Resveratrol-loaded exosome-liposome hybrid nanoparticles, methods of making and using the same

Abstract

The present application belongs to the technical field of anti-inflammatory and immunomodulatory pharmaceutical preparations, and particularly relates to an exosome-liposome mixed nanoparticle loaded with resveratrol as well as a preparation method and application thereof. Raw materials of the mixed nanoparticle include lipids, resveratrol and exosomes. The mixed nanoparticle of the present application can solve the technical problems of short drug half-life of resveratrol and difficulty in reaching an effective therapeutic concentration at a target site, and has advantages in terms of in-vitro and in-vivo antioxidant, change of macrophage polarization phenotype and anti-inflammatory; can also reduce alveolar bone loss of periodontitis rats and inhibit periodontal inflammation. Safety evaluation in vivo also shows that the mixed nanoparticle has no damage to main organs at a therapeutic dose. Therefore, the exosome-liposome mixed nanoparticle loaded with resveratrol can be used for preparing an antioxidant, an anti-inflammatory drug or an immunomodulatory drug, and has an ideal popularization and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of anti-inflammatory and immunomodulatory drug formulation technology, specifically relating to resveratrol-loaded exosome-liposome hybrid nanoparticles, their preparation methods, and applications. Background Technology

[0002] Resveratrol is a non-flavonoid polyphenolic organic compound. In vitro and animal experiments have shown that resveratrol has antioxidant, anti-inflammatory, anticancer, and cardiovascular protective effects. It mainly exerts its antioxidant effect through mechanisms such as scavenging or inhibiting free radical generation, inhibiting lipid peroxidation, and regulating the activity of antioxidant-related enzymes; it also achieves anti-inflammatory effects by reducing platelet adhesion and altering platelet activity during the anti-inflammatory process. However, resveratrol is a lipid-soluble drug with poor water solubility, poor oral absorption, lack of targeting, and almost impossible to reach effective therapeutic concentrations at the target site. Furthermore, its metabolism in vivo is unstable, thus greatly limiting its clinical application. How to prolong the half-life of resveratrol and enhance its clinical application is an urgent problem to be solved in this field. Although the combined use of resveratrol and liposome nanoparticles can improve the above problems to some extent, the efficacy of resveratrol liposome nanoparticles still cannot meet the requirements. Summary of the Invention

[0003] The present invention aims to provide exosome-liposome hybrid nanoparticles loaded with resveratrol to solve the technical problems of resveratrol's short drug half-life and difficulty in achieving effective therapeutic concentrations at the target site.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] Resveratrol-loaded exosome-liposome hybrid nanoparticles, the raw materials of which include lipids, resveratrol, and exosomes; the raw materials of the lipids include PBA-COOH, EDC, NHS, and DSPE-PEG. 2K -NH2.

[0006] This technical solution also provides a method for preparing resveratrol-loaded exosome-liposome hybrid nanoparticles, comprising the following steps performed sequentially:

[0007] S1: Dissolve PBA-COOH, EDC, and NHS in DMSO, react, and then add DSPE-PEG. 2K -NH2; after reaction, dialysis and lyophilization, Lip lyophilized powder is obtained;

[0008] S2: Dissolve Lip lyophilized powder and resveratrol in DMSO, then add triethylamine; after reaction, dialysis and lyophilization, obtain Lip-Res lyophilized powder;

[0009] S3: Dissolve Lip-Res lyophilized powder in DMSO, dialyze to obtain Lip-Res solution, mix with exosome solution and react to obtain Exo@Lip-Res.

[0010] This technical solution also provides the application of resveratrol-loaded exosome-liposome hybrid nanoparticles in the preparation of antioxidants, anti-inflammatory drugs, or immunomodulatory drugs.

[0011] This technical solution also provides the application of resveratrol-loaded exosome-liposome hybrid nanoparticles in the preparation of drugs for treating periodontitis.

[0012] Experiments have demonstrated that the resveratrol-loaded exosome-liposome hybrid nanoparticles of this scheme exhibit significant effects in antioxidation, anti-inflammation, and immunomodulation, and can be used alone for these purposes. The inventors further tested the therapeutic effect in a periodontitis model, where the resveratrol-loaded exosome-liposome hybrid nanoparticles also showed good efficacy. The pathological mechanism of periodontitis is complex, and the resveratrol-loaded exosome-liposome hybrid nanoparticles can improve the condition of periodontitis, particularly by preventing alveolar bone loss, while exerting antioxidant, anti-inflammatory, and immunomodulatory effects. Although this technical solution only conducted animal experiments on the resveratrol-loaded exosome-liposome hybrid nanoparticles in a periodontitis model, this does not mean that the nanoparticles of this scheme only have a therapeutic effect on periodontitis. Rather, since periodontitis involves various complex pathological mechanisms such as inflammation, oxidative stress, and immune dysregulation, researching methods for treating periodontitis has strong application value. Based on the in vitro experimental results of antioxidant, anti-inflammatory, and immunomodulatory (macrophage polarization) effects presented in this protocol, we can undoubtedly confirm the antioxidant, anti-inflammatory, and immunomodulatory effects of the nanoparticles in this protocol. In other types of diseases, varying degrees of inflammation, oxidative stress, and immune dysregulation also occur, and the nanoparticles in this protocol can be used to address these issues accordingly.

[0013] Furthermore, PBA-COOH, EDC, NHS, and DSPE-PEG... 2K The ratio of -NH2 used is 1:1.2:1.2:1.

[0014] Furthermore, in S1, PBA-COOH, EDC, NHS, and DSPE-PEG are present. 2K The ratio of -NH2 used is 1:1.2:1.2:1; PBA-COOH, EDC and NHS are dissolved in DMSO and reacted at 40℃ for 4-6 hours under magnetic stirring.

[0015] Furthermore, DSPE-PEG is added to S1. 2K After -NH2, react at 40℃ for 10-12 hours.

[0016] Furthermore, in S2, the mass ratio of Lip lyophilized powder to resveratrol is 14:1.

[0017] Furthermore, in S2, the amount of DMSO used is 2 mL of DMSO for every 5 mg of resveratrol; the volume ratio of DMSO to triethylamine is 200:1.

[0018] Furthermore, in S3, the volume ratio of Lip-Res solution to exosome solution was 1:1, and the mixture was incubated at 37°C for 4-6 hours.

[0019] The principle behind this solution is:

[0020] Resveratrol (RSV) is a non-flavonoid polyphenol known for its anti-inflammatory and immunomodulatory effects. However, its medical applications are limited by its poor stability, water solubility, and low bioavailability. Currently, there is considerable development of resveratrol liposome systems (Lipo-RSV) to expand the applications of resveratrol, demonstrating more effective therapeutic effects than resveratrol raw materials alone.

[0021] Therefore, unlike traditional methods of directly loading resveratrol onto exosomes, this study prepared resveratrol-liposome hybrid nanoparticles to further enhance therapeutic effects. Results showed that the hybrid nanoparticles exhibited advantages in in vitro and in vivo antioxidant activity, alteration of macrophage polarization phenotype, and anti-inflammatory properties. Due to these advantages, resveratrol-loaded exosome-liposome hybrid nanoparticles can be used to prepare antioxidants, anti-inflammatory drugs, or immunomodulatory drugs, offering diverse applications.

[0022] Using a specific rat model of periodontitis as an example, the inventors conducted preliminary in vivo studies and found that the nanoparticles in this formulation can reduce alveolar bone loss and inhibit periodontal inflammation in rats with periodontitis. In addition to addressing periodontal inflammation, the nanoparticles in this formulation possess antioxidant, anti-inflammatory, and macrophage polarization-regulating functions, making them applicable to diseases related to oxidative stress, inflammation, and immunity. For example, using immunomodulatory drugs: macrophages are innate immune cells that respond to their microenvironment with different activation states. Macrophage activation is edited to inhibit inflammatory diseases by promoting the repolarization of inflammatory (M1) macrophages into anti-inflammatory (M2) macrophages. In vivo safety assessments also showed that the mixed nanoparticles caused no damage to major organs at therapeutic doses.

[0023] The periodontal tissue consists of three parts: the periodontal ligament, alveolar bone, and gingiva. Its main functions are to support, fix, and nourish the teeth. Periodontitis is a chronic inflammatory disease affecting approximately 90% of the world's population. The chronic inflammation of periodontitis primarily damages the alveolar bone and the connective tissue supporting the teeth, which can lead to the gradual detachment of teeth from the periodontal ligament (PDL), forming periodontal pockets, and ultimately resulting in tooth loss. In clinical practice, standard mechanical methods are typically used to remove subgingival biofilm and calculus, which can reduce clinical attachment loss in a short period. However, in the case of periodontitis, bone repair and reconstruction are impaired. Traditional clinical treatments cannot fully repair damaged periodontal tissues. Periodontal tissue repair remains an ongoing challenge. How to alleviate periodontal inflammation and reconstruct damaged periodontal bone tissue is a difficult problem in the treatment of periodontitis. The EXO@Lip-Res technology in this study has shown good therapeutic and restorative effects on periodontitis, especially the alveolar bone loss it induces. Attached Figure Description

[0024] Figure 1 The images show a schematic diagram of the nanoparticle structure, an electron microscope image, and a WB surface antigen detection image of Example 2 of the present invention.

[0025] Figure 2 This is an immunofluorescence image of Example 2 of the present invention.

[0026] Figure 3 The images show the particle size and potential statistics of the nanoparticles in Example 2 of this invention, as well as the ultraviolet absorption diagrams of various raw materials and nanoparticles scanned across the entire wavelength range using an ultraviolet spectrophotometer.

[0027] Figure 4 The results are from the DPPH free radical scavenging ability determination experiment of Example 3 of the present invention.

[0028] Figure 5 The results are from the ABTS free radical scavenging ability determination experiment of Example 3 of the present invention.

[0029] Figure 6 The results are from the hydrogen peroxide scavenging capacity test of Example 3 of the present invention.

[0030] Figure 7 The results of the FRAP measurement experiment in Example 3 of this invention are shown.

[0031] Figure 8 This is a fluorescence image of PDLSCs cells from Example 4 of the present invention.

[0032] Figure 9 The results of fluorescence microplate reader detection of PDLSCs cells in Example 4 of this invention are shown.

[0033] Figure 10The results are obtained by flow cytometry analysis of PDLSCs cells in Example 4 of this invention.

[0034] Figure 11 This is a fluorescence image of RAW264.7 cells from Example 4 of the present invention.

[0035] Figure 12 The results of fluorescence microplate reader detection of RAW264.7 cells in Example 4 of this invention are shown.

[0036] Figure 13 This is the result of a study on the effect of nanoparticles on macrophage polarization in Example 5 of the present invention (qRT-PCR).

[0037] Figure 14 The results of the study on the effect of nanoparticles on LPS-induced inflammation in PDLSCs in Example 6 of this invention (qRT-PCR).

[0038] Figure 15 The results of the study (WB) on the effect of nanoparticles on LPS-induced inflammation in PDLSCs in Example 6 of this invention are shown.

[0039] Figure 16 The images show tissue staining of experimental rats in Example 7 of this invention and micro-CT scans showing alveolar bone repair.

[0040] Figure 17 The results of the in vivo safety assessment experiment (HE staining of major organs) of Example 8 of the present invention are shown.

[0041] Figure 18 The results of the in vivo safety assessment experiment (blood physiological and biochemical detection) of Example 8 of the present invention are shown. Detailed Implementation

[0042] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods; and the materials, reagents, etc. used are all commercially available.

[0043] Example 1: Preparation method of EXO@Lip-Res

[0044] (1) Lipid (Lip) preparation

[0045] Carboxyphenylboronic acid (PBA-COOH), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) were weighed and dissolved in dimethyl sulfoxide (DMSO) at a mass ratio of 1:1.2:1.2. The mixture was magnetically stirred and reacted at 40°C for 4-6 hours (5 hours was used in subsequent experimental studies). Then, phospholipid polyethylene glycol amino (DSPE-PEG) was added in a mass ratio of 1:1 to PBA-COOH. 2K -NH2), react at 40℃ overnight (optional range 10-12h, preferred 12h), dialyze in a conventional 1000KDa dialysis bag in the dark for 12h, and freeze dry to obtain Lip freeze-dried powder.

[0046] (2) Preparation of resveratrol liposomes (Lip-Res)

[0047] Lip lyophilized powder and resveratrol (Res) were mixed at a mass ratio of 14:1, dissolved in DMSO (2 mL DMSO for every 5 mg Res), and triethylamine (added at a volume ratio of 200:1 to DMSO) was added. The mixture was reacted overnight (12 h in an oil bath at 37 °C), dialyzed for 12 h in the dark using a standard 1000 kDa dialysis bag, and then lyophilized to obtain Lip-Res lyophilized powder.

[0048] (3) Preparation of resveratrol-loaded exosome-liposome hybrid nanoparticles (EXO@Lip-Res)

[0049] Weigh out lyophilized Lip-Res powder and dissolve it in DMSO (e.g., 2 mL DMSO for every 50 mg of lyophilized Lip-Res powder; this can be adjusted according to the actual required Lip-Res concentration, e.g., to prepare a 500 μg / mL Lip-Res solution). After dialysis in a 1000 kDa dialysis bag in the dark for 12 hours, obtain the Lip-Res solution. Mix the Lip-Res solution with exosomes (Exo) (exosome solution, Exo concentration 1 mg / mL; for subsequent use, the EXO can be diluted to a specified final concentration, e.g., 10 μg / mL) at a volume ratio of 1:1 and incubate overnight at 37°C (incubation can be 4-6 hours, but approximately 5 hours is usually used in subsequent cases) to prepare Exo@Lip-Res.

[0050] In addition, exosomes (Exo) are prepared according to the following method:

[0051] Dental pulp stem cells (DPSCs) were obtained from wisdom teeth extracted from healthy donors with informed consent for this study. Pulp tissue was extracted from freshly extracted teeth, minced, and digested at 37°C with 3 mg / mL type I collagenase (Sigma, USA) for 40 min. The tissue suspension was seeded in T25 culture flasks and cultured in α-minimum essential medium (α-MEM, HyClone, USA) containing 10% fetal bovine serum (FBS, Lonsera, Uruguay) and 100 U / mL penicillin-streptomycin (HyClone, USA) at 37°C and 5% CO2. Primary cells were induced to differentiate into osteogenic and adipogenic cells at passages three to four. Alizarin Red and Oil Red staining was performed on day 21 of induction to observe their multi-lineage differentiation capacity. Cell surface antibody markers were identified by flow cytometry.

[0052] The obtained DPSCs were induced and exosomes were prepared as follows: When the DPSCs reached 60–80% confluence, serum-free medium was used instead of conventional medium, and the cells were cultured for another 48 hours. Then, centrifugation was performed at 4°C: 300g for 10 min, supernatant discarded; 2000g for 10 min, supernatant discarded; 10000g for 30 min, supernatant discarded; 100000g for 70 min, supernatant discarded, the precipitate was retained at the bottom of the tube and resuspended in PBS; the previous steps were repeated to obtain an exosome suspension. The total concentration of exosomes was determined using a bicinchoninic acid (BCA) kit (Beyotime, Shanghai, China). DPSC-EXO was stored at -80°C for subsequent experiments.

[0053] Example 2: Characterization of EXO@Lip-Res

[0054] For the EXO@Lip-Res synthesis process and structural diagram, please refer to [link / reference]. Figure 1 A, See electron microscope image. Figure 1 B, WB images of surface antigen detection are shown. Figure 1 C. Using a UV spectrophotometer to scan the entire wavelength range, Lip-Res (resveratrol liposomes) and EXO@Lip-Res (resveratrol-loaded exosome-liposome hybrid nanoparticles) showed a characteristic peak of Res (resveratrol) at 306 nm, while EXO and Lip showed no characteristic peak. This indicates successful loading of resveratrol onto liposomes and exosome-liposome hybrid nanoparticles. See the experimental results below. Figure 3 Phospholipids were labeled with FITC, and EXO was labeled with PKH26. The successful fusion of the mixed nanoparticles was verified by immunofluorescence imaging. See the experimental results below. Figure 2 .

[0055] The obtained product was subjected to particle size and potential measurements to characterize the nanoparticle properties. The experimental results are shown in [reference needed]. Figure 3 The particle size of exosomes (EXO) is 150.34 nm, the particle size of Lip-Res is 163.38 nm, and the particle size of EXO@Lip-Res is 330.1 nm. The particle size of exosomes (EXO) is -16.6 mV, the particle size of Lip-Res is -11.78 mV, and the particle size of EXO@Lip-Res is -15.13 mV.

[0056] Example 3: Determination of the antioxidant capacity of EXO@Lip-Res using a chemical antioxidant method

[0057] During respiration, organisms produce energy called ATP. In this process, about 2% of the inhaled oxygen is converted into reactive oxygen species, commonly known as free radicals. Free radicals cause cell membrane oxidation and promote aging and DNA damage. Recent research indicates that arteriosclerosis, brain and cardiovascular diseases, and cancer are all related to oxidative reactions. This experiment uses a spectrophotometer to measure the ability of a sample to scavenge free radicals.

[0058] (1) DPPH free radical scavenging capacity determination

[0059] Pipette 3 ml of a 0.1 mmol / L DPPH solution (absorbance at 517 nm = 0.8) and mix it with 1 ml of sample / water in a centrifuge tube. Incubate at 25°C in the dark for 30 min. Measure the absorbance (A) of the sample at 517 nm using a UV spectrophotometer. 样品 The absorbance of water at 517 nm is A 对照 DPPH free radical scavenging rate (%) = (A 对照 -A 样品 ) / A 对照 ×100%.

[0060] DPPH is a stable free radical in organic solvents. In the presence of free radical scavengers, the single electrons of DPPH are captured, causing its color to lighten and its absorbance at the wavelength of maximum absorption to decrease linearly. The decrease in absorbance indicates an increase in antioxidant activity, thus evaluating the antioxidant capacity of the test samples. The results showed that exosomes possessed only weak antioxidant capacity, while Lip-Res exhibited good antioxidant capacity at all concentrations. The exosome-liposome hybrid nanoparticles loaded with resveratrol retained the antioxidant capacity of Lip-Res, and this modification of exosomes improved their antioxidant capacity. For detailed experimental results, please refer to [link to relevant documentation]. Figure 4 .

[0061] (2) Determination of ABTS free radical scavenging ability

[0062] Mix 0.0384 g ABTS with 0.0134 g potassium persulfate solution and incubate at room temperature in the dark for 16 h to prepare an ABTS solution. Dilute the ABTS solution with water to an absorbance of 0.70 (±0.02) at 734 nm. Mix 1 ml of appropriately diluted sample / water with 3 ml of ABTS solution and react at 30 °C in the dark for 6 min. The absorbance A at 734 nm is measured using a UV spectrophotometer. 样品 The absorbance of water at 734 nm is A 对照 ABTS free radical scavenging rate (%) = (A 对照 -A 样品 ) / A 对照 ×100%.

[0063] If a substance exhibits a decrease in absorbance at 734 nm after being added to an ABTS radical scavenging solution, it indicates that the substance possesses free radical scavenging activity and is classified as an antioxidant. This method, known as the ABTS free radical scavenging method, can be used to evaluate the antioxidant capacity of plant (or herbal extracts) and pure compounds. Results showed that exosomes only exhibit antioxidant capacity at high concentrations, while Lip-Res demonstrates excellent antioxidant capacity even at lower concentrations. Exosome-liposome hybrid nanoparticles loaded with resveratrol retained the antioxidant capacity of Lip-Res, thus modifying exosomes in this way improved their antioxidant capacity. For detailed experimental results, please refer to [link to relevant documentation]. Figure 5 .

[0064] (3) Hydrogen peroxide scavenging capacity determination

[0065] Prepare a 40 mmol / L H₂O₂ solution: Dissolve H₂O₂ in 0.1 mmol / L phosphate buffer (pH 7.4). Add 3.4 mL of sample and 0.6 mL of H₂O₂ solution to each test tube, and measure the absorbance at 230 nm. Hydrogen peroxide scavenging rate (%) = (A 对照 -A 样品 ) / A 对照 ×100%.

[0066] H2O2, as an oxidant, has a toxic effect on cells. Studies have shown that when nerve cells are cultured with H2O2 for 24 hours, the cell viability drops to 60% when the H2O2 concentration reaches 250 μM; and when the H2O2 concentration reaches 500 μM, the cells can hardly survive. The scavenging effect of H2O2 can reflect the antioxidant capacity of a drug. The results show that exosomes have only weak antioxidant capacity, while Lip-Res has good antioxidant capacity at all concentrations, and the antioxidant capacity increases with increasing concentration. The exosome-liposome hybrid nanoparticles loaded with resveratrol retain the antioxidant capacity of Lip-Res, and this modification of exosomes improves their antioxidant capacity. See the experimental results below. Figure 6 .

[0067] (4) FRAP determination

[0068] FRAP working solution preparation: Sodium diacetate solution (300 mmol / L): TPTZ solution (10 mmol / L, prepared with 40 mmol / L HCl solution): FeCl3 aqueous solution (20 mmol / L) in a 10:1:1 ratio. Accurately weigh 1.87 g of anhydrous sodium acetate, dissolve it in 16 mL of anhydrous acetic acid, and then add water to a final volume of 1 L. Prepare 10 mmol / L TPTZ solution: Weigh 0.1594 g of TPTZ and dissolve it in 50 mL of 40 mmol / L HCl. Prepare FRAP working solution: Take 25 mL of acetate-sodium acetate buffer, 2.5 mL of TPTZ solution, and 2.5 mL of 20 mmol / L FeCl3 solution, and mix well. Pipette 300 μL of water and 100 μL of sample into a centrifuge tube, add 3 mL of FRAP working solution, incubate at 37 °C in the dark for 4 min, and measure the absorbance at 593 nm using UV.

[0069] The principle behind the FRAP method for determining total antioxidant capacity is that, under acidic conditions, antioxidants can reduce Fe. 3+ -TPTZ produces blue-violet Fe 2+ Fe-TPTZ was then measured at 593 nm to obtain the total antioxidant capacity of the sample. The results showed that exosomes possessed only extremely weak antioxidant capacity, while the antioxidant capacity of Lip-Res increased with increasing concentration. The exosome-liposome hybrid nanoparticles loaded with resveratrol retained the antioxidant capacity of Lip-Res, thus modifying exosomes in this way improved their antioxidant capacity. Experimental results are shown below. Figure 7 .

[0070] Example 4: Determination of the antioxidant capacity of EXO@Lip-Res using a cellular antioxidant method

[0071] The Beyotime Reactive Oxygen Species Assay Kit (ROSAssay Kit) is a kit for detecting reactive oxygen species using the fluorescent probe DCFH-DA. DCFH-DA itself is non-fluorescent and can freely cross the cell membrane. Once inside the cell, it is hydrolyzed by intracellular esterases to generate DCFH. DCFH is impermeable to the cell membrane, making it easy for the probe to be loaded into the cell. Intracellular reactive oxygen species oxidize the non-fluorescent DCFH to generate fluorescent DCF. Detecting the fluorescence of DCF indicates the level of intracellular reactive oxygen species.

[0072] Periodontal ligament stem cells (PDLSCs) or RAW264.7 cells were pretreated with the drug overnight. Experimental groupings: CON group: blank control CON was treated with PBS; EXO group: final EXO concentration was 10 μg / mL (10 μL of 1 mg / mL EXO solution was added per mL of culture medium, resulting in a final cell concentration of 10 μg / mL); Lip-RES group: final Lip-Res concentration was 5 μg / mL (10 μL of 500 μg / mL Lip-Res solution was added per mL of culture medium, resulting in a final cell concentration of 5 μg / mL). This concentration was based on preliminary experiments, and CCK8 analysis showed this to be a safe concentration with no significant toxicity to cells; EXO@Lip-RES group: obtained by incubation of 1 mg / mL EXO solution and 500 μg / mL Lip-Res solution at a 1:1 volume ratio, following the method in Example 1, with 10 μL added per mL of culture medium; drug treatment time was approximately 12 h for all groups. Then, add hydrogen peroxide to stimulate for 30 min; dilute DCFH-DA with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10 μmol / L. Remove the cell culture medium and add an appropriate volume of diluted DCFH-DA. The volume added should be sufficient to fully cover the cells; generally, at least 1 mL of diluted DCFH-DA should be added to each well of a six-well plate. Incubate at 37°C for 20 min. Wash the cells three times with serum-free cell culture medium to thoroughly remove any DCFH-DA that has not yet entered the cells. Detect reactive oxygen species levels using a fluorescence microscope, a fluorescence microplate reader, and a flow cytometer.

[0073] See fluorescence images of PDLSCs cells. Figure 8 For the results of the fluorescence microplate reader test, please refer to [link / reference]. Figure 9 ROS fluorescence staining showed that EXO@Lip-Res effectively eliminated ROS in periodontal ligament stem cells. Fluorescent microplate reader analysis also showed that EXO@Lip-Res effectively eliminated ROS in periodontal ligament stem cells. Flow cytometry results for RAW264.7 cells can be found in [link to relevant documentation]. Figure 10ROS flow cytometry showed that EXO@Lip-Res could effectively scavenge ROS, with this effect largely attributed to Lip-Res. EXO may enhance its ROS scavenging ability by increasing cellular drug uptake. See fluorescence images of RAW264.7 cells. Figure 11 For the results of the fluorescence microplate reader test, please refer to [link / reference]. Figure 12 ROS fluorescence staining showed that EXO@Lip-Res could effectively remove ROS from RAW264.7 cells, and fluorescence microplate reader readings also showed that EXO@Lip-Res could effectively remove ROS from RAW264.7 cells.

[0074] Example 5: Study on the effect of EXO@Lip-Res on macrophage polarization

[0075] Macrophage functional polarization plays a crucial role in the quality and efficiency of the body's resistance to pathogenic infection and damage repair. Macrophages exhibit different polarization modes. For example, when the body encounters intracellular pathogens, macrophages exhibit classical polarization (M1) and express corresponding pro-inflammatory factors such as TNF-α, IL-12, and iNOS to clear pathogens. Simultaneously, M1 cells produce inducible nitric oxide synthase (iNOS) to synthesize large amounts of NO intracellularly. Typical M1 cells are effector cells in type I responses, killing microorganisms and tumor cells and secreting large amounts of inflammatory factors. On the other hand, M2 macrophages limit inflammatory responses and type I adaptive immunity, clear residues, promote angiogenesis, and participate in tissue remodeling and repair. Therefore, precise regulation of macrophage polarization is essential for maintaining the body's health. Because the M2 subtype of cells can limit inflammatory responses, we generally pay more attention to the expression of the M2 subtype during tissue repair.

[0076] This technical approach detected the mRNA levels of CD86, a marker molecule in M1 macrophages, and iNOS, a marker molecule (reflecting the effect of M1 macrophages on promoting the secretion of inflammatory factors). The mRNA levels of CD206, a marker molecule in M2 macrophages, and Arg1, a marker molecule, were also detected (increased Arg1 levels indicate a stronger anti-inflammatory effect). Inducing M2 macrophage polarization and increasing the ARG1 / iNOS ratio can thereby alleviate various inflammatory conditions.

[0077] In this technical solution, the experimental procedure is as follows: RAW264.7 cells were cultured to 60%-70% confluence, treated with 10 μg / ml LPS for 8 hours, the medium was changed, and each group was treated with drugs. The blank control group was treated with PBS. The final concentration of EXO in the other groups was 10 μg / mL, and the final concentration of Lip-Res was 5 μg / mL. The EXO@Lip-Res group was prepared by mixing the two in a 1:1 ratio (refer to Example 1), and the final concentration was the same as above. For the specific grouping and drug treatment, please refer to Example 4. Total RNA was extracted from the cells 24 hours after treatment and detected. The experimental results are shown below. Figure 13 While EXO@Lip-Res treatment, compared to Lip-Res and EXO treatments, did not significantly affect the macrophage surface marker CD86 (the increase in the number of M1 macrophages was not significant), it significantly inhibited the expression of inducible nitric oxide synthase (iNOS), thus suppressing the activity of M1 macrophages and alleviating the inflammatory response. Furthermore, for M2 macrophages, EXO@Lip-Res treatment significantly increased the mRNA levels of Arg1 and the surface marker CD206, indicating a significant increase in both the number and activity of M2 macrophages.

[0078] Figure 13 The specific data corresponding to this are shown in the table below (Table 1):

[0079] Table 1: Experimental results of the study on the effect of EXO@Lip-Res on macrophage polarization (with CON data as the baseline, i.e., "1", the relative mRNA expression level was calculated; N=3; data are presented in mean±SEM format).

[0080] markers CON EXO Lip-RES EXO@Lip-RES CD86 1.00±0.08 0.40±0.01 0.55±0.04 0.39±0.06 iNOS 1.00±0.10 0.96±0.12 0.95±0.07 0.31±0.06 Arg1 1.00±0.03 2.54±0.15 2.85±0.15 4.22±0.12 CD206 1.00±0.07 1.58±0.12 1.47±0.16 2.43±0.09

[0081] Regarding the expression of nitric oxide synthase (iNOS), the addition of EXO resulted in a decrease of 0.04 (1-0.96=0.04) in iNOS expression, the addition of Lip-RES resulted in a decrease of 0.05 (1-0.95=0.05) in iNOS expression, and the addition of EXO@Lip-RES resulted in a decrease of 0.61 (1-0.31=0.61) in iNOS expression. The effect of EXO@Lip-RES was stronger than the sum of the effects of EXO and Lip-RES, indicating that in specific EXO@Lip-RES lipid nanoparticles, EXO and Res have a synergistic effect on inhibiting macrophage M1, thereby reducing the level of inflammatory response.

[0082] Regarding the mRNA level of the surface marker CD206, the addition of EXO increased CD206 expression by 0.58 (1.58-1=0.58), the addition of Lip-RES increased CD206 expression by 0.47 (1.47-1=0.47), and the addition of EXO@Lip-RES increased CD206 expression by 1.43 (2.43-1=1.43). The effect of EXO@Lip-RES is stronger than the sum of the effects of EXO and Lip-RES, indicating that in specific EXO@Lip-RES lipid nanoparticles, EXO and Res have a synergistic effect on activating macrophage M2, thereby reducing the level of inflammatory response.

[0083] Existing technologies have attempted to combine exosomes and resveratrol, but none have explored the combined use of dental pulp stem cell exosomes and resveratrol. The composition of exosomes produced by different cells varies significantly, resulting in different effects when combined with resveratrol. Existing technologies have not reported the phenomenon that dental pulp stem cell exosomes and resveratrol can jointly influence macrophage polarization; this technical solution is the first to report this phenomenon. Furthermore, in this technical solution, dental pulp stem cell exosomes and resveratrol are not simply mixed, nor is it an attempt to load resveratrol onto exosomes; the aforementioned methods of combined use cannot effectively combine the two substances and achieve the desired effect. Due to the poor water solubility of resveratrol, ordinary combined methods are ineffective. The inventors have previously attempted to directly mix resveratrol and exosomes without preparing a Lip-Res solution. Although exosomes are also a type of lipid vesicle, they have a poor adsorption effect on resveratrol, which means that exosomes cannot fully bind with resveratrol, and the dissolution effect of resveratrol is difficult to improve effectively.

[0084] In this technical solution, resveratrol is first loaded onto liposome nanoparticles, and then exosomes are loaded onto the liposome particles through incubation. During incubation, the resveratrol is loaded onto the exosomes via the liposomes, allowing for better absorption and utilization by cells. This ensures the effective binding of resveratrol and exosomes, resulting in a synergistic effect, rather than simply the two drugs acting independently. The inventors also attempted to use a mixture of EXO and Lip-Res solutions directly on macrophages, without incubation at 37°C, building upon the EXO@Lip-Res group in this experiment. In this case, resveratrol and exosomes were not fully loaded, and the exosome vesicles did not fully fuse with the lipid nanoparticles, reducing the cell utilization of both active ingredients and decreasing the effect on macrophages. The results showed a significant decrease in Arg1 and CD206 mRNA levels compared to the EXO@Lip-RES group formed after incubation, indicating that the synergistic effect between EXO and Lip-Res was no longer present. For EXO@Lip-Res to exert its immunomodulatory effects, appropriate nanoparticle loading is required.

[0085] Example 6: Study on the effect of EXO@Lip-Res on LPS-induced inflammation in PDLSCs

[0086] The specific process for establishing the LPS-induced PDLSC inflammation model was as follows: PDLSCs were cultured to 60%-70% confluence and treated with 10 μg / ml LPS for 12 hours. Each group was treated with medication; the blank control group received PBS, while the final concentrations of EXO and Lip-Res in the other groups were 10 μg / mL and 5 μg / mL, respectively. EXO@Lip-Res was prepared by mixing the two at a 1:1 ratio, with the final effective concentrations as described above. For details on grouping and drug treatment, please refer to Example 4. Total RNA was extracted from cells 24 hours after treatment and total protein was extracted from cells 48 hours after treatment.

[0087] TGF-β is an immunosuppressant that inhibits T and B cell proliferation, reduces the expression of major histocompatibility complex (MHC) class II antigens, and alleviates local inflammatory responses. IL-8 is a chemotactic inflammatory cytokine that is produced under conditions of infection, inflammation, local ischemia, and trauma, and plays a role in chemotactically attracting neutrophil aggregation in tissues. Using conventional qRT-PCR to detect the mRNA levels of these two factors, EXO@Lip-Res significantly promoted the level of the anti-inflammatory factor TGF-β and significantly reduced the level of the pro-inflammatory factor IL-8. The experimental results are shown below. Figure 14 .

[0088] IL-1β, a key pro-inflammatory cytokine, participates in various autoimmune inflammatory responses and multiple cellular activities, including cell proliferation, differentiation, and apoptosis. TNF-α, a pleiotropic systemic inflammatory mediator, is also a pro-inflammatory cytokine involved in immune inflammatory responses. Using conventional Western blotting to detect the protein levels of these two factors, EXO@Lip-Res significantly reduced the levels of the pro-inflammatory factors IL-1β and TNF-α. (See attached results). Figure 15 .

[0089] Figure 14 and Figure 15 The specific data corresponding to this are shown in the following tables (Table 2 and Table 3):

[0090] Table 2: Results of the study on the effect of EXO@Lip-Res on inflammation in PDLSCs by qRT-PCR (with CON data as the baseline, i.e., "1", the relative mRNA expression level was calculated; N=3; data are presented in mean±SEM format).

[0091]

[0092]

[0093] Regarding TGF-β expression, the addition of EXO increased TGF-β expression by 0.27 (1.27-1=0.27), the addition of Lip-RES increased it by 0.43 (1.43-1=0.43), and the addition of EXO@Lip-RES increased it by 1.27 (2.27-1=1.27). The effect of EXO@Lip-RES was stronger than the sum of the effects of EXO and Lip-RES, indicating that in specific EXO@Lip-RES lipid nanoparticles, EXO and Res have a synergistic effect on increasing the level of the anti-inflammatory factor TGF-β in PDLSCs, thereby reducing the level of inflammatory response and alleviating local inflammatory response.

[0094] Table 3: Results of the study on the effect of EXO@Lip-Res on inflammation in PDLSCs by Western blotting (N=3; data are presented in mean ± SEM format).

[0095] markers CON EXO Lip-RES EXO@Lip-RES IL-1β 1.13±0.03 1.18±0.05 0.83±0.02 0.63±0.02 TNF-α 1.14±0.10 1.08±0.04 0.83±0.09 0.70±0.02

[0096] Regarding IL-1β protein levels, the addition of EXO resulted in a decrease of -0.05 (1.13 - 1.18 = -0.05), the addition of Lip-RES resulted in a decrease of 0.05 (1.13 - 0.83 = 0.3), and the addition of EXO@Lip-RES resulted in a decrease of 0.61 (1.13 - 0.63 = 0.5). The effect of EXO@Lip-RES was stronger than the combined effect of EXO and Lip-RES, indicating that in specific EXO@Lip-RES lipid nanoparticles, EXO and Res have a synergistic effect in reducing the level of the pro-inflammatory factor IL-1β, thereby reducing the level of inflammatory response.

[0097] Regarding TNF-α protein levels, the addition of EXO resulted in a decrease of 0.06 (1.14-1.08=0.06), the addition of Lip-RES resulted in a decrease of 0.31 (1.14-0.83=0.31), and the addition of EXO@Lip-RES resulted in a decrease of 0.44 (1.14-0.70=0.44). The effect of EXO@Lip-RES was stronger than the combined effect of EXO and Lip-RES, indicating that in specific EXO@Lip-RES lipid nanoparticles, EXO and Res have a synergistic effect in reducing the level of the pro-inflammatory factor TNF-α, thereby reducing the level of inflammatory response.

[0098] Example 7: Animal experiments to determine the therapeutic effect of EXO@Lip-Res on periodontitis

[0099] Establishment of a rat model of periodontitis: A rat was placed in a sealed container with a 4% (v / v) isoflurane flow rate until fully anesthetized. A 0.20 mm diameter stainless steel wire was tied around the bilateral maxillary first molars of the isoflurane-anesthetized rat. LPS (10 μg / mL) was injected into the periodontal pocket every other day for 5 consecutive days. The pocket was ligated for 30 days to induce periodontitis. Twenty-four SD rats were then randomly divided into four groups (n=6 per group): a control group (n=6), in which stainless steel ligatures were removed after periodontitis was induced, and PBS was injected into the periodontal pockets every other day; an EXO group, in which stainless steel ligatures were removed after periodontitis was induced, and EXO (100 μg / mL) was injected into the periodontal pockets every other day; a Lip-Res group, in which EXO in the EXO group was replaced with Lip-Res 50 μg / mL; and an EXO@Lip-Res group, in which 100 μg / mL EXO solution and 50 μg / mL Lip-Res solution were mixed at a 1:1 volume ratio and prepared according to Example 1, and then injected into the periodontal pockets. The experimental and control groups received equal volumes of injections. After 30 days, the rats were sacrificed, the maxillary alveolar bone was dissected, and fixed with 4% paraformaldehyde.

[0100] See the experimental results. Figure 16 EXO@Lip-Res significantly reduced alveolar bone loss (CEJ-ABC). Figure 16 E and D) show that the periodontal ligament fibers are neatly arranged, indicating that the drug we prepared can effectively treat periodontitis in rats. Figure 16 In D, the EXO concentration in the EXO@Lip-Res group was half that of the EXO group, and the Lip-Res concentration in the EXO@Lip-Res group was half that of the Lip-Res group. If there were no synergistic effect, and only the additive effects of individual components, theoretically, the combined use of half-concentration EXO and EXO@Lip-Res would not be more effective than using the full concentration alone. However, experimental data showed that the CEJ-ABC value of the EXO@Lip-Res group was significantly lower than that of the EXO and Lip-Res groups. This indicates that the EXO@Lip-Res prepared using this method ensured the synergistic effect of EXO and Res in reducing alveolar bone loss and treating periodontitis, exceeding the expectations of the technicians and achieving unexpected technical results. Micro-CT showed the alveolar bone repair status, such as... Figure 16 As shown in A, B, and C. Figure 16 BV / TV is the relative bone volume or bone volume fraction (in %). Figure 16 Tb.Th in C refers to trabecular thickness, which is the average thickness of the trabecular bone. The EXO@Lip-Res group showed a more significant effect in inhibiting alveolar bone loss.

[0101] Example 8: In vivo safety assessment

[0102] HE staining of major organs and physiological and biochemical tests of blood in experimental rats; experimental results are shown in [reference needed]. Figure 17 and Figure 18 The experimental results show that the nanoparticles in this formulation did not significantly affect HE staining of major organs or blood physiological and biochemical indicators in rats, indicating high safety. In vivo safety assessments showed that the mixed nanoparticles caused no damage to major organs at therapeutic doses.

[0103] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. Resveratrol-loaded exosome-liposome hybrid nanoparticles for treating periodontitis, characterized by: [Further details needed] Its raw materials include lipids, resveratrol, and exosomes derived from dental pulp stem cells; It is prepared by the following method: S1: Dissolve PBA-COOH, EDC, and NHS in DMSO, react, and then add DSPE-PEG. 2K -NH2; after reaction, dialysis and lyophilization, Lip lyophilized powder is obtained; S2: Dissolve Lip lyophilized powder and resveratrol in DMSO, then add triethylamine; after reaction, dialysis and lyophilization, obtain Lip-Res lyophilized powder; S3: Dissolve Lip-Res lyophilized powder in DMSO, dialyze to obtain Lip-Res solution, mix with exosome solution and react to obtain Exo@Lip-Res.

2. The resveratrol-loaded exosome-liposome hybrid nanoparticles for treating periodontitis, as described in claim 1, characterized in that: PBA-COOH, EDC, NHS and DSPE-PEG 2K The ratio of -NH2 used is 1:1.2:1.2:

1.

3. The method for preparing resveratrol-loaded exosome-liposome hybrid nanoparticles for treating periodontitis with the effect of preventing alveolar bone loss, as described in any one of claims 1 or 2, characterized in that: The following steps are performed sequentially: S1: Dissolve PBA-COOH, EDC, and NHS in DMSO, react, and then add DSPE-PEG. 2K -NH2; after reaction, dialysis and lyophilization, Lip lyophilized powder is obtained; S2: Dissolve Lip lyophilized powder and resveratrol in DMSO, then add triethylamine; after reaction, dialysis and lyophilization, obtain Lip-Res lyophilized powder; S3: Dissolve Lip-Res lyophilized powder in DMSO, dialyze to obtain Lip-Res solution, mix with exosome solution and react to obtain Exo@Lip-Res.

4. The method for preparing resveratrol-loaded exosome-liposome hybrid nanoparticles for treating periodontitis with the effect of preventing alveolar bone loss according to claim 3, characterized in that: In S1, PBA-COOH, EDC, NHS, and DSPE-PEG are present. 2K The ratio of -NH2 used is 1:1.2:1.2:1; PBA-COOH, EDC, and NHS are dissolved in DMSO and reacted at 40℃ for 4-6 hours under magnetic stirring.

5. The method for preparing resveratrol-loaded exosome-liposome hybrid nanoparticles for treating periodontitis with the effect of preventing alveolar bone loss according to claim 4, characterized in that: In S1, add DSPE-PEG 2K After -NH2, react at 40℃ for 10-12 hours.

6. The method for preparing resveratrol-loaded exosome-liposome hybrid nanoparticles for treating periodontitis with the effect of preventing alveolar bone loss according to claim 5, characterized in that: In S2, the mass ratio of Lip lyophilized powder to resveratrol is 14:

1.

7. The method for preparing resveratrol-loaded exosome-liposome hybrid nanoparticles for treating periodontitis with the effect of preventing alveolar bone loss according to claim 6, characterized in that: In S2, the amount of DMSO used is 2 mL for every 5 mg of resveratrol; the volume ratio of DMSO to triethylamine is 200:

1.

8. The method for preparing resveratrol-loaded exosome-liposome hybrid nanoparticles for treating periodontitis with the effect of preventing alveolar bone loss according to claim 7, characterized in that: In S3, the volume ratio of Lip-Res solution to exosome solution was 1:1, and the mixture was incubated at 37°C for 4-6 hours.

9. The use of resveratrol-loaded exosome-liposome hybrid nanoparticles for treating periodontitis, as described in any one of claims 1 or 2, for the purpose of preventing alveolar bone loss, in the preparation of a medicament for treating periodontitis.

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