A ruthenium-dihydromyricetin, an apoptosis vesicle loaded with ruthenium-dihydromyricetin, and a preparation method and application thereof

Abstract

The application provides a ruthenium-dihydroquercetin, an apoptosis vesicle loaded with the ruthenium-dihydroquercetin and a preparation method and application thereof, and belongs to the technical field of antibacterial and anti-inflammatory product preparation.The application provides a ruthenium-dihydroquercetin, which comprises the following raw materials: 50-80 parts of polyvinylpyrrolidone, 20-30 parts of ruthenium trichloride and 5-15 parts of dihydroquercetin.The ruthenium-dihydroquercetin obtained by the method has good dispersity, and the method is simple and the product morphology is uniform.In addition, the ruthenium-dihydroquercetin has antibacterial and anti-inflammatory effects, has low toxicity and high biological safety in the antibacterial and anti-inflammatory process.The apoptosis vesicle loaded with the ruthenium-dihydroquercetin prepared by the application can further improve the treatment effect on bacteria and inflammation in cooperation with the ruthenium-dihydroquercetin, and can reduce the use dosage of nanometer drugs.The apoptosis vesicle loaded with the ruthenium-dihydroquercetin has a wide application prospect in the preparation of antibacterial and anti-inflammatory products.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of antibacterial and anti-inflammatory product preparation, and particularly relates to a ruthenium-dihydroquercetin, an apoptosis vesicle loaded with the ruthenium-dihydroquercetin, and a preparation method and application thereof. BACKGROUND

[0002] Bacterial infected wounds have a high incidence in clinical practice. Whether it is surgical incisions, skin abrasions, or burns, different types of trauma are prone to bacterial invasion. When the integrity of the skin or tissue is damaged, bacteria have the opportunity to invade the body, leading to the occurrence of infection. These infections not only affect the healing rate of the wound, but also can trigger systemic inflammatory responses such as sepsis, septicemia, and even threaten the life of the patient. The pathogenic bacteria of bacterial infected wounds are diverse, and have different drug resistance and pathogenicity. Common pathogenic bacteria include Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, etc., which can enter the body through different routes such as wound contamination, blood circulation, etc. These pathogenic bacteria produce toxins and enzymes during the infection process, which destroy the normal structure and function of the tissue, leading to symptoms such as redness, swelling, pain, and exudation of the wound. Antibiotics are the main treatment choice for bacterial infection, however, antibiotics are removed by blood in the body circulation, the physical barrier of bacterial cell wall and cell membrane hinders the penetration of antibiotics, leading to low accumulation of antibiotics in bacterial cells. Unfortunately, because there is no effective vaccine for popular bacterial pathogens such as Escherichia coli and Staphylococcus aureus, antibiotic prophylaxis is the only clinically available option to prevent most bacterial infections after surgery. Although antibiotics are crucial in combating bacterial infections and eradicating drug-resistant strains, their development is hindered by factors such as long cycle, unpredictable side effects, and the emergence of drug resistance. Therefore, there is an urgent need in the art to provide a strategy to address the clinical challenges of antibiotic use (unsatisfactory therapeutic effect, long-term toxicity, and induction of antibiotic resistance). SUMMARY

[0003] Therefore, one of the purposes of the present application is to provide a ruthenium-dihydroquercetin with antibacterial and anti-inflammatory effects and a preparation method thereof. The ruthenium-dihydroquercetin prepared by the present application has lower toxicity and higher biological safety in the process of antibacterial and anti-inflammatory.

[0004] The second purpose of the present application is to provide an apoptosis vesicle loaded with ruthenium-dihydroquercetin and a preparation method thereof. The apoptosis vesicle cooperates with the ruthenium-dihydroquercetin to further improve the treatment effect on bacteria and inflammation, and has high biological safety.

[0005] The third purpose of the present application is to provide the application of the ruthenium-dihydroquercetin or the apoptosis vesicle loaded with the ruthenium-dihydroquercetin in the preparation of antibacterial and anti-inflammatory products.

[0006] To achieve the above object, the present application provides the following technical solutions.

[0007] The present application provides a kind of ruthenium-dihydro myricetin, including the following raw materials according to weight fraction: polyvinylpyrrolidone 50~80 parts, ruthenium trichloride 20~30 parts, dihydro myricetin 5~15 parts.

[0008] The present application also provides the preparation method of the above-mentioned ruthenium-dihydro myricetin, comprising the following steps: polyvinylpyrrolidone, ruthenium trichloride and dihydro myricetin are dispersed into liquid solvent respectively, polyvinylpyrrolidone solution, ruthenium trichloride solution and dihydro myricetin solution are obtained;Ruthenium trichloride solution is added to polyvinylpyrrolidone solution, and mixed liquid is obtained;Dihydro myricetin solution is added to mixed liquid and stirred, dialysis is taken after dialysis solution, purification is taken to precipitate and wash, and freeze-drying is obtained ruthenium-dihydro myricetin.

[0009] Preferably, the liquid solvent is at least one of water, ethanol, methanol, acetone, N-methylpyrrolidone, dimethyl sulfoxide, N,N-dimethylformamide, dichloromethane, chloroform, ethylene glycol, glycerol, isopropyl alcohol and polyethylene glycol 200.

[0010] Preferably, the weight-volume ratio of polyvinylpyrrolidone to liquid solvent is 50~80 mg:1~20 mL;The weight-volume ratio of ruthenium trichloride to liquid solvent is 10~50 mg:1~20 mL;The weight-volume ratio of dihydro myricetin to liquid solvent is 5~15 mg:1~5 mL.

[0011] Preferably, the stirring time is more than 1 h;The dialysis method is dialysis bag dialysis, the molecular weight of the dialysis bag is 6000~12000 daltons, and the dialysis time is 12~24 h.

[0012] Preferably, the purification method includes centrifugation, ultrafiltration or rotary evaporation;The rotation speed of centrifugation is 8000~20000 rpm, and the centrifugation time is 5~30 min.

[0013] The present application also provides a kind of apoptosis vesicle loaded with ruthenium-dihydro myricetin, which is prepared by the following method: in the process of inducing mesenchymal stem cell apoptosis, the above-mentioned ruthenium-dihydro myricetin or the ruthenium-dihydro myricetin prepared by the above preparation method is added to culture, the supernatant is centrifuged, and the apoptosis vesicle loaded with ruthenium-dihydro myricetin is obtained.

[0014] Preferably, the agent for inducing mesenchymal stem cell apoptosis includes staurosporine;The ruthenium-dihydro myricetin is added when mesenchymal stem cell apoptosis is induced for 2~10 h;The culture time is 1~10 h;The centrifugal force of centrifugation is 10000~20000 g, and the centrifugation time is 10~30 min.

[0015] Preferably, the mesenchymal stem cells comprise umbilical cord mesenchymal stem cells.

[0016] The application also provides the use of the above-mentioned ruthenium-dihydroquercetin, the ruthenium-dihydroquercetin prepared by the above-mentioned preparation method or the above-mentioned apoptotic vesicles in the preparation of an antibacterial and anti-inflammatory product.

[0017] The application has the following beneficial effects:

[0018] The application prepares ruthenium-dihydroquercetin with good dispersity, and the method is simple and the product has uniform morphology. In addition, the ruthenium-dihydroquercetin prepared by the application has antibacterial and anti-inflammatory effects, and has low toxicity and high biological safety in the antibacterial and anti-inflammatory process.

[0019] The apoptotic vesicles loaded with ruthenium-dihydroquercetin prepared by the application can further improve the treatment effect on bacteria and inflammation in cooperation with ruthenium-dihydroquercetin, and can reduce the use dose of nanomedicine. The apoptotic vesicles are derived from cells and also have high biological safety, so the apoptotic vesicles loaded with ruthenium-dihydroquercetin prepared by the application have wide application prospects in the preparation of antibacterial and anti-inflammatory products. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 It is a transmission electron microscope observation result of ruthenium-dihydroquercetin;

[0021] Figure 2 It is a transmission electron microscope observation result of umbilical cord mesenchymal stem cell-derived apoptotic vesicles;

[0022] Figure 3 It is a transmission electron microscope observation result of apoptotic vesicles loaded with ruthenium-dihydroquercetin;

[0023] Figure 4 It is a verification result of the enzyme-like activity of ruthenium-dihydroquercetin, wherein the left graph is the TMB result and the right graph is the OPD result;

[0024] Figure 5 It is the free radical scavenging effect of ruthenium-dihydroquercetin under weak alkaline conditions, wherein the left graph is the DPPH result and the right graph is the ABTS result;

[0025] Figure 6 It is the catalase activity result of ruthenium-dihydroquercetin;

[0026] Figure 7 It is the agar plate photographing result of the in-vitro antibacterial effect, wherein 1 is the Control group, 2 is the Apovs group, 3 is the Ru-DMY group, and 4 is the Apovs@Ru-DMY group;

[0027] Figure 8Statistical results of in vitro antibacterial effect, in which *** represents P<0.001;

[0028] Figure 9 MI index of macrophages, from top to bottom, the first row to the third row are the staining results of DAPI, CD86 and Merge, respectively, in which 1 represents the Control group, 2 represents the LPS group, 3 represents the Apovs group, 4 represents the Ru-DMY group, and 5 represents the Apovs@Ru-DMY group;

[0029] Figure 10 M2 index of macrophages, from top to bottom, the first row to the third row are the staining results of DAPI, CD206 and Merge, respectively, in which 1 represents the Control group, 2 represents the LPS group, 3 represents the Apovs group, 4 represents the Ru-DMY group, and 5 represents the Apovs@Ru-DMY group. DETAILED DESCRIPTION

[0030] The application provides a ruthenium-dihydroquercetin, which comprises the following raw materials in parts by weight: polyvinylpyrrolidone (PVP) 50-80 parts, ruthenium trichloride (RuCl3) 20-30 parts and dihydroquercetin 5-15 parts.

[0031] The application does not have special limitations on the specific sources of the above raw materials. The ruthenium-dihydroquercetin provided by the application is a product of coordination of a metal and an organic substance, which combines the antibacterial and anti-inflammatory abilities of the metal and the natural product, and has good biological safety. In the ruthenium-dihydroquercetin provided by the application, the amount of PVP is preferably 55-75 parts, more preferably 60-70 parts, and further preferably 66 parts; the amount of RuCl3 is preferably 22-28 parts, more preferably 24-26 parts; and the amount of dihydroquercetin is preferably 6-12 parts, more preferably 8-10 parts.

[0032] The application further provides a preparation method of the above ruthenium-dihydroquercetin, which comprises the following steps: dispersing PVP, RuCl3 and dihydroquercetin into liquid solvents respectively to obtain PVP solution, RuCl3 solution and dihydroquercetin solution; adding the RuCl3 solution into the PVP solution to obtain a mixed solution; adding the dihydroquercetin solution into the mixed solution and stirring, dialyzing to obtain a dialyzed solution, purifying to obtain a precipitate, washing, and freeze-drying to obtain the ruthenium-dihydroquercetin.

[0033] In the present application, the liquid solvent is preferably at least one of water, ethanol, methanol, acetone, N-methylpyrrolidone, dimethyl sulfoxide, N,N-dimethylformamide, dichloromethane, chloroform, ethylene glycol, glycerol, isopropyl alcohol and polyethylene glycol 200. In the present application, the weight volume ratio of PVP to the liquid solvent is preferably 50-80 mg: 1-20 mL, more preferably 60-70 mg: 3-15 mL, and most preferably 66 mg: 5 mL; the weight volume ratio of RuCl3 to the liquid solvent is preferably 10-50 mg: 1-20 mL, more preferably 20-40 mg: 1-10 mL, and most preferably 30 mg: 1 mL; and the weight volume ratio of dihydromyricetin to the liquid solvent is preferably 5-15 mg: 1-5 mL, more preferably 8-12 mg: 1-3 mL, and most preferably 10 mg: 1 mL. In the present application, the RuCl3 solution is preferably added dropwise to the PVP solution. In the present application, the stirring time is preferably more than 1 h, and more preferably 1-100 h; the dialysis method is preferably dialysis in water, and the dialysis is preferably performed using a dialysis bag with a molecular weight of 6000-12000 Dalton, more preferably 7000-10000 Dalton, and most preferably 8000 Dalton, and the dialysis time is preferably 12-24 h, and more preferably 15-20 h. After the impurities are removed by dialysis, the solution in the dialysis bag is purified, and the purification method preferably includes centrifugation, ultrafiltration or rotary evaporation; the centrifugation is preferably 8000-20000 rpm for 5-30 min, and more preferably 10000-15000 rpm for 10-15 min, and the precipitate is collected and washed. The washing is preferably performed using anhydrous ethanol and water in sequence. In the present application, the freeze-drying is preferably performed using a vacuum drying machine, and the temperature is preferably -60°C.

[0034] The ruthenium-dihydromyricetin prepared in the present application has good water dispersibility, and can be dispersed in water, physiological saline, PBS buffer, cell culture medium and the like, or can be dispersed in a sodium alginate hydrogel, a F127 hydrogel and the like.

[0035] The present application also provides an apoptosis vesicle loaded with ruthenium-dihydromyricetin, which is prepared by adding the above-mentioned ruthenium-dihydromyricetin or the ruthenium-dihydromyricetin prepared by the above-mentioned preparation method to the mesenchymal stem cells during the induction of apoptosis, culturing, centrifuging the supernatant, and obtaining the apoptosis vesicle loaded with ruthenium-dihydromyricetin.

[0036] In the present application, the agent for inducing mesenchymal stem cell apoptosis preferably comprises staurosporine (STS), and the concentration of the STS is preferably 500 nM; the ruthenium-dihydroquercetin is added when the mesenchymal stem cells are induced to apoptosis for 2-10 h. In the present application, the mesenchymal stem cells preferably comprise umbilical cord mesenchymal stem cells, which have the advantages of convenient sampling and wide sources. The culture medium used in the present application is preferably α-MEM medium, and the present application does not have special limitations on the specific temperature for culture, and the conventional culture temperature in the art can be used. In the present application, the amount of the cell apoptosis inducer added is preferably sufficient to cover all the cells on the bottom of the culture dish. In the present application, when the umbilical cord mesenchymal stem cells are induced to culture by adding the ruthenium-dihydroquercetin, the culture time is preferably 1-10 h, and more preferably 2-6 h, and the final concentration of the ruthenium-dihydroquercetin is 100 mg / mL. The supernatant is collected and centrifuged, the purpose of the centrifugation is to extract the umbilical cord mesenchymal stem cell-derived apoptosis vesicles loaded with ruthenium-dihydroquercetin, the centrifugal force of the centrifugation is preferably 10,000-20,000 g, and more preferably 12,000-18,000 g, and the centrifugation time is preferably 10-30 min, and more preferably 15-20 min.

[0037] The umbilical cord mesenchymal stem cell-derived apoptosis vesicles provided by the present application have a certain therapeutic effect on antibacterial and anti-inflammatory aspects, and the umbilical cord mesenchymal stem cell-derived apoptosis vesicles loaded with ruthenium-dihydroquercetin have a good therapeutic effect on bacteria and inflammation through synergistic effect, while reducing the dosage of the nanodrug and the bacteriostatic agent, thereby reducing the toxic side effects of the bacteriostatic agent.

[0038] The present application also provides the use of the above-mentioned ruthenium-dihydroquercetin, the ruthenium-dihydroquercetin prepared by the above-mentioned preparation method, or the above-mentioned apoptosis vesicles in the preparation of antibacterial and anti-inflammatory products. In the present application, the product preferably comprises a drug.

[0039] The technical solutions provided by the present application will be described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present application.

[0040] In the following examples, if not otherwise specified, all are conventional methods.

[0041] In the following examples, the materials, reagents, etc. used, if not otherwise specified, can be obtained from commercial channels.

[0042] Example 1

[0043] A kind of ruthenium-dihydroquercetin is prepared from 66 mg PVP, 30 mg ruthenium trichloride, 10 mg dihydroquercetin and methanol;The preparation method is:

[0044] 66mg PVP was dispersed into 5mL methanol to form a PVP solution; 30mg ruthenium trichloride was dispersed into 1mL methanol to form a ruthenium trichloride solution; the ruthenium trichloride solution was added dropwise into the PVP solution to form a mixture; 10mg dihydromyricetin was dispersed into 1mL methanol to form a dihydromyricetin solution; the dihydromyricetin solution was added into the mixture and stirred for 12h, then dialyzed in water for 12h using a dialysis bag with a molecular weight of 8000 daltons, the solution in the dialysis bag was taken and centrifuged at a high speed of 10000rpm for 5min, the precipitate was washed with anhydrous ethanol and water for 3 times respectively, and then freeze-dried in a vacuum drying machine at-60℃ to obtain ruthenium-dihydromyricetin.

[0045] The obtained ruthenium-dihydromyricetin was observed under a transmission electron microscope, and the results are shown in Figure 1 .

[0046] Example 2

[0047] A kind of ruthenium-dihydromyricetin, which was prepared from 80mg PVP, 20mg ruthenium trichloride, 15mg dihydromyricetin and methanol; the preparation method is as follows:

[0048] 80mg PVP was dispersed into 10mL acetone to form a PVP solution; 20mg ruthenium trichloride was dispersed into 10mL acetone to form a ruthenium trichloride solution; the ruthenium trichloride solution was added dropwise into the PVP solution to form a mixture; 15mg dihydromyricetin was dispersed into 5mL acetone to form a dihydromyricetin solution; the dihydromyricetin solution was added into the mixture and stirred for 24h, then dialyzed in water for 24h using a dialysis bag with a molecular weight of 10000 daltons, the solution in the dialysis bag was taken and centrifuged at a high speed of 20000rpm for 10min, the precipitate was washed with anhydrous ethanol and water for 2 times respectively, and then freeze-dried in a vacuum drying machine at-60℃ to obtain ruthenium-dihydromyricetin.

[0049] Example 3

[0050] A kind of apoptosis vesicle loaded with ruthenium-dihydromyricetin, which was prepared by the following method:

[0051] Umbilical cord mesenchymal stem cells were cultured in α-MEM medium, and then STS was added (the addition of STS ensured that all the cells on the bottom of the culture dish were covered) to induce apoptosis for 4h. Transmission electron microscopy observation showed that apoptosis vesicles were obtained, as shown in Figure 2 . Then, the ruthenium-dihydromyricetin prepared in Example 1 was added (so that the final concentration of ruthenium-dihydromyricetin was 100mg / mL) and cultured for 2h. The supernatant was collected and centrifuged at 10000g for 20min to obtain apoptosis vesicles loaded with ruthenium-dihydromyricetin. The observation results under a transmission electron microscope are shown in Figure 3 .

[0052] Example 4

[0053] A kind of apoptosis vesicle of loading ruthenium-dihydroquercetin is prepared by the following method:

[0054] Umbilical cord mesenchymal stem cells are cultured in α-MEM medium, then STS is added (add STS to ensure that all cells on the bottom of the culture dish are covered) to induce apoptosis for 6h, then ruthenium-dihydroquercetin prepared in Example 2 is added (the final concentration of ruthenium-dihydroquercetin is 100mg / mL) and cultured for 4h, and the supernatant is collected and centrifuged at 18000g for 10min to obtain apoptosis vesicles loaded with ruthenium-dihydroquercetin.

[0055] Example 5

[0056] Verification of the enzyme-like activity of ruthenium-dihydroquercetin obtained in Example 1

[0057] Different concentrations (0, 40, 80, 120, 160, 200 μg / mL) of ruthenium-dihydroquercetin and o-phenylenediamine (OPD) were mixed in PBS salt buffer at pH = 5.5, incubated at room temperature (25°C) for 0.5h, the color of the solution was imaged, and the ultraviolet-visible-near infrared spectrum was measured, and the results are shown in the right graph of Figure 4 .

[0058] Different concentrations (0, 20, 40, 60, 80, 100 μg / mL) of ruthenium-dihydroquercetin and 3,3,5,5-tetramethylbenzidine (TMB) were mixed in PBS salt buffer at pH = 5.5, incubated at room temperature (25°C) for 0.5h, the color of the solution was imaged, and the ultraviolet-visible-near infrared spectrum was measured, and the results are shown in the left graph of Figure 4 .

[0059] It can be seen that ruthenium-dihydroquercetin has enzyme-like activity and can generate hydroxyl radicals, and has antibacterial properties.

[0060] Example 6

[0061] Effect of ruthenium-dihydroquercetin obtained in Example 1 on free radical scavenging under weak alkaline conditions

[0062] DPPH was dissolved in anhydrous ethanol to obtain a DPPH free radical test solution, and its absorption peak was at 519nm. The absorbance at 519nm was adjusted to 0.6-1.0 and immediately used. Different concentrations (0, 1, 2, 3, 4, 5 μg / mL) of ruthenium-dihydroquercetin solution (ruthenium-dihydroquercetin solution was neutral PBS) were added to the DPPH free radical test solution. Then the absorbance was measured at 519nm. The results are shown in the left graph of Figure 5 .

[0063] ABTS free radical working solution was diluted with 0.01M PBS (pH 7.4), and the absorbance at 734nm was determined and adjusted to be 0.6-1.0. Different concentrations (0, 10, 20, 30, 40, 50 μg / mL) of ruthenium-dihydroquercetin solution were added, and the absorbance at 734nm was determined. The results are shown in the right graph of Fig. Figure 5 .

[0064] It can be seen that the ruthenium-dihydroquercetin prepared in the present application can scavenge nitrogen free radicals and has anti-inflammatory properties.

[0065] Example 7

[0066] Catalase (CAT enzyme) activity of the ruthenium-dihydroquercetin obtained in Example 1

[0067] The CAT enzyme activity of the ruthenium-dihydroquercetin was determined using a CAT enzyme kit. Different concentrations (20, 40, 60, 80, 100 μg / mL) of ruthenium-dihydroquercetin solution were reacted with the test solution at 37°C for 20 min. The absorbance at 550nm was determined to determine the scavenging efficiency of H2O2. The results are shown in Fig. Figure 6 . It shows that the ruthenium-dihydroquercetin can scavenge hydrogen peroxide to produce oxygen.

[0068] Example 8

[0069] In vitro antibacterial effect of the loaded ruthenium-dihydroquercetin apoptotic vesicles obtained in Example 3

[0070] The in vitro antibacterial activity of the umbilical cord mesenchymal stem cell-derived loaded ruthenium-dihydroquercetin apoptotic vesicles was evaluated by plate counting method. The methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli) used were typical gram-positive and gram-negative bacteria, respectively. The experiment was divided into the following four groups:

[0071] Control group (referred to as group 1): PBS was added to the bacteria for treatment;

[0072] Apovs group (referred to as group 2): apoptotic vesicles (preparation method same as Example 3, except that no ruthenium-dihydroquercetin was added, and the rest were the same as Example 3.) were added (dissolved in PBS, concentration 1.0×10 10 particles / mL);

[0073] Ru-DMY group (referred to as group 3): the ruthenium-dihydroquercetin obtained in Example 1 was added (dissolved in PBS, concentration 100 mg / mL);

[0074] Apovs@Ru-DMY group (denoted as group 4): adding the apoptosis vesicles loaded with ruthenium-dihydroquercetin obtained in Example 3 (dissolved with PBS, in which the concentration of the apoptosis vesicles is 1.0 x 10 10 particles / mL, and the concentration of ruthenium-dihydroquercetin is 100 mg / mL);

[0075] Taking the MRSA and E. coli bacterial suspensions, adjusting the OD value to about 0.6 (1.0 x 10 7 CFU / mL), respectively preparing working solutions of different groups, respectively adding 20 μL of the MRSA bacterial suspension and 20 μL of the E. coli bacterial suspension into a 96-well plate, and then adding 180 μL of the working solution of different groups into each well, incubating at 37°C for 4 h, diluting the bacterial suspension after incubation by 10 3 times, and taking 20 μL of the diluted bacterial suspension and dropping it onto a bouillon agar plate, uniformly coating it with glass beads, and then placing it in a 37°C incubator for incubation for 12 h, and then taking out the agar plate to take a photo.

[0076] The results, as shown in Figure 7 and Figure 8 , show that the ruthenium-dihydroquercetin and the apoptosis vesicles loaded with ruthenium-dihydroquercetin both have significant antibacterial effects, and the antibacterial effect of the apoptosis vesicles loaded with ruthenium-dihydroquercetin is significantly better than that of the ruthenium-dihydroquercetin group and the simple apoptosis vesicles group, and the apoptosis vesicles loaded with ruthenium-dihydroquercetin of the present application has a synergistic effect.

[0077] Example 9

[0078] Immunofluorescence staining of the umbilical cord mesenchymal stem cell-derived apoptosis vesicles loaded with ruthenium-dihydroquercetin prepared in Example 3 on mouse monocyte macrophage leukemia cells (RAW264.7)

[0079] The experiment is divided into the following five groups:

[0080] Control group (denoted as group 1): no treatment is given to the cells;

[0081] LPS group (denoted as group 2): adding the LPS group with a concentration of 100 ng / mL to the cells;

[0082] Apovs group (denoted as group 3): adding the LPS group with a concentration of 100 ng / mL to the cells, and adding the apoptosis vesicles (the preparation method is the same as in Example 3, except that no ruthenium-dihydroquercetin is added, and the rest is the same as in Example 3.) (dissolved with PBS);

[0083] Ru-DMY group (denoted as group 4): adding the LPS group with a concentration of 100 ng / mL to the cells, and adding the ruthenium-dihydroquercetin obtained in Example 1 (dissolved with PBS);

[0084] Apovs@Ru-DMY group (referred to as group 5): cells were treated with 100 ng / mL LPS and apoptotic vesicles loaded with ruthenium-dihydromyricetin prepared in Example 3 (dissolved in PBS).

[0085] The results are as follows Figure 9 and Figure 10 As shown, in Figure 9 The color lightens from left to right, indicating fewer macrophages are transforming into M1 cells, thus demonstrating a good anti-inflammatory effect. Figure 10 The color deepens from left to right, representing an increasing number of macrophages transforming into M2 cells, thus demonstrating a good anti-inflammatory effect. CD86 is an indicator of M1, and CD206 is an indicator of M2. A higher proportion of macrophages transforming into M2 cells and a lower proportion into M1 cells indicates a good anti-inflammatory effect. Figure 9 and Figure 10 It can be seen that the apoptotic vesicle group alone, the ruthenium-dihydromyricetin group alone, and the apoptotic vesicle group loaded with ruthenium-dihydromyricetin can all promote macrophage polarization towards M2 rather than towards M1, indicating that the ruthenium-dihydromyricetin group and the apoptotic vesicles loaded with ruthenium-dihydromyricetin provided in this invention have anti-inflammatory effects.

[0086] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A ruthenium-dihydromyricetin loaded apoptotic vesicle, characterized in that, The preparation method of the ruthenium-dihydromyricetin comprises the following steps: dispersing polyvinylpyrrolidone, ruthenium trichloride and dihydromyricetin into liquid solvents respectively to obtain polyvinylpyrrolidone solution, ruthenium trichloride solution and dihydromyricetin solution; adding the ruthenium trichloride solution into the polyvinylpyrrolidone solution to obtain a mixed solution; adding the dihydromyricetin solution into the mixed solution and stirring; dialyzing to obtain a dialyzed solution; purifying to obtain a precipitate; washing, freezing and drying to obtain the ruthenium-dihydromyricetin.

2. The apoptotic vesicle of claim 1, wherein, The liquid solvent is at least one of water, ethanol, methanol, acetone, N-methylpyrrolidone, dimethyl sulfoxide, N,N-dimethylformamide, dichloromethane, chloroform, ethylene glycol, glycerol, isopropyl alcohol and polyethylene glycol 200.

3. The apoptotic vesicle of claim 2, wherein, The weight-volume ratio of polyvinylpyrrolidone to the liquid solvent is 50-80 mg: 1-20 mL; the weight-volume ratio of ruthenium trichloride to the liquid solvent is 10-50 mg: 1-20 mL; and the weight-volume ratio of dihydromyricetin to the liquid solvent is 5-15 mg: 1-5 mL.

4. The apoptotic vesicle of claim 2, wherein, The stirring time is more than 1 h; the dialysis method is dialysis bag dialysis, the molecular weight of the dialysis bag is 6000-12000 Dalton, and the dialysis time is 12-24 h.

5. The apoptotic vesicle of claim 2, wherein, The purification method comprises centrifugation, ultrafiltration or rotary evaporation; the centrifugation speed is 8000-20000 rpm, and the centrifugation time is 5-30 min.

6. The apoptotic vesicle of claim 2, wherein, The reagent for inducing mesenchymal stem cell apoptosis comprises staurosporine; the ruthenium-dihydromyricetin is added when the mesenchymal stem cells are induced to undergo apoptosis for 2-10 h; the culture time is 1-10 h; the centrifugation force is 10000-20000 g, and the centrifugation time is 10-30 min.

7. The apoptotic vesicle of claim 1, wherein, 8. Use of the apoptotic vesicle according to any one of claims 1-7 in the preparation of an antibacterial and anti-inflammatory product. ​

Images