Stem cell biological preparation, preparation method and application of preparation as rhinitis treatment medicine

Abstract

The invention relates to the field of stem cell biomedicine, and provides a stem cell biological preparation, a preparation method and application of the preparation as a rhinitis treatment medicine in order to solve the problem that when a Syk inhibitor is independently used, the recovery time of rhinitis is long, and the preparation method comprises the steps that S100, a lipidosome entrapped with the Syk inhibitor is prepared through a film dispersion method; s200, mixing the monoclonal antibody with N-hydroxysuccinimide ester according to a molar ratio of 1: (20-40), and obtaining a micelle solution through an incubation process; s300, mixing the liposome and the micelle solution according to a volume ratio of (40-70): 1 to obtain a first effective preparation; and S400, mixing the first effective preparation with the stem cell solution to obtain the stem cell biological preparation. Through cooperation of the Syk inhibitor and the stem cell repair function, the rhinitis recovery effect is improved, the Syk inhibitor is coated with the isolation layer, the action time and the action position of the Syk inhibitor are regulated and controlled, and the influence on the stem cells is reduced; due to modification of the lipidosome, the lipidosome has targeting property, and the influence on the stem cells can be reduced.

Description

Technical Field

[0001] The present invention relates to the field of stem cell biomedicine, and in particular to a stem cell biological preparation, a preparation method and application of the preparation as a drug for treating rhinitis. Background Art

[0002] Rhinitis is an inflammation of the nasal mucosa caused by viruses, bacteria, allergens, etc., which may be caused by various physical and chemical factors or certain systemic diseases. Among the many types, the most common is allergic rhinitis, which is caused by allergens such as dust mites, mold, pets, and pollen. Currently, Syk inhibitors are common rhinitis treatment drugs, which have a good effect in relieving rhinitis symptoms, but the recovery time of rhinitis is relatively long, especially for chronic rhinitis. The treatment effect is not ideal. Summary of the invention

[0003] The purpose of the present invention is to provide a stem cell biological preparation to solve the problem of long recovery time of rhinitis when Syk inhibitor is used alone in the prior art.

[0004] Another object of the present invention is to provide a method for preparing a stem cell biological preparation, regulating the action time of Syk inhibitors and stem cells, and reducing the influence of Syk inhibitors on the repair effect of stem cells.

[0005] The present invention also aims to provide a stem cell biological preparation for use as a rhinitis treatment drug, and enhances the therapeutic effect of rhinitis by combining Syk inhibitors with stem cells.

[0006] The embodiments of the present invention are achieved through the following technical solutions:

[0007] A stem cell biological preparation comprises: a therapeutically effective amount of stem cells and a therapeutically effective amount of Syk inhibitor; the Syk inhibitor is coated with an isolation layer.

[0008] A therapeutically effective amount refers to the dosage of a drug given to a patient in medicine or pharmacology to achieve the desired therapeutic effect. This dosage should be sufficient to have a positive therapeutic effect on the disease while minimizing side effects and toxic reactions. A variety of factors need to be considered when determining a therapeutically effective amount, including but not limited to the patient's weight, age, liver and kidney function status, and the presence of other diseases or other drugs being used. The therapeutically effective amount is not fixed, and for different individuals and different diseases, even for the same drug, the therapeutically effective amount may vary. The therapeutically effective amount can be determined by a person skilled in the art through conventional technical means.

[0009] Syk is involved in signal transduction processes in multiple cell types, including mast cells, eosinophils, and macrophages, which play a key role in the occurrence and development of allergic rhinitis. By inhibiting Syk, the inflammatory response mediated by these cells can be reduced, thereby alleviating the symptoms of rhinitis. Allergic rhinitis is associated with the IgE-mediated response of the immune system, which leads to the release of chemical mediators such as histamine, causing symptoms such as sneezing and runny nose. Inhibiting Syk helps regulate this process and reduce the immune system's overreaction to harmless substances.

[0010] Rhinitis can easily lead to damage to the nasal mucosa. Under the action of inflammation, the self-repair of the nasal mucosa is inhibited. The nasal mucosa is the first line of defense for the nasal cavity. Its damage can easily increase the exposure to allergens. At the same time, nasal mucosal damage will also affect the absorption rate of drugs during nasal administration, making the absorption rate uncontrollable and the drug concentration in the blood difficult to reach the optimal value. In order to avoid overdose, the dosage will be controlled during administration, making it difficult to achieve the best therapeutic effect. The applicant hopes to promote the repair of damaged nasal tissues through the repair function of stem cells, and then cooperate with Syk inhibitors to jointly promote the recovery of allergic rhinitis. However, it was found during the experiment that after increasing the dosage of Syk inhibitors, the repair effect of the tissue will show a downward trend. The applicant speculates that Syk inhibitors will simultaneously affect the signal transduction pathway of stem cells, affecting the differentiation direction of stem cells, secreted factors, and homing ability of stem cells. In order to reduce the impact of Syk inhibitors on the repair function of stem cells, the applicant added an isolation layer on the basis of the Syk inhibitor to separate the homing of stem cells from the action time of the Syk inhibitor. This can not only reduce the impact of the Syk inhibitor on stem cells, but also make full use of inflammatory mediators to promote the homing of stem cells before the Syk inhibitor takes effect and reduces inflammatory factors.

[0011] Preferably, the isolation layer comprises liposomes, the surface of the liposomes is modified with monoclonal antibodies, and the monoclonal antibodies are at least specific markers for one or more cells among mast cells, eosinophils, macrophages, dendritic cells or T lymphocytes.

[0012] In order to prevent the Syk inhibitor from acting on stem cells during and before administration, the present invention encapsulates the active ingredient of the Syk inhibitor by liposomes, and the active ingredient can exist in solid form or in liquid form. The antibody on the surface of the liposome can increase the targeting of the Syk inhibitor, thereby preventing it from acting on stem cells. A specific marker for a cell means that the antibody can bind to the marker or to the marker receptor.

[0013] Preferably, the monoclonal antibodies include: one or more of Omalizumab and Barzolvolimab.

[0014] A method for preparing a stem cell biological preparation, comprising:

[0015] S100, preparation of Syk inhibitor-loaded liposomes by thin film dispersion method;

[0016] S200, mixing the monoclonal antibody with N-hydroxysuccinimide ester at a molar ratio of 1:(20-40), and obtaining a micellar solution through an incubation process;

[0017] S300, mixing the liposomes and the micelle solution in a volume ratio of (40-70):1 to obtain a first effective preparation;

[0018] S400, mixing the first effective agent with the stem cell solution to obtain a stem cell biological agent.

[0019] The liposomes in S300 were mixed with the micelle solution and incubated at 37°C for 3-6h.

[0020] Preferably, the S100 includes:

[0021] A100, dissolving egg yolk phosphatidylcholine, cholesterol and DSPE-PEG-Maleimide in an organic solvent at a molar ratio of (60-80):(15-20):(1-3) to obtain a mixed solution;

[0022] A200, the mixed solution is evaporated under reduced pressure to remove the organic solvent to obtain a liposome film;

[0023] A300 and liposome films were hydrated with physiological saline containing Syk inhibitors, and then liposomes encapsulating Syk inhibitors were obtained by ultrasonic treatment and polycarbonate membrane extrusion.

[0024] The organic solvent may be chloroform, methanol, anhydrous ethanol, etc., or a mixed organic solvent may be used. The preparation of conventional liposomes is mostly focused on the encapsulation rate of Syk inhibitors and the modification rate of monoclonal antibodies, but the present invention focuses on the release time of Syk inhibitors during the preparation process. Therefore, in the selection of the method and its parameters, if the encapsulation rate of Syk inhibitors and the modification rate of monoclonal antibodies are considered, it is not optimal, but the release time of Syk inhibitors and the homing time of stem cells have a better coordination effect, reducing the effect of Syk on stem cell repair. The present invention is based on most water-soluble Syk inhibitors for experiments, and the release time of Syk inhibitors is regulated by the selection of liposome composition and the ratio.

[0025] Preferably, the frequency of ultrasonic treatment in the A300 is 18-25 kHz, each interval is 30-60 seconds, the ultrasonic treatment is 20-30 seconds, and the number of treatments is 4-6 times.

[0026] Ultrasonic treatment can affect the structure and particle size distribution of liposomes, and thus affect the release time of Syk inhibitors. By controlling the average number of liposomes through ultrasonic treatment, the release time of Syk inhibitors can be effectively regulated.

[0027] Preferably, the A100 further comprises DSPE-SS-PEG, and the molar ratio of the egg yolk phosphatidylcholine to DSPE-SS-PEG is (60-80):(4-8).

[0028] Adding DSPE-SS-PEG to the liposome component can increase the responsiveness of Syk inhibitor release. When stem cells home to and repair tissues, they release Syk inhibitors in response to antioxidant enzymes, further regulating the release time of Syk inhibitors.

[0029] Preferably, the incubation process comprises: incubating at 5-15°C for 10-15h, then heating to 25-37°C at a heating rate of 2-5°C / min, and then incubating for 5-10h.

[0030] Preferably, the volume ratio of the first effective agent to the stem cell solution is (15-20): (6-10), and the concentration of stem cells in the stem cell solution is 1 10 7 -2 10 7 / ml.

[0031] A use of the stem cell biological preparation as a drug for treating rhinitis.

[0032] The preparation can be in the form of a spray or an injection, etc. The preparation can be frozen for later use.

[0033] The present invention has at least the following beneficial effects:

[0034] The present invention can effectively improve the recovery effect of rhinitis by cooperating the Syk inhibitor with the stem cell repair function. At the same time, after the Syk inhibitor is coated with an isolation layer, the action time and action position of the Syk inhibitor can be regulated to reduce its influence on stem cell repair. The preparation method provided by the present invention not only makes the liposome encapsulating the Syk inhibitor targeted, but also reduces its influence on stem cells through the modification of the liposome. DETAILED DESCRIPTION

[0035] In order to make the purpose, method scheme and advantages of the embodiments of the present invention clearer, the method scheme in the embodiments of the present invention is clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0036] Example 1: A stem cell biological preparation, comprising: a therapeutically effective amount of stem cells and a therapeutically effective amount of Syk inhibitor; the Syk inhibitor is coated with an isolation layer; the isolation layer comprises liposomes, the surface of the liposomes is modified with a monoclonal antibody, and the monoclonal antibody is Omalizumab.

[0037] Example 2: A stem cell biological preparation, comprising: a therapeutically effective amount of stem cells and a therapeutically effective amount of Syk inhibitor; the Syk inhibitor is coated with an isolation layer; the isolation layer comprises liposomes, the surface of the liposomes is modified with a monoclonal antibody, and the monoclonal antibody is Barzolvolimab.

[0038] Embodiment 3: A method for preparing a stem cell biological preparation, comprising:

[0039] S100, preparation of Syk inhibitor-loaded liposomes by thin film dispersion method;

[0040] S200, Omalizumab and N-hydroxysuccinimide ester were mixed in a molar ratio of 1:20, and micellar solution was obtained by incubation process;

[0041] S300, mixing the liposomes and the micelle solution at a volume ratio of 40:1, and incubating at 37° C. for 5 h to obtain a first effective preparation;

[0042] S400, mixing the first effective agent with the stem cell solution to obtain a stem cell biological agent;

[0043] The S100 includes:

[0044] A100, dissolve egg yolk phosphatidylcholine, cholesterol and DSPE-PEG-Maleimide in 2 ml of chloroform at a molar ratio of 60:15:1 to obtain a mixed solution;

[0045] A200, the mixed solution is evaporated under reduced pressure to remove the organic solvent to obtain a liposome film;

[0046] A300, liposome film was hydrated with 1 ml of normal saline containing Syk inhibitor, and then liposomes encapsulating Syk inhibitor were obtained by ultrasonic treatment and extrusion through polycarbonate membrane; 2 mg of Syk inhibitor was dissolved in each ml of normal saline;

[0047] The frequency of the ultrasonic treatment in the A300 is 18 kHz, the ultrasonic treatment is performed for 20 s at an interval of 30 s, and the number of treatments is 4 times;

[0048] The incubation process comprises: incubating at 5°C for 10 hours, then heating to 25°C at a heating rate of 2°C / min, and then incubating for 5 hours;

[0049] The volume ratio of the first effective agent to the stem cell solution is 15:6, and the concentration of stem cells in the stem cell solution is 1 10 7 / ml.

[0050] Embodiment 4: A method for preparing a stem cell biological preparation, comprising:

[0051] S100, preparation of Syk inhibitor-loaded liposomes by thin film dispersion method;

[0052] S200, mixing Omalizumab and N-hydroxysuccinimide ester at a molar ratio of 1:40, and obtaining a micellar solution through an incubation process;

[0053] S300, mixing the liposomes and the micelle solution at a volume ratio of 70:1, and incubating at 37° C. for 5 h to obtain a first effective preparation;

[0054] S400, mixing the first effective agent with the stem cell solution to obtain a stem cell biological agent;

[0055] The S100 includes:

[0056] A100, dissolve egg yolk phosphatidylcholine, cholesterol and DSPE-PEG-Maleimide in 2 ml of chloroform at a molar ratio of 80:20:3 to obtain a mixed solution;

[0057] A200, the mixed solution is evaporated under reduced pressure to remove the organic solvent to obtain a liposome film;

[0058] A300, liposome film was hydrated with 1 ml of normal saline containing Syk inhibitor, and then liposomes encapsulating Syk inhibitor were obtained by ultrasonic treatment and extrusion through polycarbonate membrane; 2 mg of Syk inhibitor was dissolved in each ml of normal saline;

[0059] The frequency of the ultrasonic treatment in the A300 is 25 kHz, the ultrasonic treatment is performed for 30 s every 60 s, and the number of treatments is 6 times;

[0060] The incubation process comprises: incubating at 15°C for 15 hours, then heating to 37°C at a heating rate of 5°C / min, and then incubating for 10 hours;

[0061] The volume ratio of the first effective agent to the stem cell solution is 20:6, and the concentration of stem cells in the stem cell solution is 2 10 7 / ml.

[0062] Embodiment 5: A method for preparing a stem cell biological preparation, comprising:

[0063] S100, preparation of Syk inhibitor-loaded liposomes by thin film dispersion method;

[0064] S200, Omalizumab and N-hydroxysuccinimide ester were mixed at a molar ratio of 1:30, and micellar solution was obtained by incubation process;

[0065] S300, mixing the liposomes and the micelle solution at a volume ratio of 60:1, and incubating at 37° C. for 5 h to obtain a first effective preparation;

[0066] S400, mixing the first effective agent with the stem cell solution to obtain a stem cell biological agent;

[0067] The S100 includes:

[0068] A100, dissolve egg yolk phosphatidylcholine, cholesterol and DSPE-PEG-Maleimide in 2 ml of chloroform at a molar ratio of 70:16:2.5 to obtain a mixed solution;

[0069] A200, the mixed solution is evaporated under reduced pressure to remove the organic solvent to obtain a liposome film;

[0070] A300, liposome film was hydrated with 1 ml of normal saline containing Syk inhibitor, and then liposomes encapsulating Syk inhibitor were obtained by ultrasonic treatment and extrusion through polycarbonate membrane; 2 mg of Syk inhibitor was dissolved in each ml of normal saline;

[0071] The frequency of the ultrasonic treatment in the A300 is 22 kHz, the ultrasonic treatment is performed for 25 s every 45 s, and the number of treatments is 4 times;

[0072] The incubation process comprises: incubating at 10°C for 12 hours, then heating to 37°C at a heating rate of 3°C / min, and then incubating for 8 hours;

[0073] The volume ratio of the first effective agent to the stem cell solution is 18:8, and the concentration of stem cells in the stem cell solution is 1.5 10 7 / ml.

[0074] Example 6: A method for preparing a stem cell biological preparation, comprising:

[0075] S100, preparation of Syk inhibitor-loaded liposomes by thin film dispersion method;

[0076] S200, Omalizumab and N-hydroxysuccinimide ester were mixed at a molar ratio of 1:30, and micellar solution was obtained by incubation process;

[0077] S300, mixing the liposomes and the micelle solution at a volume ratio of 60:1, and incubating at 37° C. for 5 h to obtain a first effective preparation;

[0078] S400, mixing the first effective agent with the stem cell solution to obtain a stem cell biological agent;

[0079] The S100 includes:

[0080] A100, dissolve egg yolk phosphatidylcholine, cholesterol, DSPE-PEG-Maleimide and DSPE-SS-PEG in 2 ml of chloroform at a molar ratio of 70:16:2.5:4 to obtain a mixed solution;

[0081] A200, the mixed solution is evaporated under reduced pressure to remove the organic solvent to obtain a liposome film;

[0082] A300, liposome film was hydrated with 1 ml of normal saline containing Syk inhibitor, and then liposomes encapsulating Syk inhibitor were obtained by ultrasonic treatment and extrusion through polycarbonate membrane; 2 mg of Syk inhibitor was dissolved in each ml of normal saline;

[0083] The frequency of the ultrasonic treatment in the A300 is 22 kHz, the ultrasonic treatment is performed for 25 s every 45 s, and the number of treatments is 4 times;

[0084] The incubation process comprises: incubating at 10°C for 12 hours, then heating to 37°C at a heating rate of 3°C / min, and then incubating for 8 hours;

[0085] The volume ratio of the first effective agent to the stem cell solution is 18:8, and the concentration of stem cells in the stem cell solution is 1.5 10 7 / ml.

[0086] Embodiment 7: A method for preparing a stem cell biological preparation, comprising:

[0087] S100, preparation of Syk inhibitor-loaded liposomes by thin film dispersion method;

[0088] S200, Omalizumab and N-hydroxysuccinimide ester were mixed at a molar ratio of 1:30, and micellar solution was obtained by incubation process;

[0089] S300, mixing the liposomes and the micelle solution at a volume ratio of 60:1, and incubating at 37° C. for 5 h to obtain a first effective preparation;

[0090] S400, mixing the first effective agent with the stem cell solution to obtain a stem cell biological agent;

[0091] The S100 includes:

[0092] A100, dissolve egg yolk phosphatidylcholine, cholesterol, DSPE-PEG-Maleimide and DSPE-SS-PEG in 2 ml of chloroform at a molar ratio of 70:16:2.5:8 to obtain a mixed solution;

[0093] A200, the mixed solution is evaporated under reduced pressure to remove the organic solvent to obtain a liposome film;

[0094] A300, liposome film was hydrated with 1 ml of normal saline containing Syk inhibitor, and then liposomes encapsulating Syk inhibitor were obtained by ultrasonic treatment and extrusion through polycarbonate membrane; 2 mg of Syk inhibitor was dissolved in each ml of normal saline;

[0095] The frequency of the ultrasonic treatment in the A300 is 22 kHz, the ultrasonic treatment is performed for 25 s every 45 s, and the number of treatments is 4 times;

[0096] The incubation process comprises: incubating at 10°C for 12 hours, then heating to 37°C at a heating rate of 3°C / min, and then incubating for 8 hours;

[0097] The volume ratio of the first effective agent to the stem cell solution is 18:8, and the concentration of stem cells in the stem cell solution is 1.5 10 7 / ml.

[0098] Embodiment 8: A method for preparing a stem cell biological preparation, comprising:

[0099] S100, preparation of Syk inhibitor-loaded liposomes by thin film dispersion method;

[0100] S200, Omalizumab and N-hydroxysuccinimide ester were mixed at a molar ratio of 1:30, and micellar solution was obtained by incubation process;

[0101] S300, mixing the liposomes and the micelle solution at a volume ratio of 60:1, and incubating at 37° C. for 5 h to obtain a first effective preparation;

[0102] S400, mixing the first effective agent with the stem cell solution to obtain a stem cell biological agent;

[0103] The S100 includes:

[0104] A100, dissolve egg yolk phosphatidylcholine, cholesterol, DSPE-PEG-Maleimide and DSPE-SS-PEG in 2 ml of chloroform at a molar ratio of 70:16:2.5:6 to obtain a mixed solution;

[0105] A200, the mixed solution is evaporated under reduced pressure to remove the organic solvent to obtain a liposome film;

[0106] A300, liposome film was hydrated with 1 ml of normal saline containing Syk inhibitor, and then liposomes encapsulating Syk inhibitor were obtained by ultrasonic treatment and extrusion through polycarbonate membrane; 2 mg of Syk inhibitor was dissolved in each ml of normal saline;

[0107] The frequency of the ultrasonic treatment in the A300 is 22 kHz, the ultrasonic treatment is performed for 25 s every 45 s, and the number of treatments is 4 times;

[0108] The incubation process comprises: incubating at 10°C for 12 hours, then heating to 37°C at a heating rate of 3°C / min, and then incubating for 8 hours;

[0109] The volume ratio of the first effective agent to the stem cell solution is 18:8, and the concentration of stem cells in the stem cell solution is 1.5 10 7 / ml.

[0110] Comparative Example 1: The difference from Example 8 is that the molar ratio of cholesterol in A100 is 10.

[0111] Comparative Example 2: The difference from Example 8 is that the molar ratio of cholesterol in A100 is 25.

[0112] Comparative Example 3: The difference from Example 8 is that the frequency of ultrasonic treatment in A300 is 30 kHz.

[0113] Comparative Example 4: The difference from Example 8 is that the number of treatments in A300 is 8 times.

[0114] Comparative Example 5: The difference from Example 8 is that the incubation process includes incubating at 10°C for 12 hours.

[0115] Comparative Example 6: The difference from Example 8 is that the first effective preparation is directly used as a stem cell biological preparation.

[0116] Comparative Example 7: The difference from Example 8 is that the stem cell solution is directly used as a stem cell biological preparation.

[0117] The preparation method of the stem cell solution in Examples 3-8 and Comparative Examples 1-7 includes:

[0118] Primary umbilical cord mesenchymal stem cells were inoculated into MSCBM culture medium. When the cell confluence reached 80%, the old culture medium was discarded. After washing the cells with D-hanks solution, trypsin digestion was performed, and D-hanks solution was added and centrifuged. The cell pellet was resuspended in DMEM / F12 culture medium to obtain P1 mesenchymal stem cells. After subculturing P1 mesenchymal stem cells in DMEM / F12 culture medium environment for 8 generations, they were resuspended in physiological saline to obtain stem cell solution. During the subculture process, when the cell confluence reached 90%, the old culture medium was discarded. Culture environment: 37°C, 5% CO 2 , 21% O 2 .

[0119] test:

[0120] Materials: Syk inhibitor was purchased from Shanghai Zeye Biotechnology Co., Ltd., and the product name is spleen tyrosine kinase (Syk) inhibitor (TAK-659 hydrochloride).

[0121] Omalizumab was purchased from Novartis Pharmaceuticals of Switzerland and the product name is Omalizumab.

[0122] MSCBM culture medium was purchased from Shanghai Dakoway Biotechnology Co., Ltd. and the product name is Mesenchymal StemCell Basal Medium (MSCBM).

[0123] DMEM / F12 culture medium was purchased from Shanghai QiDa Biotechnology Co., Ltd., and the product name is DMEM / F12 complete culture medium.

[0124] Test 1: Liposomes were prepared according to the preparation methods provided in Examples 3-8 and Comparative Examples 1-4, wherein the Syk inhibitor was labeled with Annexin V-FITC. The encapsulation efficiency of the liposomes was tested, and the specific test steps were as follows: the prepared liposomes were centrifuged for 30 minutes, and the supernatant was taken. The supernatant of the liposomes without the Syk inhibitor was used as a control, and the encapsulation efficiency of the Syk inhibitor in the liposomes was determined by fluorescence spectrophotometry. Each group was tested 5 times, and the test results were averaged. The test results are shown in Table 1.

[0125] Table 1

[0126] Example 3 Example 4 Example 5 Example 6 Example 7 Encapsulation efficiency 75.18% 82.64% 78.07% 77.12% 79.94% Example 8 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Encapsulation efficiency 80.36% 70.53% 86.41% 85.08% 83.94%

[0127] It can be seen from the test results of Examples 1-8 that the encapsulation effect of the Syk inhibitor in the liposome prepared by the preparation method provided by the present invention is better.

[0128] From the test results of Example 6 and Example 5, it can be seen that the encapsulation efficiency is slightly reduced after adding DSPE-SS-PEG to the liposome component, but from the comparison of Examples 7-8 with Examples 5-6, it can be seen that the encapsulation effect can be improved by adjusting the proportion of DSPE-SS-PEG.

[0129] From the comparison between Comparative Examples 1-2 and Example 8, it can be seen that the proportion of cholesterol will greatly affect the encapsulation efficiency, and the proportion of cholesterol used in the present invention is not optimal in terms of encapsulation efficiency.

[0130] From the comparison between Comparative Examples 3-4 and Example 8, it can be seen that excessive ultrasonic treatment and treatment times will reduce the encapsulation efficiency.

[0131] Experiment 2: Liposome stability test: After the liposomes were placed in PBS buffer for 45 days, the particle size change of the liposomes was tested. The initial particle size of the liposomes prepared according to the preparation method provided in Example 3-8 was about 219.56 nm. After 45 days, the initial particle size was almost unchanged, and the liposome stability was good.

[0132] Experiment 3: Effect verification

[0133] Establishment of animal model

[0134] Healthy SD rats weighing 265-300 g were selected, half male and half female. On the first day of modeling, 1 ml of isotonic saline, 1.5 mg OVA, 3 mg aluminum hydroxide sol and 1×10 10 The mice were injected with a mixture of inactivated Bordetella pertussis into the subcutaneous part of the rats' forefoot and hindfoot, with 0.2 ml / mouse and 0.3 ml / mouse injections. On the 5th day, 1 ml of isotonic saline, 1.0 mg OVA and 2 mg aluminum hydroxide sol were mixed and injected into the subcutaneous part of the rats' backs, divided into 5 points, with 0.2 ml injected at each point. From the 14th day, the mice were stimulated: 1% ovalbumin saline solution was dripped into the nose once a day, 30 μl in each nostril, for 10 days.

[0135] The model was successfully established if the behavioral (number of sneezes and nose scratching) and symptom manifestations (nasal secretions, etc.) within 30 minutes after nasal drop stimulation were greater than or equal to 2 points.

[0136] Scoring criteria: (1) 0 points: no sneezing, scratching the nose, sneezing and nasal secretions; (2) 1 point: less than 4 sneezes, mild nose scratching, and nasal secretions in the anterior nares; (3) 2 points: 4 to 10 sneezes, frequent nose scratching, and nasal secretions exceeding the anterior nares; (4) 3 points: more than 11 sneezes, continuous nose scratching and rubbing, and face covered with nasal secretions.

[0137] The stem cell biological preparations in Examples 3-8 and Comparative Examples 1-7 were administered to model rats, 120 μl per nasal drop, twice a day, and the control group was administered with distilled water for 7 days. 30 minutes after the last administration, the rat behavior was observed. Ten rats were selected in each group, and the scoring results were averaged. The test results are shown in Table 2.

[0138] Table 2

[0139] Example 3 Example 4 Example 5 Example 6 Example 7 Example 8 Comparative Example 1 Scratching (times) 2.8 3.1 2.3 1.8 2.1 1.5 2.3 Sneeze (times) 8.3 8.0 7.1 5.9 6.4 5.2 6.2 Runny nose (points) 1.5 1.7 1.6 1.1 0.9 0.8 0.9 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 Comparative Example 6 Comparative Example 7 Model control group Scratching (times) 2.6 3.1 2.8 2.0 2.9 5.8 6.7 Sneeze (times) 7.4 7.8 7.6 6.7 8.7 15.3 18.9 Runny nose (points) 1.6 1.3 1.3 1.0 1.4 2.2 2.6

[0140] Normal control group: scratching 0.6 times, sneezing 0.9 times, runny nose 0 points.

[0141] Scoring criteria for runny nose: 1 point if the nose flows to the anterior nasal aperture, 2 points if the nose flows beyond the anterior nasal aperture, and 3 points if the nose is completely runny.

[0142] From the comparison between Examples 3-8 and the model control group, it can be seen that the stem cell biological preparation provided by the present invention has a good therapeutic effect.

[0143] From the comparison between Example 5 and Example 6, it can be seen that adding DSPE-SS-PEG to the liposome component can improve the therapeutic effect of the stem cell biological preparation.

[0144] From the comparison between Comparative Examples 2-4 and Example 8, it can be seen that although the encapsulation rate of Comparative Examples 2-4 is significantly increased, the therapeutic effect is significantly decreased.

[0145] From the comparison between Comparative Examples 1-5 and Example 8, it can be seen that the cholesterol content, ultrasonic treatment operation and incubation process all significantly affect the effect of the stem cell biological preparation.

[0146] From the comparison between Comparative Examples 6-7 and Example 8, it can be seen that the therapeutic effect of rhinitis can be improved by compounding the stem cell solution with the first effective preparation.

[0147] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A stem cell biological preparation, characterized in that: The invention comprises: a therapeutically effective amount of stem cells and a therapeutically effective amount of Syk inhibitor; the Syk inhibitor is coated with an isolation layer.

2. The stem cell biological preparation according to claim 1, characterized in that: The isolation layer comprises liposomes, the surface of which is modified with monoclonal antibodies, which are at least directed to specific markers of one or more cells among mast cells, eosinophils, macrophages, dendritic cells or T lymphocytes.

3. The stem cell biological preparation according to claim 2, characterized in that: The monoclonal antibodies include: one or more of Omalizumab and Barzolvolimab.

4. A method for preparing the stem cell biological preparation according to claim 2 or 3, characterized in that: include: S100, preparation of Syk inhibitor-loaded liposomes by thin film dispersion method; S200, mixing the monoclonal antibody with N-hydroxysuccinimide ester at a molar ratio of 1:(20-40), and obtaining a micellar solution through an incubation process; S300, mixing the liposomes and the micelle solution in a volume ratio of (40-70):1 to obtain a first effective preparation; S400, mixing the first effective agent with the stem cell solution to obtain a stem cell biological agent.

5. The preparation method according to claim 4, characterized in that: The S100 includes: A100, dissolving egg yolk phosphatidylcholine, cholesterol and DSPE-PEG-Maleimide in an organic solvent at a molar ratio of (60-80):(15-20):(1-3) to obtain a mixed solution; A200, the mixed solution is evaporated under reduced pressure to remove the organic solvent to obtain a liposome film; A300 and liposome films were hydrated with physiological saline containing Syk inhibitors, and then liposomes encapsulating Syk inhibitors were obtained by ultrasonic treatment and polycarbonate membrane extrusion.

6. The preparation method according to claim 5, characterized in that: The frequency of ultrasonic treatment in the A300 is 18-25 kHz, the ultrasonic treatment is performed for 20-30 s at intervals of 30-60 s, and the number of treatments is 4-6 times.

7. The preparation method according to claim 5, characterized in that: The A100 also includes DSPE-SS-PEG, and the molar ratio of the egg yolk phosphatidylcholine to DSPE-SS-PEG is (60-80):(4-8).

8. The preparation method according to claim 4, characterized in that: The incubation process includes: incubating at 5-15°C for 10-15h, then heating to 25-37°C at a heating rate of 2-5°C / min, and then incubating for 5-10h.

9. The preparation method according to claim 4, characterized in that: The volume ratio of the first effective agent to the stem cell solution is (15-20): (6-10), and the concentration of stem cells in the stem cell solution is 1 10 7 -2 10 7 / ml.

10. Use of the stem cell biological preparation according to any one of claims 1 to 3 as a drug for treating rhinitis.