenesis, and regulation of osteogenic differentiation, which are highly consistent with the pathological damage of diabetic periodontitis.

[0011] Another system for carrying ligustilide includes Schwann extracellular vesicles and ligustilide, with ligustilide contained within the Schwann extracellular vesicles.

[0012] Schwann extracellular vesicles were prepared by enzymatic digestion to isolate and culture Schwann cells, collecting the supernatant of P3–P8 generation cell culture, and extracting Schwann extracellular vesicles (SCs-EVs) by differential ultracentrifugation (removing cells and debris at 300 g, 2,000 g and 10,000 g in sequence, and precipitating the extracellular vesicles at 100,000 g).

[0013] The obtained Schwann extracellular vesicles were characterized and identified by nanoparticle tracking analysis (NTA), surface potential was detected by Zeta potentiometer, and expression of extracellular vesicle marker proteins was detected by Western blotting.

[0014] The preparation method of the system loaded with scutellarin is as follows: The obtained SCs-EVs are mixed with a scutellarin (Scu) solution, and the scutellarin is loaded into the SCs-EVs by intermittent sonication (200W, sonication for 3s / intermittent for 5s, 3min). After sonication, the free scutellarin is removed by ultracentrifugation (100,000 g for 30min), and the scutellarin-loaded Schwann extracellular vesicle complex (SC-EVs@Scu) is obtained.

[0015] Verification has shown that the system provided by this invention can target and accumulate in damaged periodontal microvascular areas. The vasodilatory activity of *Ligustrum lucidum* extract synergistically works with the angiogenic factors (such as VEGF and miR-126) carried by Schwann extracellular vesicles to effectively repair capillary basement membrane thickening and endothelial dysfunction caused by diabetes, significantly reversing degenerative vascular changes in periodontal tissues, restoring local blood supply, and providing the necessary nutritional and oxygen supply basis for periodontal tissue regeneration.

[0016] By inhibiting osteoclast activity and promoting osteogenic differentiation of periodontal ligament stem cells (PDLSCs), it effectively improves the alveolar bone resorption status of patients with diabetic periodontitis, restores alveolar bone resorption, significantly increases alveolar bone height and bone density, and achieves structural repair of bone tissue.

[0017] Schwann extracellular vesicles, as naturally derived nanocarriers, have extremely low immunogenicity and high biocompatibility, effectively avoiding foreign body reactions and immune clearance that may be caused by artificially synthesized carriers in vivo, thus exhibiting high safety.

[0018] Leveraging the natural structural advantages of external vesicles, this treatment fully preserves a variety of endogenous bioactive molecules, including proteins, mRNAs, and miRNAs, derived from Schwann cells. These molecules, along with the loaded scutellarin, form a multi-target synergistic system of "traditional Chinese medicine monomer + bio-nanocarrier," overcoming the limitations of single-component therapy. The system provided by this invention not only acts on bone tissue and the vascular system, but its retained bioactive components also have the potential to repair the surrounding soft tissues such as periodontal ligament and nerve endings, which helps to achieve functional regeneration of periodontal tissues and demonstrates good multi-tissue repair characteristics.

[0019] The system provided by this invention, when combined with pharmaceutical excipients, can be formulated into a drug suitable for the treatment of periodontitis, especially periodontitis associated with diabetes.

[0020] The system provided by this invention can be used to create medical devices such as gels or microneedles by loading other biomaterials, such as gels or PGA, for the treatment of periodontitis, especially periodontitis with diabetes. Attached Figure Description

[0021] Figure 1 Schematic diagram of the preparation of SCs-EVs@Scu; Figure 2Figures show the characterization and identification results of SCs-EVs; where A is a transmission electron microscope view of SCs-EVs (showing that the morphology of SCs-EVs presents a classic disc-like vesicle), B is a Western blotting result of external vesicle markers HSP70, CD63, and TSG101, C is a result of NTA particle size distribution, and D is a fluorescence view of the phagocytosis of SCs-EVs by periodontal ligament cells. Figure 3 The curve of scutellarin release from SCs-EVs@Scu over 48 hours; Figure 4 A statistical chart showing the results of bioactivity assessment of the proliferative capacity of human periodontal ligament cells at various concentrations of SCs-EVs. Figure 5 The images show the morphological and particle size analysis results of SCs-EVs and SCs-EVs@Scu. A is a transmission electron microscope (TEM) field of view of SCs-EVs, B is the NTA particle size measurement result of SCs-EVs, C is a TEM field of view of SCs-EVs@Scu, and D is the NTA particle size measurement result of SCs-EVs@Scu (average particle size approximately 163 nm). Figure 6 The images show the results of Micro-CT 3D reconstruction and bone morphometric analysis; where A is the Micro-CT image, B is the BV statistical graph, C is the TV statistical graph, D is the BV / TV statistical graph, and E is the bone loss statistical graph. Figure 7 The image shows the results of CD31 and VEGF immunofluorescence staining. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. Unless otherwise specified, the processes, conditions, experimental methods, etc., involved in implementing the present invention employ conventional techniques in the art, which are well known to those skilled in the art and are not specifically limited by the present invention.

[0023] Example 1: Isolation, purification and identification of Schwann extracellular vesicles Schwann cells were isolated from the sciatic nerve tissue of neonatal SD rats using a tissue block combined with enzymatic digestion. Phenotypic identification of the obtained cells was performed by S100β immunofluorescence staining. The supernatant from P3–P8 cell cultures was collected, and impurities were removed stepwise by a gradient centrifugation method: sequential centrifugation at 300 g for 10 min, 2,000 g for 10 min, and 10,000 g for 30 min, followed by ultracentrifugation at 100,000 g for 90 min to precipitate Schwann cell extracellular vesicles (SCs-EVs). The concentration of extracellular vesicle proteins was determined using the BCA method, and particle size distribution was detected by nanoparticle tracking analysis (NTA). The results are as follows: Figure 2 The main particle size peak was located in the 100–150 nm range. Western blotting detected the extracellular vesicle marker proteins HSP70, CD63, and TSG101, all of which showed positive expression, indicating that the obtained product was a typical Schwann cell extracellular vesicle. The CCK-8 assay was used to detect the effect of 10 μg / mL and 100 μg / mL concentrations of Schwann cell extracellular vesicles on the proliferation capacity of human periodontal ligament cells. (See details) Figure 4 ) Example 2: Preparation and Characterization of Ligustrum lucidum Loading and SC-EVs@Scu The preparation process of SCs-EVs@Scu is as follows: Figure 1 As shown. Specifically, scutellarin was dissolved in phosphate-buffered saline (PBS) containing 10% DMSO to prepare a stock solution of appropriate concentration. 100 μg of SCs-EVs was mixed with a 50 μM scutellarin solution, and PBS was added to a total volume of 1 mL. The mixture was incubated in a 4°C shaker in the dark for 4 hours, followed by intermittent sonication (200W, 3 s sonication / 5 s intermittent sonication, 3 min). After incubation, free scutellarin was removed by high-speed centrifugation, and the eluent was collected to obtain the Schwann extracellular vesicle complex loaded with scutellarin (SC-EVs@Scu). The drug loading was determined by high-performance liquid chromatography-mass spectrometry (HPLC-MS), and the encapsulation efficiency was calculated to be 35%. Transmission electron microscopy analysis showed that, compared with Schwann cell vesicles, the SC-EVs@Scu loaded with scutellarin still exhibited a vesicle-like structure with a nanoparticle size of 160 nm. (See...) Figure 5 ) Example 3: Verification of the therapeutic effect and angiogenesis reversal in a rat model of diabetic periodontitis. 3.1 Model Establishment: A type 2 diabetic rat model was established using a high-fat, high-sugar diet combined with intraperitoneal injection of streptozotocin (STZ). After successful establishment of the diabetic model, ligatures were placed around the periodontal tissues of the bilateral maxillary first molars, and periodontal pathogens (Porphyromonas gingivalis, P. gingivalis) were locally applied for 4 weeks to establish a rat model of diabetic periodontitis.

[0024] 3.2 Grouping and Administration: Rats that successfully modeled the disease were randomly divided into four groups: model control group (administered PBS), free scutellarin group (Scu), blank Schwann extracellular vesicle group (SCs-EVs), and SC-EVs@Scu group. All groups received local multi-point injections into the periodontal zone twice weekly for four consecutive weeks. Figure 3 The curve of scutellarin release from SCs-EVs@Scu shows that scutellarin is continuously and steadily released within 48 hours, indicating that scutellarin is retained in the periodontium and continuously provides scutellarin to the periodontium.

[0025] 3.3 Alveolar bone repair detection: After treatment, the mandibles of rats in each group were subjected to Micro-CT scans. The results are as follows: Figure 6 The results showed that significant new bone formation was observed in the alveolar bone resorption area of ​​the SC-EVs@Scu group, and the bone volume fraction (BV / TV) and trabecular bone thickness were significantly higher than those of the model control group, the free scutellarin group, and the blank SCs-EVs group, indicating that SC-EVs@Scu can effectively promote alveolar bone regeneration in patients with diabetic periodontitis.

[0026] 3.3 Vascular endothelial function detection: The expression levels of vascular endothelial growth factor (VEGF) and CD31 in periodontal tissues were detected by immunofluorescence staining. Results are as follows: Figure 7 The results showed that the positive expression signals of VEGF and CD31 were significantly enhanced in the SC-EVs@Scu group, suggesting that SC-EVs@Scu can promote the repair of vascular endothelial function and improve the local microcirculation status of periodontium.

Claims

1. A system for carrying scutellarin, characterized in that, It includes extracellular vesicles and scutellarin, with scutellarin contained within the extracellular vesicles.

2. The system for carrying scutellaria baicalensis according to claim 1, characterized in that, The extracellular vesicles are Schwann extracellular vesicles and scutellarin.

3. The system for carrying scutellarin according to claim 1, characterized in that, Efficient loading of extracellular vesicles and ligustrazine was achieved using intermittent ultrasound.

4. The system for carrying scutellarin according to claim 1, characterized in that, Intermittent ultrasound was performed at 200W for 3 seconds followed by a 5-second interval, for a total of 3 minutes.

5. The system for carrying scutellarin according to claim 1, characterized in that, Schwann cells were isolated and cultured using enzymatic digestion. The supernatant of P3–P8 generation cells was collected, and extracellular vesicles of Schwann cells were extracted using differential ultracentrifugation.

6. The system for carrying scutellarin according to claim 5, characterized in that, Differential ultracentrifugation was used to remove cells and debris at 300 g, 2,000 g and 10,000 g in sequence, and the exovesicles were precipitated at 100,000 g.

7. The use of the system for carrying scutellarin according to claim 1 in the preparation of a medicament or medical device for treating periodontitis.

8. The application according to claim 7, characterized in that, The periodontitis mentioned is periodontitis associated with diabetes.

9. The application according to claim 7, characterized in that, The drug also includes pharmaceutical excipients.

10. The application according to claim 7, characterized in that, The medical device in question is a gel or a microneedle.

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