Preparation method and application of human umbilical cord mesenchymal stem cell membrane biomimetic nanovesicles

Abstract

A preparation method and application of biomimetic nanovesicles of human umbilical cord mesenchymal stem cell membranes, which relate to the technical field of cell therapy and liposome drug precision delivery. The present invention aims to solve the problems that the biocompatibility of nanovesicles synthesized by artificial liposomes is not high, artificial nanovesicles do not have a mitigating and therapeutic effect on diseases, and generally do not have active targeting of lesion sites. The present invention extracts the mesenchymal stem cell membrane and mixes it with phospholipid liposomes, and prepares biomimetic nanovesicles containing mesenchymal stem cell membranes through a thin film method and a co-extrusion method. Due to the surface protein of the human umbilical cord mesenchymal stem cell membrane, it can target the lesion site of idiopathic pulmonary fibrosis and alleviate the degree of fibrosis in idiopathic pulmonary fibrosis. The present invention is applied to the field of diagnosis and treatment of idiopathic pulmonary fibrosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell therapy and liposome drug precision delivery, and specifically relates to a preparation method and application of human umbilical cord mesenchymal stem cell membrane biomimetic nanovesicles. Background Art

[0002] Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disease characterized by progressive lung scarring characterized histologically by usual interstitial pneumonia (UIP). It is associated with cough, increased dyspnea, and impaired quality of life. IPF affects approximately 3 million people worldwide, with incidence increasing dramatically with age. IPF is thought to begin at the bases and peripheries (edges) of the lungs and gradually spread to all lung tissues. The disease can be sporadic or familial and is associated with increased cough and dyspnea (shortness of breath), with a devastating impact on patients' quality of life. Improved diagnostic and treatment methods are increasingly important. For many years, IPF was considered a primarily inflammatory disease due to the increase in inflammatory cells in the lungs. However, increasing evidence suggests that IPF is an epithelial-driven disease, in which abnormally activated lung epithelium produces mediators that promote fibroblast migration, proliferation, and differentiation into active myofibroblasts, which secrete large amounts of extracellular matrix (ECM), thereby remodeling the lung's architecture.

[0003] Human umbilical cord mesenchymal stem cells (hUC-MSCs) are a type of mesenchymal stem cell. They have been found to lack MHC-II expression but moderately express MHC-I. They also express immunosuppressants such as HLA-G, IDO, and PEG-2, indicating very low immunogenicity. MSCs in birth-related tissues exhibit superior proliferation, lifespan, and differentiation potential compared to bone marrow mesenchymal stem cells (BM-MSCs). Their intermediate state between adult and embryonic stem cells makes them ideal candidates for reprogramming to a pluripotent state. Human umbilical cord mesenchymal stem cells (HUMSCs) are very attractive for the widespread application of regenerative medicine. Wharton's jelly (WJ) from the umbilical cord has satisfactory characteristics: (1) Umbilical cord mesenchymal stem cells are not ethically controversial and can provide a sufficient source of stem cells for treatment; (2) Compared with adult mesenchymal stem cells, HUMSCs have a faster proliferation rate and greater expansion capacity, have a wide range of differentiation pluripotency, and do not induce teratomas; (3) HUMSCs are considered to be more primitive than mesenchymal stem cells from other tissue sources; (4) The process of collecting HUMSCs is non-invasive.

[0004] The drug delivery system for current drug-targeted lesions includes liposomes, emulsions, microspheres, nanoparticles, etc., wherein the liposome drug delivery system is loaded in an ultramicroscopic spherical vesicle wrapped in lipid molecules (liposomes), the inner lipid molecule layer is a hydrophilic space that can be loaded with hydrophilic drugs, i.e., a hydrophilic drug loading space, and the outer lipid molecule layer can be loaded with lipophilic drugs, i.e., a lipophilic drug loading space, and the size is generally a few nm to a few μm, and is divided into two types of active targeting and passive targeting. The various types of liposomes that people have invented at present have relatively low randomness in fusion with target cells. Due to the type and modification of liposome lipids and the lack of mediation of protein factors, there is a large difference between their surface components and the surface components of target cells, resulting in a relatively low probability of fusion between such artificial liposomes and target cells, and increased nonspecific fusion with non-target cells. In addition, traditional liposomes are easily phagocytosed by the reticuloendothelial system in the body and cleared, resulting in a greatly reduced half-life and effectiveness of the drug. Therefore, how to stably and efficiently transport the drug to the lesion site is the key to effectively exerting drug efficacy. Although relevant research on bionic nanovesicles has appeared, the therapeutic effect of the selected cell membrane on the disease itself is not obvious. Therefore, whether the selected cell membrane has a certain therapeutic effect on the disease itself is also an important research goal of bionic nanovesicles. Summary of the Invention

[0005] The purpose of the present invention is to solve the problems that the current artificial liposome-synthesized nanovesicles have low biocompatibility, artificial nanovesicles do not have a mitigating and therapeutic effect on diseases, and do not have the ability to actively target the lesion site. It provides a targeted, personalized human umbilical cord mesenchymal stem cell cell membrane biomimetic nanovesicle drug and its preparation method and application in idiopathic pulmonary fibrosis. Figure 1 As shown, the present invention extracts mesenchymal stem cell membranes, mixes them with phospholipid liposomes, and then prepares biomimetic nanovesicles containing mesenchymal stem cell membranes through ultrasound and co-extrusion. Due to the surface proteins of human umbilical cord mesenchymal stem cell membranes, these vesicles can target idiopathic pulmonary fibrosis lesions and alleviate the degree of fibrosis in idiopathic pulmonary fibrosis.

[0006] The method for preparing human umbilical cord mesenchymal stem cell membrane biomimetic nanovesicles of the present invention is carried out according to the following steps:

[0007] 1) extracting the cell membrane of human umbilical cord mesenchymal stem cells (MSC) after subculture and suspending them in a cell suspension;

[0008] 2) adding cholesterol to the artificial lipid and then dissolving it in an organic solvent;

[0009] 3) Rotary evaporation of the organic solvent containing the artificial lipid in step 2) and vacuum drying;

[0010] 4) hydrating, ultrasonicating, magnetically stirring, and co-extruding the mixed film containing the human umbilical cord mesenchymal stem cell membrane in step 1) and the lipid after drying in step 3) to obtain the human umbilical cord mesenchymal stem cell membrane biomimetic nanovesicles;

[0011] The hydration is to hydrate the lipid mixed film after drying in step 3);

[0012] Among them, the mass ratio of human umbilical cord mesenchymal stem cell membrane to industrial lipid is 5:6-9;

[0013] The mass ratio of artificial lipids to cholesterol is 6 to 8:1;

[0014] The organic solvents are chloroform and methanol.

[0015] Furthermore, the human umbilical cord mesenchymal stem cells are stem cells transfected with TGFBRII.

[0016] Furthermore, in step 4), after the dried mixed film of cell membrane and lipid is hydrated, ultrasonication is performed in a water bath at 48 to 55° C. for 20 to 25 minutes; ultrasonication is performed in a water bath at 35 to 48° C. for 10 to 15 minutes; and then, probe ultrasonication is performed for 9 to 10 minutes, with a power of 140 to 155 W.

[0017] Furthermore, the cell membrane suspension in step 4) is mixed with the mixed thin film at 37° C. to 30° C. and magnetically stirred at a stirring speed of 200 to 300 rpm for 2 to 3 hours.

[0018] Furthermore, the organic solvent in step 1) is chloroform and methanol.

[0019] The present invention discloses an application of human umbilical cord mesenchymal stem cell membrane bionic nanovesicles, wherein the bionic nanovesicles are used as a medicine for alleviating and inhibiting pulmonary fibrosis; the bionic nanovesicles contain the cell membrane of human umbilical cord mesenchymal stem cells.

[0020] Furthermore, the membrane protein of the stem cell is a membrane protein extracted from human umbilical cord mesenchymal stem cells.

[0021] Furthermore, the stem cell membrane protein is a membrane protein extracted from human umbilical cord mesenchymal stem cells transfected with TGFBRII, and the TGFBRII is a transmembrane membrane protein of the TGF-β1 receptor.

[0022] Furthermore, the biomimetic nanovesicles are delivered through the respiratory tract.

[0023] Furthermore, the bionic nanocapsules can be loaded with chemical drugs or biological agents.

[0024] Furthermore, the chemical drug is a hydrophilic, hydrophobic or amphiphilic chemical drug; the biological agent is a mixture of one or more of siRNA drugs, mRNA drugs, DNA drugs, protein drugs, polypeptide drugs, immunopotentiators, immunosuppressants, immune adjuvants and imaging contrast agents.

[0025] Compared with the prior art, the present invention has the following outstanding beneficial technical effects:

[0026] (1) Compared with umbilical cord mesenchymal stem cell transplantation, biomimetic nanovesicles derived from human umbilical cord mesenchymal stem cell membranes are smaller in size and less likely to cause pulmonary embolism during delivery;

[0027] (2) The membrane protein components on the surface of biomimetic nanovesicles based on mesenchymal stem cell membranes can have a certain immune escape ability from the body's immune system;

[0028] (3) Due to the abundant membrane proteins expressed on the surface of human umbilical cord mesenchymal stem cells, biomimetic nanovesicles derived from human umbilical cord mesenchymal stem cells have a certain effect on alleviating pulmonary fibrosis and inhibiting the progression of fibrosis;

[0029] (4) Delivery of biomimetic nanovesicles derived from human umbilical cord mesenchymal stem cells through the respiratory tract can better enable the nanovesicles to reach the lesion site, maximize the accumulation of biomimetic nanovesicles derived from human umbilical cord mesenchymal stem cells in the lesion site of pulmonary fibrosis and exert the greatest therapeutic effect;

[0030] (5) The present invention is based on the unique discovery of the alleviation and inhibition of pulmonary fibrosis by proteins on the surface of human umbilical cord mesenchymal stem cells;

[0031] (6) Compared with ordinary cell membrane biomimetic liposomes, the biomimetic nanovesicles derived from human umbilical cord mesenchymal stem cells involved in the present invention have the ability to inhibit and alleviate pulmonary fibrosis, and have the multifunctionality of both encapsulating drugs and targeting lesions.

[0032] The present invention's mesenchymal stem cell-based nano-bionic nanovesicles contain membrane protein components derived from human umbilical cord mesenchymal stem cells. These nanovesicles can actively target pulmonary fibrosis lesions, evade immune surveillance, and reduce the immune response induced by the biomimetic nanovesicles within the body. Human umbilical cord mesenchymal stem cells are genetically transfected with TGFBRII, enhancing the liposomes' own ability to alleviate pulmonary fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a diagram of the appearance of the liquid of the present invention;

[0034] Figure 2Transmission electron microscopy (TEM) image of human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles;

[0035] Figure 3 The figure shows the particle size comparison of human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) prepared at different probe ultrasonic powers;

[0036] Figure 4 Particle size distribution of human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles (Size);

[0037] Figure 5 Zeta potential diagram of human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles; Figure a is a single time potential diagram, and Figure b is a multi-day potential diagram;

[0038] Figure 6 The dispersion index (PDI) of human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles;

[0039] Figure 7 Long-term stability of human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles;

[0040] Figure 8 Comparison of dry-wet ratio in mouse lung;

[0041] Figure 9 Comparison of dry-wet ratio of mouse lungs after treatment with human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles prepared at different probe ultrasound powers;

[0042] Figure 10 Figure 2 is the weight test chart of mice: A is the treatment group, B is the pulmonary fibrosis group, and C is the healthy control group;

[0043] Figure 11 Micro CT scan of mice;

[0044] Figure 12 This is a graph showing the total amount of all cells in the alveolar lavage fluid of mice in Example 1;

[0045] Figure 13 This is a graph showing the total amount of all cells in the bronchoalveolar lavage fluid of mice treated with human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles prepared at different probe ultrasonic powers in Example 1;

[0046] Figure 14 This is a graph showing the total amount of all cells in the alveolar lavage fluid of mice in Example 2;

[0047] Figure 15 This is a graph showing the total amount of all cells in the bronchoalveolar lavage fluid of mice treated with human umbilical cord mesenchymal stem cell membrane (MSC)-dipalmitoylphosphatidylcholine (DPPC) biomimetic nanovesicles prepared at different probe ultrasonic powers in Example 2;

[0048] Figure 16 This is a comparison of the particle sizes of human umbilical cord mesenchymal stem cell membrane (MSC)-dipalmitoylphosphatidylcholine (DPPC) prepared at different probe ultrasonic powers in Example 2;

[0049] Figure 17 This is a graph showing the total amount of all cells in the alveolar lavage fluid of mice in Example 3;

[0050] Figure 18 This is a graph showing the total amount of all cells in the bronchoalveolar lavage fluid of mice treated with human umbilical cord mesenchymal stem cell membrane (MSC)-egg yolk phosphatidylcholine (EPC) biomimetic nanovesicles prepared at different probe ultrasonic powers in Example 3;

[0051] Figure 19 This is a comparison of the particle sizes of human umbilical cord mesenchymal stem cell membrane (MSC)-egg yolk phosphatidylcholine (EPC) prepared at different probe ultrasonic powers in Example 3;

[0052] Figure 20 This is a graph showing the total amount of all cells in the alveolar lavage fluid of mice in Example 4;

[0053] Figure 21 This is a graph showing the total amount of all cells in the bronchoalveolar lavage fluid of mice treated with human umbilical cord mesenchymal stem cell membrane (MSC)-hydrogenated soy lecithin (HSPC) biomimetic nanovesicles prepared at different probe ultrasonic powers in Example 4;

[0054] Figure 22 This is a comparison chart of the particle sizes of human umbilical cord mesenchymal stem cell membrane (MSC)-hydrogenated soy lecithin (HSPC) prepared at different probe ultrasonic powers in Example 2. DETAILED DESCRIPTION

[0055] In order to make the objectives, technical solutions and advantages of the embodiments of the present invention more clear, the spirit of the contents disclosed in the present invention will be described in detail below. After understanding the embodiments of the contents of the present invention, any technician in the relevant technical field can change and modify the contents of the present invention based on the techniques taught by the contents of the present invention without departing from the spirit and scope of the contents of the present invention.

[0056] The exemplary embodiments of the present invention and the description thereof are used to explain the present invention but are not intended to limit the present invention.

[0057] Implementation Case 1

[0058] A method for preparing human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles and its application in pulmonary fibrosis, the method comprising the following steps:

[0059] S1: culture about 6.7×10 8 6.7×10 human umbilical cord mesenchymal stem cells were isolated by liquid nitrogen freeze-thaw method. 8 Extraction of cell membranes and membrane proteins from individual umbilical cord mesenchymal stem cells;

[0060] S2: The protein quantitative BCA method was used to quantify the membrane protein of the extracted human umbilical cord mesenchymal stem cells;

[0061] (When the cell membrane is extracted, the BCA protein concentration measurement method is needed to measure the protein in the cell membrane. Finally, the total mass of the membrane protein calculated after the measurement is used to estimate the mass of membrane protein that needs to be extracted.)

[0062] S3: The extracted human umbilical cord mesenchymal stem cell membranes were stored at 4°C to prepare for the subsequent synthesis of biomimetic nanovesicles;

[0063] S4: Dissolve 16 mg of soybean lecithin (SPC) and 2 mg of cholesterol (CHOL) in chloroform;

[0064] S5: 12 mg of human umbilical cord mesenchymal stem cell membrane (MSC) was suspended in deionized water or PBS;

[0065] S6: The organic solvent containing soybean lecithin (SPC) in S4 was mixed in a glass test tube and rotary evaporated at 45-55°C and 20 rpm until a uniform thin film appeared at the bottom of the glass test tube. The glass test tube was removed and vacuum dried for 6-8 hours to prepare a dry film.

[0066] S7: Hydrate the dried film prepared in S6 using 894 μL of deionized water or PBS.

[0067] S8: The liposome emulsion prepared in S7 was sonicated in a water bath at 45°C for 22 min;

[0068] S9: The cell membrane suspension prepared in S5 was sonicated in a water bath at 38°C for 14 min;

[0069] S10: The S8 liposome emulsion and the S9 cell membrane suspension were mixed and magnetically stirred at 37°C and 200 rpm for 2 h.

[0070] S11: Perform probe ultrasound on the S10 mixed solution, set the output power to 184W, and ultrasound for 10 minutes;

[0071] S12: The mixed solution of S11 was co-extruded at 42°C to prepare biomimetic nanovesicles of uniform size;

[0072] S13: Western blot detection of CD44, CD73, and CD90 on the cell membrane biomimetic nanovesicles prepared in S12;

[0073] S14: The cell membrane biomimetic nanovesicles of S12 were sealed by injecting argon gas and stored at 4°C;

[0074] S15: Human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles were delivered to the lungs of diseased mice through the respiratory tract to monitor the therapeutic effect of the biomimetic nanovesicles themselves on pulmonary fibrosis.

[0075] The step S15 includes the following steps:

[0076] S151: C57BL / 6N mice (8-week-old, male) were divided into three groups: healthy control group, pulmonary fibrosis group, and treatment group.

[0077] S152: BLM was delivered to the lungs via the respiratory tract. The dose of BLM was 3.5 mg / kg, 50 μL / time.

[0078] S153: Human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles were delivered to the lungs of mice via the respiratory tract on days 8, 10, 12, 14, 16, 18, and 20 after bleomycin (BLM) instillation. The body weights of the mice were recorded, and the efficacy of the treatment was monitored by Micro CT on days 7 and 14 after bleomycin (BLM) instillation.

[0079] S154: On day 21, lungs were removed from mice in the three groups (healthy control group, pulmonary fibrosis group, and treatment group), and the alveolar lavage fluid was obtained and its cell content was counted;

[0080] In order to improve the targeting and therapeutic ability of human umbilical cord mesenchymal stem cell biomimetic nanovesicles on diseased areas, TGFBRII can be transfected with lentiviral genes during the culture of human umbilical cord mesenchymal stem cells. After successful transfection, they can be passaged normally and the preparation steps S1-S14 can be repeated.

[0081] The appearance of the human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) liquid prepared in this example is shown in the figure below: Figure 1 As shown, it can be seen that this example successfully prepared a human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) in good condition.

[0082] The transmission electron microscopy (TEM) image of the human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) prepared in this example is as follows: Figure 2 As shown in FIG, it can be seen that this embodiment successfully prepared spherical human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) nanovesicles

[0083] The particle size comparison of human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles prepared by the probe ultrasonic power of this embodiment and the human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles prepared by different probe ultrasonic powers is shown in the figure. Figure 3 As shown, it can be seen that the ultrasonic treatment of this embodiment can significantly improve the particle size of nanovesicles.

[0084] The particle size distribution (Size) of the human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles of this embodiment is shown in the figure below: Figure 4 As shown, from Figure 4 It can be seen that the prepared biomimetic nanovesicles have small and good particle size.

[0085] The potential diagram (Zeta) and potential stability diagram of the human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles of this embodiment are shown in FIG. Figure 5 As shown by Figure 5 It can be seen that Zeta has better stability and expresses stronger negative charge, indicating that it contains cell membrane components.

[0086] The stability of the dispersion index (PDI) of the human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles of this embodiment is as follows Figure 6 As shown, this shows that the prepared biomimetic nanovesicles have good dispersibility.

[0087] The long-term stability results of human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles in this example are as follows Figure 7 As shown, it can be seen that the prepared biomimetic nanovesicles have good stability within 14 days.

[0088] The dry-wet ratio of mouse lungs after 21 days of human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles administration in this example is shown in FIG. Figure 8As shown, this proves that the pulmonary edema of mice with pulmonary fibrosis induced by bleomycin (BLM) is more severe than that of the lungs of mice treated with human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles. Therefore, the dry and wet lung weights of the bleomycin (BLM)-induced pulmonary fibrosis group are greater than those of the human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) treatment group, indicating that the human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles of this example have therapeutic effects on pulmonary fibrosis.

[0089] The dry-wet ratio of mouse lungs after 21 days was as follows: human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles prepared by the probe ultrasonic power of this embodiment and human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles prepared by different probe ultrasonic powers. Figure 9 As shown, it is proved that the human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles prepared by the probe ultrasonic power parameters adopted in this embodiment have significantly improved the severity of pulmonary edema in mice with pulmonary fibrosis induced by bleomycin (BLM) compared with the pulmonary edema of mice treated with human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles prepared by other probe ultrasonic power parameters.

[0090] The weight monitoring data of mice treated with human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles in this example are as follows: Figure 10 As shown, the weight of mice treated with the human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles of this embodiment was greater than that of the pulmonary fibrosis group, proving that the growth status of mice treated with the human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles of this embodiment was better than that of mice with pulmonary fibrosis.

[0091] The micro CT scan images of the mouse lungs using human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles in this example are shown in FIG. Figure 11 As shown, it is proved that the human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles of this example have a significant therapeutic effect compared with the pulmonary fibrosis mice.

[0092] The statistical chart of the total number of cells in the alveolar lavage fluid of the mouse lungs containing human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles of this embodiment is as follows: Figure 12As shown in the figures, the total number of cells in the alveolar lavage fluid of mice treated with human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) was significantly less than the total number of cells in the alveolar lavage fluid of mice with pulmonary fibrosis, demonstrating that the human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles of this example have a therapeutic effect on pulmonary fibrosis.

[0093] The statistical graph of the total number of cells in the alveolar lavage fluid of the mouse lung containing human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles prepared with the probe ultrasonic power of this embodiment and human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles prepared with different probe ultrasonic powers is shown in the figure. Figure 13 As shown, the total number of cells in the bronchoalveolar lavage fluid of mice with pulmonary fibrosis induced by bleomycin (BLM) treated with human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles prepared by the probe ultrasonic power parameters adopted in this embodiment is much smaller than the total number of cells in the bronchoalveolar lavage fluid of mice with pulmonary fibrosis induced by bleomycin (BLM) treated with human umbilical cord mesenchymal stem cell membrane (MSC)-soy lecithin (SPC) biomimetic nanovesicles prepared by other probe ultrasonic power parameters.

[0094] Implementation Case 2

[0095] A method for preparing human umbilical cord mesenchymal stem cell membrane (MSC)-dipalmitoylphosphatidylcholine (DPPC) biomimetic nanovesicles, the method comprising the following steps:

[0096] S1: culture about 6.7×10 8 6.7×10 human umbilical cord mesenchymal stem cells were obtained by freeze-thaw method. 8 Extraction of cell membranes and membrane proteins from individual umbilical cord mesenchymal stem cells;

[0097] S2: The protein quantitative BCA method was used to quantify the membrane protein of the extracted human umbilical cord mesenchymal stem cells;

[0098] S3: The extracted human umbilical cord mesenchymal stem cell membranes were stored at 4°C to prepare for the subsequent synthesis of biomimetic nanovesicles;

[0099] S4: Dissolve appropriate amounts of dipalmitoylphosphatidylcholine (DPPC) and cholesterol (CHOL) in chloroform;

[0100] S5: 11 mg of human umbilical cord mesenchymal stem cell membrane (MSC) was suspended in deionized water or PBS;

[0101] S6: The organic solvent containing dipalmitoylphosphatidylcholine (DPPC) in S4 was mixed in a glass test tube and rotary evaporated at 45°C and 120 rpm until a uniform film appeared at the bottom of the glass test tube. The glass test tube was removed and vacuum dried for 6-8 hours to prepare a dry film.

[0102] S7: Hydrate the dried film prepared in S6 with 894 μL of deionized water or PBS.

[0103] S8: The liposome emulsion of S7 was sonicated in a water bath at 54°C for 24 min;

[0104] S9: The cell membrane suspension prepared in S5 was sonicated in a water bath at 38°C for 14 min;

[0105] S10: S8 liposome emulsion and S9 cell membrane suspension were mixed and magnetically stirred at 37°C and 200 rpm for 2 h.

[0106] S11: Perform probe ultrasound on the mixed solution of S10, set the output power to 150W, and ultrasound for 15 minutes;

[0107] S12: The mixed solution of S11 was co-extruded at 32°C to prepare biomimetic nanovesicles of uniform size;

[0108] S13: Western Blot detection of CD44, CD73, and CD90 in the cell membrane biomimetic nanovesicles prepared in S12;

[0109] S14: The cell membrane biomimetic nanovesicles of S12 were sealed by injecting argon gas and stored at 4°C.

[0110] S15: Human umbilical cord mesenchymal stem cell membrane (MSC)-dipalmitoylphosphatidylcholine (DPPC) biomimetic nanovesicles were delivered to the lungs of diseased mice through the respiratory tract to monitor the therapeutic effect of the biomimetic nanovesicles themselves on pulmonary fibrosis.

[0111] The step S15 includes the following steps:

[0112] S151: C57BL / 6N mice (8-week-old, male) were divided into three groups: healthy control group, pulmonary fibrosis group, and treatment group.

[0113] S152: BLM was delivered to the lungs via the respiratory tract. The dose of BLM was 3.5 mg / kg, 50 μL / time.

[0114] S153: Human umbilical cord mesenchymal stem cell membrane (MSC)-dipalmitoylphosphatidylcholine (DPPC) biomimetic nanovesicles were delivered to the lungs of mice via the respiratory tract on days 8, 10, 12, 14, 16, 18, and 20 after bleomycin (BLM) was instilled into the mice via the respiratory tract. The therapeutic efficacy was monitored by mouse body weight and lung micro-CT.

[0115] S154: On day 21, lungs were removed from mice in the three groups (healthy control group, pulmonary fibrosis group, and treatment group), and the alveolar lavage fluid was obtained and its cell content was counted;

[0116] In order to improve the targeting and therapeutic ability of human umbilical cord mesenchymal stem cell biomimetic nanovesicles on diseased areas, human umbilical cord mesenchymal stem cells can be lentivirally transfected with TGFBRII during the culture process. After successful transfection, they can be passaged normally and the preparation steps S1-S13 can be repeated.

[0117] The statistical chart of the total number of cells in the alveolar lavage fluid of the mouse lungs containing human umbilical cord mesenchymal stem cell membrane (MSC)-dipalmitoylphosphatidylcholine (DPPC) biomimetic nanovesicles of this embodiment is as follows: Figure 14 As shown, the total number of cells in the alveolar lavage fluid of mice treated with human umbilical cord mesenchymal stem cell membrane (MSC)-dipalmitoylphosphatidylcholine (DPPC) was significantly less than the total number of cells in the alveolar lavage fluid of mice with pulmonary fibrosis, demonstrating that the human umbilical cord mesenchymal stem cell membrane (MSC)-dipalmitoylphosphatidylcholine (DPPC) biomimetic nanovesicles of this example have a therapeutic effect on pulmonary fibrosis.

[0118] The statistical graph of the total number of cells in the alveolar lavage fluid of the mouse lung containing human umbilical cord mesenchymal stem cell membrane (MSC)-dipalmitoylphosphatidylcholine (DPPC) biomimetic nanovesicles prepared by the probe ultrasonic power of this embodiment and human umbilical cord mesenchymal stem cell membrane (MSC)-dipalmitoylphosphatidylcholine (DPPC) biomimetic nanovesicles prepared by different probe ultrasonic powers is shown in the figure. Figure 15 As shown, the total number of cells in the bronchoalveolar lavage fluid of mice with pulmonary fibrosis induced by bleomycin (BLM) treated with human umbilical cord mesenchymal stem cell membrane (MSC)-dipalmitoylphosphatidylcholine (DPPC) biomimetic nanovesicles prepared by the probe ultrasonic power parameters adopted in this embodiment is much smaller than the total number of cells in the bronchoalveolar lavage fluid of mice with pulmonary fibrosis induced by bleomycin (BLM) treated with human umbilical cord mesenchymal stem cell membrane (MSC)-dipalmitoylphosphatidylcholine (DPPC) biomimetic nanovesicles prepared by other probe ultrasonic power parameters.

[0119] The particle size comparison of human umbilical cord mesenchymal stem cell membrane (MSC)-dipalmitoylphosphatidylcholine (DPPC) biomimetic nanovesicles prepared by the probe ultrasonic power of this embodiment and the human umbilical cord mesenchymal stem cell membrane (MSC)-dipalmitoylphosphatidylcholine (DPPC) biomimetic nanovesicles prepared by different probe ultrasonic powers is shown in the figure. Figure 16 As shown, it can be seen that the ultrasonic preparation process parameters of the probe in this embodiment are the best preparation process parameters compared with other process parameters.

[0120] Implementation Case 3:

[0121] A method for preparing human umbilical cord mesenchymal stem cell membrane-egg yolk phosphatidylcholine (EPC) biomimetic nanovesicles, the method comprising the following steps:

[0122] S1: culture about 6.7×10 8 6.7×10 human umbilical cord mesenchymal stem cells were isolated by freeze-thaw method. 8 Extraction of cell membranes and membrane proteins from individual umbilical cord mesenchymal stem cells;

[0123] S2: The protein quantitative BCA method was used to quantify the membrane protein of the extracted human umbilical cord mesenchymal stem cells;

[0124] S3: The extracted human umbilical cord mesenchymal stem cell membrane (MSC) was stored at 4°C to prepare for the subsequent synthesis of biomimetic nanovesicles;

[0125] S4: Take appropriate amount of egg yolk phosphatidylcholine (EPC) and cholesterol (CHOL) and dissolve them in chloroform;

[0126] S5: Take 8.4 mg of human umbilical cord mesenchymal stem cell membranes and suspend them in deionized water or PBS;

[0127] S6: The organic solvent containing egg yolk phosphatidylcholine (EPC) in S4 was mixed in a glass test tube and rotary evaporated at 45°C and 120 rpm until a uniform thin film appeared at the bottom of the glass test tube. The glass test tube was removed and vacuum dried for 6-8 hours to prepare a dry film.

[0128] S7: Hydrate the dried film prepared in S6 with 894 μL of deionized water or PBS.

[0129] S8: The liposome emulsion of S7 was sonicated in a water bath at 52°C for 23 min.

[0130] S9: The cell membrane suspension prepared in S5 was sonicated in a water bath at 38°C for 14 min;

[0131] S10: S8 liposome emulsion and S9 cell membrane suspension were mixed and magnetically stirred at 37°C and 200 rpm for 2 h.

[0132] S11: The mixed solution of S10 was subjected to probe ultrasound, with the output power set to 179 W and ultrasound for 13 min;

[0133] S12: The mixed solution of S11 was co-extruded at 45°C to prepare biomimetic nanovesicles of uniform size;

[0134] S13: Western Blot detection of CD44, CD73, and CD90 in the cell membrane biomimetic nanovesicles prepared in S12;

[0135] S14: The cell membrane biomimetic nanovesicles of S12 were sealed by injecting argon gas and stored at 4°C;

[0136] S15: Human umbilical cord mesenchymal stem cell membrane (MSC)-egg yolk phosphatidylcholine (EPC) biomimetic nanovesicles were delivered to the lungs of diseased mice through the respiratory tract to monitor the therapeutic effect of the biomimetic nanovesicles themselves on pulmonary fibrosis.

[0137] The step S15 includes the following steps:

[0138] S151: C57BL / 6N (8-week-old, male mice) were divided into three groups: healthy control group, pulmonary fibrosis group, and treatment group.

[0139] S152: BLM was delivered to the lungs via the respiratory tract. The dose of BLM was 3.5 mg / kg, 50 μL / time.

[0140] S153: Human umbilical cord mesenchymal stem cell membrane (MSC)-egg lecithin (EPC) biomimetic nanovesicles were delivered to the lungs of mice via the respiratory tract on days 8, 10, 12, 14, 16, 18, and 20 after bleomycin (BLM) was instilled into the mice via the respiratory tract, and the efficacy was monitored by mouse body weight and Micro CT.

[0141] S154: On day 21, lungs were removed from the three groups of mice (healthy control group, pulmonary fibrosis group, and treatment group), and the alveolar lavage fluid was obtained and its cell content was counted.

[0142] In order to improve the targeting and therapeutic ability of human umbilical cord mesenchymal stem cell biomimetic nanovesicles on diseased areas, human umbilical cord mesenchymal stem cells can be lentivirally transfected with TGFBRII during the culture process. After successful transfection, they can be passaged normally and the preparation steps S1-S13 can be repeated.

[0143] The statistical chart of the total number of cells in the alveolar lavage fluid of the mouse lungs containing human umbilical cord mesenchymal stem cell membrane (MSC)-egg yolk phosphatidylcholine (EPC) biomimetic nanovesicles of this embodiment is as follows: Figure 17 As shown, the total number of cells in the alveolar lavage fluid of mice treated with human umbilical cord mesenchymal stem cell membrane (MSC)-egg yolk lecithin (EPC) biomimetic vesicles was significantly less than the total number of cells in the alveolar lavage fluid of mice with pulmonary fibrosis, proving that the human umbilical cord mesenchymal stem cell membrane (MSC)-egg yolk lecithin (EPC) biomimetic nanovesicles of this example have a therapeutic effect on pulmonary fibrosis.

[0144] The statistical graph of the total number of cells in the alveolar lavage fluid of the mouse lung containing human umbilical cord mesenchymal stem cell membrane (MSC)-egg yolk phosphatidylcholine (EPC) biomimetic nanovesicles prepared by the probe ultrasonic power of this embodiment and human umbilical cord mesenchymal stem cell membrane (MSC)-egg yolk phosphatidylcholine (EPC) biomimetic nanovesicles prepared by different probe ultrasonic powers is shown in the figure. Figure 18 As shown, the total number of cells in the alveolar lavage fluid of mice with pulmonary fibrosis induced by bleomycin (BLM) treated with human umbilical cord mesenchymal stem cell membrane (MSC)-egg yolk phosphatidylcholine (EPC) biomimetic nanovesicles prepared by the probe ultrasonic power parameters adopted in this embodiment is much smaller than the total number of cells in the alveolar lavage fluid of mice with pulmonary fibrosis induced by bleomycin (BLM) treated with human umbilical cord mesenchymal stem cell membrane (MSC)-egg yolk phosphatidylcholine (EPC) biomimetic nanovesicles prepared by other probe ultrasonic power parameters.

[0145] The particle size comparison of human umbilical cord mesenchymal stem cell membrane (MSC)-egg yolk lecithin (EPC) biomimetic nanovesicles prepared by the probe ultrasonic power of this embodiment and the human umbilical cord mesenchymal stem cell membrane (MSC)-egg yolk lecithin (EPC) biomimetic nanovesicles prepared by different probe ultrasonic powers is shown in the figure. Figure 19 As shown, it can be seen that the ultrasonic preparation process parameters of the probe in this embodiment are the best preparation process parameters compared with other process parameters.

[0146] Implementation Case 4:

[0147] A method for preparing human umbilical cord mesenchymal stem cell membrane (MSC)-hydrogenated soy lecithin (HSPC) biomimetic nanovesicles and its application in pulmonary fibrosis, the method comprising the following steps:

[0148] S1: culture about 6.7×10 8 6.7×10 human umbilical cord mesenchymal stem cells were obtained by freeze-thaw method. 8 Extraction of cell membranes and membrane proteins from individual umbilical cord mesenchymal stem cells;

[0149] S2: The protein quantitative BCA method was used to quantify the membrane protein of the extracted human umbilical cord mesenchymal stem cells;

[0150] S3: The extracted human umbilical cord mesenchymal stem cell membranes were stored at 4°C to prepare for the subsequent synthesis of biomimetic nanovesicles;

[0151] S4: Dissolve appropriate amounts of hydrogenated soybean lecithin (HSPC) and cholesterol (CHOL) in chloroform;

[0152] S5: Take 7 mg of human umbilical cord mesenchymal stem cell membrane and suspend it in deionized water or PBS;

[0153] S6: The organic solvent containing hydrogenated soybean lecithin (HSPC) in S4 was mixed in a glass test tube and rotary evaporated at 45°C and 120 rpm until a uniform thin film appeared at the bottom of the glass test tube. The glass test tube was removed and vacuum dried for 6-8 hours to prepare a dry film.

[0154] S7: Hydrate the dried film prepared in S6 with 894 μL of deionized water or PBS;

[0155] S8: The liposome solution of S7 was sonicated in a water bath at 55°C for 25 min;

[0156] S9: The cell membrane suspension prepared in S5 was sonicated in a water bath at 38°C for 14 min;

[0157] S10: The S8 liposome emulsion and the S9 cell membrane suspension were mixed and magnetically stirred at 37°C and 200 rpm for 2 h.

[0158] S11: The mixed solution of S10 was subjected to probe ultrasound, with the output power set to 155 W and ultrasound for 16 minutes;

[0159] S12: The mixed solution of S11 was co-extruded at 42°C to prepare biomimetic nanovesicles of uniform size;

[0160] S13: Western blot detection of CD44, CD73, and CD90 on the cell membrane biomimetic nanovesicles prepared in S11;

[0161] S14: The cell membrane biomimetic nanovesicles of S12 were sealed by injecting argon gas and stored at 4°C;

[0162] S15: Human umbilical cord mesenchymal stem cell membrane-hydrogenated soy lecithin (HSPC) biomimetic nanovesicles were delivered to the lungs of diseased mice through the respiratory tract to monitor the therapeutic effect of the biomimetic nanovesicles themselves on pulmonary fibrosis.

[0163] The step S15 includes the following steps:

[0164] S151: C57BL / 6N mice (8-week-old, male) were divided into three groups: healthy control group, pulmonary fibrosis group, and treatment group.

[0165] S152: BLM was delivered to the lungs via the respiratory tract. The dose of BLM was 3.5 mg / kg, 50 μL / time.

[0166] S153: Human umbilical cord mesenchymal stem cell membrane-hydrogenated soy lecithin (HSPC) biomimetic nanovesicles were delivered to the lungs of mice via the respiratory tract on days 8, 10, 12, 14, 16, 18, and 20 after bleomycin (BLM) was instilled into the mice via the respiratory tract. The body weight of the mice was recorded, and the efficacy was monitored by Micro CT.

[0167] S154: On day 21, lungs were removed from mice in the three groups (healthy control group, pulmonary fibrosis group, and treatment group), and the alveolar lavage fluid was obtained and its cell content was counted;

[0168] In order to improve the targeting and therapeutic ability of human umbilical cord mesenchymal stem cell biomimetic nanovesicles on diseased areas, TGFBRII can be transfected with lentiviral genes during the culture of human umbilical cord mesenchymal stem cells. After successful transfection, they can be passaged normally and the preparation steps S1-S14 can be repeated.

[0169] The statistical chart of the total number of cells in the alveolar lavage fluid of the mouse lungs containing human umbilical cord mesenchymal stem cell membrane (MSC)-hydrogenated soy lecithin (HSPC) biomimetic nanovesicles of this embodiment is as follows: Figure 20 As shown, the total number of cells in the alveolar lavage fluid of mice treated with human umbilical cord mesenchymal stem cell membrane (MSC)-hydrogenated soy lecithin (HSPC) biomimetic vesicles was significantly less than the total number of cells in the alveolar lavage fluid of mice with pulmonary fibrosis, demonstrating that the human umbilical cord mesenchymal stem cell membrane (MSC)-hydrogenated soy lecithin (HSPC) biomimetic nanovesicles of this example have a therapeutic effect on pulmonary fibrosis.

[0170] The statistical graph of the total number of cells in the alveolar lavage fluid of the mouse lung containing human umbilical cord mesenchymal stem cell membrane (MSC)-hydrogenated soy lecithin (HSPC) biomimetic nanovesicles prepared by the probe ultrasonic power of this embodiment and human umbilical cord mesenchymal stem cell membrane (MSC)-hydrogenated soy lecithin (HSPC) biomimetic nanovesicles prepared by different probe ultrasonic powers is shown in the figure. Figure 21 As shown, the total number of cells in the bronchoalveolar lavage fluid of mice with pulmonary fibrosis induced by bleomycin (BLM) treated with human umbilical cord mesenchymal stem cell membrane (MSC)-hydrogenated soy lecithin (HSPC) biomimetic nanovesicles prepared by the probe ultrasonic power parameters adopted in this embodiment is much smaller than the total number of cells in the bronchoalveolar lavage fluid of mice with pulmonary fibrosis induced by bleomycin (BLM) treated with human umbilical cord mesenchymal stem cell membrane (MSC)-hydrogenated soy lecithin (HSPC) biomimetic nanovesicles prepared by other probe ultrasonic power parameters.

[0171] The particle size comparison of human umbilical cord mesenchymal stem cell membrane (MSC)-hydrogenated soy lecithin (HSPC) biomimetic nanovesicles prepared by the probe ultrasonic power of this embodiment and the human umbilical cord mesenchymal stem cell membrane (MSC)-hydrogenated soy lecithin (HSPC) biomimetic nanovesicles prepared by different probe ultrasonic powers is shown in the figure. Figure 22 As shown, it can be seen that the ultrasonic preparation process parameters of the probe in this embodiment are the best preparation process parameters compared with other process parameters.

Claims

1. An application of human umbilical cord mesenchymal stem cell membrane biomimetic nanovesicles, characterized in that: The bionic nanovesicles are used to prepare drugs for alleviating and inhibiting pulmonary fibrosis; the bionic nanovesicles contain cell membranes of human umbilical cord mesenchymal stem cells; The preparation method of the human umbilical cord mesenchymal stem cell membrane biomimetic nanovesicles is as follows: 1) extracting cell membranes of subcultured human umbilical cord mesenchymal stem cells and suspending them in a cell suspension; 2) adding cholesterol to the artificial lipid and then dissolving it in an organic solvent; 3) Rotary evaporation of the organic solvent containing the artificial lipid in step 2) and vacuum drying; 4) hydrating, ultrasonicating, magnetically stirring, and co-extruding the cell suspension containing human umbilical cord mesenchymal stem cell membranes in step 1) and the mixed film of lipids after drying in step 3) to obtain the human umbilical cord mesenchymal stem cell membrane biomimetic nanovesicles; the biomimetic nanovesicles contain stem cell membrane proteins, and the stem cell membrane proteins are membrane proteins extracted from human umbilical cord mesenchymal stem cells; Among them, the mass ratio of human umbilical cord mesenchymal stem cell membrane to artificial lipid is 5:6-9; The mass ratio of artificial lipids to cholesterol is 6 to 8:

1.

2. The use according to claim 1, characterized in that The stem cell membrane protein is a membrane protein extracted from human umbilical cord mesenchymal stem cells transfected with TGFBRII. The TGFBRII is a transmembrane membrane protein of the TGF-β1 receptor.

3. The use according to claim 1 or 2, characterized in that The biomimetic nanovesicles are delivered through the respiratory tract.

4. The use according to claim 1 or 2, characterized in that The biomimetic nanovesicles can also be loaded with chemical drugs or biological agents.

5. The use according to claim 4, characterized in that The chemical drug is a hydrophilic, hydrophobic or amphiphilic chemical drug; the biological agent is a mixture of one or more of siRNA drugs, mRNA drugs, DNA drugs, protein drugs, polypeptide drugs, immunopotentiators, immunosuppressants, immune adjuvants and imaging contrast agents.

6. The use according to claim 1, characterized in that The human umbilical cord mesenchymal stem cells are stem cells transfected with TGFBRII gene.

7. The use according to claim 1, characterized in that In step 4), after the dried mixed film is hydrated, the artificial liposome emulsion is ultrasonicated in a water bath at 48 to 55° C. for 20 to 25 minutes; the cell membrane suspension is ultrasonicated in a water bath at 35 to 48° C. for 10 to 15 minutes; then, the artificial liposome emulsion and the cell suspension are mixed and ultrasonicated using a probe for 9 to 10 minutes, with the power of the probe ultrasonication being 140 to 155 W.

8. The use according to claim 1, characterized in that The cell suspension in step 1) is deionized water or PBS; the organic solvent is chloroform and methanol.

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