Bionic vesicle preparation as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of bionic nano-drugs, and particularly relates to a bionic vesicle preparation as well as a preparation method and application thereof. The bionic vesicle preparation disclosed by the invention is prepared from the following raw materials: mesenchymal stem cell outer vesicles, nintedanib and DSPE-PEG-HA (hyaluronic acid modified pegylated phospholipid); the nintedanib is loaded in the mesenchymal stem cell outer vesicle, and the DSPE-PEG-HA is coupled to the surface of the mesenchymal stem cell outer vesicle. The bionic vesicle preparation disclosed by the invention can be used for directly delivering a drug to a pulmonary fibrosis injury part through inhalation type drug delivery, and acting on a focus part at a higher local concentration, meanwhile, the exposure of the drug in whole-body circulation is remarkably reduced, the occurrence of side effects is reduced, and the specific targeting intervention and regulation on fibroblasts are realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of bionic nanomedicine, and specifically relates to a bionic vesicle preparation and a preparation method and application thereof. Background Art

[0002] Pulmonary fibrosis (PF) is a common feature of many diseases, including fibrotic interstitial lung disease, pneumoconiosis and cystic pulmonary fibrosis, which seriously threatens human health and quality of life. In addition, some lung diseases, such as chronic obstructive pulmonary disease, acute lung injury, radiation lung injury and immune checkpoint inhibitor-related lung injury, may also induce pulmonary fibrosis. In cases of pulmonary fibrosis, the cause of the vast majority of cases is unknown. These cases of pulmonary fibrosis with unknown causes are idiopathic pulmonary fibrosis. As the incidence of idiopathic pulmonary fibrosis increases year by year, how to find an effective method to treat idiopathic pulmonary fibrosis has become a problem that needs to be solved urgently.

[0003] Nintedanib has been approved by the U.S. Food and Drug Administration for the treatment of moderate idiopathic pulmonary fibrosis, becoming one of the drugs for treating this type of disease. However, the side effects of Nintedanib are more obvious, including nausea, vomiting, diarrhea, and gastrointestinal discomfort. At the same time, the high cost and limited efficacy of Nintedanib in the treatment of pulmonary fibrosis restrict its widespread clinical application.

[0004] At present, although bionic nanomedicines have achieved certain results in the treatment of pulmonary fibrosis in the existing technology, the bionic nanomedicine intervention strategies in the existing technology are all implemented through intravenous administration. This method of administration may induce side effects on organs such as the liver and kidneys, and due to the existence of the gas-blood barrier, the drug penetration of intravenous administration is further limited. Inhalation administration can improve the delivery efficiency of drugs, but there is a lung clearance mechanism in the lungs, which is the main factor affecting the drug deposition rate during inhalation administration. The inhalation treatment strategy still faces huge challenges in drug particle size design and reducing the clearance effect of lung macrophages.

[0005] Therefore, there is an urgent need to develop a bionic nanodrug treatment strategy that is suitable for the aerodynamic diameter, can be administered by inhalation, and can improve lung deposition efficiency, in order to achieve effective treatment of pulmonary fibrosis. Summary of the invention

[0006] In order to solve the problem that the prior art lacks a bionic nano drug that is suitable for aerodynamic diameter and can be administered by inhalation, the present invention provides a bionic vesicle preparation and a preparation method and application thereof. The bionic vesicle preparation can be administered by inhalation, improve the drug delivery efficiency and lung deposition efficiency, and effectively treat pulmonary fibrosis. The specific technical scheme includes the following:

[0007] A bionic vesicle preparation, the raw materials of which include mesenchymal stem cell extracellular vesicles, nintedanib, and DSPE-PEG-HA;

[0008] The DSPE-PEG-HA is conjugated to the surface of mesenchymal stem cell extracellular vesicles to obtain mesenchymal stem cell extracellular vesicles conjugated with DSPE-PEG-HA; the nintedanib is loaded into the mesenchymal stem cell extracellular vesicles conjugated with DSPE-PEG-HA.

[0009] Preferably, the mass ratio of the DSPE-PEG-HA conjugated with the mesenchymal stem cell extracellular vesicles is 1-2:1-10.

[0010] Preferably, the mass ratio of the nintedanib to the mesenchymal stem cell extracellular vesicles conjugated with DSPE-PEG-HA is 1-2:1-10.

[0011] Preferably, the particle size of the bionic vesicle preparation is 100nm-200nm.

[0012] Preferably, the dosage form of the bionic vesicle preparation includes any one of aerosol, dry powder inhaler, or nebulized inhaler.

[0013] The present invention also discloses a preparation method of the bionic vesicle preparation as described in any one of the above, including the following steps:

[0014] Conjugate DSPE-PEG-HA to the surface of mesenchymal stem cell extracellular vesicles and incubate for 24 h to obtain mesenchymal stem cell extracellular vesicles conjugated with DSPE-PEG-HA;

[0015] Load nintedanib into the mesenchymal stem cell extracellular vesicles conjugated with DSPE-PEG-HA to obtain the bionic vesicle preparation.

[0016] Preferably, before quantifying the mesenchymal stem cell extracellular vesicles, it further includes the steps of culturing mesenchymal stem cells and extracting mesenchymal stem cell extracellular vesicles.

[0017] Preferably, the nintedanib is loaded into the mesenchymal stem cell extracellular vesicles conjugated with DSPE-PEG-HA by electropermeation technology.

[0018] Preferably, the conditions for the incubation are: overnight incubation at 4°C.

[0019] The present invention also provides the application of the bionic vesicle preparation prepared by the bionic vesicle preparation or the preparation method as described in any one of the above in the preparation of a drug for treating pulmonary fibrosis.

[0020] The beneficial effects of the present invention are as follows:

[0021] The present invention provides a bionic vesicle preparation, comprising mesenchymal stem cell extracellular vesicles, nintedanib, and DSPE-PEG-HA. The nintedanib is loaded in the mesenchymal stem cell extracellular vesicles, and the DSPE-PEG-HA is conjugated to the surface of the mesenchymal stem cell extracellular vesicles. The bionic vesicle preparation of the present invention can be administered by inhalation to directly deliver the bionic vesicle preparation to the pulmonary fibrosis injury site, act on the lesion site at a higher local concentration, and at the same time significantly reduce the exposure of the drug in the systemic circulation, reduce the occurrence of side effects, and achieve specific targeted intervention and regulation of fibroblasts. Moreover, the designed bionic vesicle preparation of the present invention can reduce the influence of the lung clearance mechanism on the drug deposition rate, improve the drug deposition rate, and further improve the drug effect. The present invention provides an effective treatment approach for pulmonary fibrosis, especially specific pulmonary fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0023] Figure 1 It is a schematic diagram of the detection results of different detection items in Example 1 and Example 3 of the present invention;

[0024] Among them, a is a schematic diagram of the result of observing extracellular vesicles with an electron microscope in Example 1 of the present invention; b is a schematic diagram of the detection result of detecting the average particle size of N@HA-MSC EVs by a Malvern particle size analyzer in Example 1 of the present invention; c is a schematic diagram of the detection result of high-performance liquid technology in Example 3 of the present invention;

[0025] Figure 2 It is the fluorescence imaging experimental result of the maximum membrane insertion amount of MSC EVs and DPSE-PEG-HA in Example 2 of the present invention;

[0026] Among them, the fluorescence imaging experimental results of HA-MSC EVs prepared from DPSE-PEG-HA with concentrations of 1 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL from left to right;

[0027] Figure 3 It is the flow cytometry detection test result of the maximum membrane insertion amount of MSC EVs and DPSE-PEG-HA in Example 2 of the present invention;

[0028] Among them, the flow cytometry detection test results of the maximum membrane insertion amount of HA-MSC EVs prepared from DPSE-PEG-HA with concentrations of 1 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL from left to right;

[0029] Figure 4This is a schematic diagram of the results of staining cell fibrosis-related proteins using immunofluorescence staining technology in Example 4 of the present invention. Detailed implementation manners

[0030] The present invention provides a bionic vesicle preparation, the raw materials of which include mesenchymal stem cell extracellular vesicles, nintedanib, and DSPE-PEG-HA; the DSPE-PEG-HA is conjugated to the surface of the mesenchymal stem cell extracellular vesicles to obtain mesenchymal stem cell extracellular vesicles conjugated with DSPE-PEG-HA; the nintedanib is loaded into the mesenchymal stem cell extracellular vesicles conjugated with DSPE-PEG-HA.

[0031] As an implementation manner, the mass ratio of the conjugation of DSPE-PEG-HA to the mesenchymal stem cell extracellular vesicles is 1-2:1-10. As another implementation manner, the mass ratio of the conjugation of DSPE-PEG-HA to the mesenchymal stem cell extracellular vesicles can be any one of 1:1, 1:5, 1:10, 1.5:1, 1.5:5, 1.5:10, 2:1, and 2:5. As an implementation manner, the mass ratio of the nintedanib to the mesenchymal stem cell extracellular vesicles conjugated with DSPE-PEG-HA is 1-2:1-10. As another implementation manner, the mass ratio of the nintedanib to the mesenchymal stem cell extracellular vesicles conjugated with DSPE-PEG-HA can be any one of 1:1, 1:4, 1:10, 1.5:1, 1.5:5, 1.5:10, 2:1, 2:5, and 2:10. As an implementation manner, the particle size of the bionic vesicle preparation is 100nm-200nm. As an implementation manner, the particle size of the bionic vesicle preparation can be any one of 100nm, 120nm, 140nm, 160nm, 180nm, and 200nm. As an implementation manner, the dosage form of the bionic vesicle preparation includes any one of aerosol, dry powder inhaler, or nebulized inhaler. In a specific embodiment, the mass ratio of the conjugation of DSPE-PEG-HA to the mesenchymal stem cell extracellular vesicles is 1:1, the loading mass ratio of the nintedanib to the mesenchymal stem cell extracellular vesicles conjugated with DSPE-PEG-HA is 1:4, the particle size of the bionic vesicle preparation is 150nm, and the dosage form of the bionic vesicle preparation is a nebulized inhaler.

[0032] The present invention also provides a method for preparing the bionic vesicle preparation as described above, which comprises the following steps: after quantifying the protein content of mesenchymal stem cell extracellular vesicles, DSPE-PEG-HA is conjugated to the surface of mesenchymal stem cell extracellular vesicles and incubated for 24 h to obtain mesenchymal stem cell extracellular vesicles conjugated with DSPE-PEG-HA (HA-MSC EVs); nintedanib is loaded into the mesenchymal stem cell extracellular vesicles conjugated with DSPE-PEG-HA to obtain the bionic vesicle preparation.

[0033] As an implementation manner, before quantifying the mesenchymal stem cell extracellular vesicles, it further comprises the steps of culturing mesenchymal stem cells and extracting mesenchymal stem cell extracellular vesicles. As an implementation manner, the present invention uses a dedicated mesenchymal stem cell medium containing 1% penicillin-streptomycin to culture mesenchymal stem cells. As an implementation manner, the culture conditions of mesenchymal stem cells are 37 °C and 5% CO2. As an implementation manner, the present invention cultures mesenchymal stem cells until the cell confluence reaches 90%.

[0034] As an implementation manner, after the culturing of mesenchymal stem cells is completed, the mesenchymal stem cell medium is removed, the cells are washed, and the cells are added to a serum-free medium and cultured for 48 h, and the culture solution is collected to obtain the culture solution. As an implementation manner, after obtaining the culture solution, the medium is subjected to gradient centrifugation to remove cell debris and particulate matter to obtain mesenchymal stem cell extracellular vesicles. As an implementation manner, the gradient centrifugation is carried out in three steps. The first centrifugation is carried out at 500 g for 10 min to remove cell debris, the second centrifugation is carried out at 23,000 g for 10 min to remove particulate matter; the third centrifugation is carried out at 100,000 g for 70 min to precipitate mesenchymal stem cell extracellular vesicles. As an implementation manner, after obtaining the mesenchymal stem cell extracellular vesicles, the mesenchymal stem cell extracellular vesicles can be resuspended with PBS or a serum-free medium and stored at -80 °C for later use.

[0035] After quantifying the mesenchymal stem cell extracellular vesicles, DSPE-PEG-HA was conjugated to the surface of the mesenchymal stem cell extracellular vesicles and incubated for 24 h to obtain mesenchymal stem cell extracellular vesicles conjugated with DSPE-PEG-HA. As an implementation manner, the present invention uses a BCA protein quantification detection kit to quantify the mesenchymal stem cell extracellular vesicles. As an implementation manner, the incubation condition is overnight incubation at 4°C. As an implementation manner, after the incubation, it further includes the step of using a 100KD ultrafiltration tube and centrifuging at 4°C and 2000g for 15 min to remove MSC EVs that did not insert DPSE-PEG-HA. In a specific embodiment, after DSPE-PEG-HA and mesenchymal stem cell extracellular vesicles are blended in the above-mentioned ratio, they are incubated for 24 h, and then a 100KD ultrafiltration tube is used to centrifuge at 4°C and 2000g for 15 min to remove MSC EVs that did not insert DPSE-PEG-HA, obtaining mesenchymal stem cell extracellular vesicles conjugated with DSPE-PEG-HA.

[0036] After obtaining the mesenchymal stem cell extracellular vesicles conjugated with DSPE-PEG-HA, the present invention loaded nintedanib into the mesenchymal stem cell extracellular vesicles conjugated with DSPE-PEG-HA to obtain the bionic vesicle preparation. As an implementation manner, the present invention uses electroporation technology to load nintedanib into the mesenchymal stem cell extracellular vesicles conjugated with DSPE-PEG-HA. In a specific embodiment, the present invention uses electroporation technology to load nintedanib into the mesenchymal stem cell extracellular vesicles conjugated with DSPE-PEG-HA at a mass ratio of 1:4 to obtain the bionic vesicle preparation N@HA-MSC EVs. The loading rate of nintedanib in N@HA-MSC EVs is 20%.

[0037] The present invention also provides the application of the bionic vesicle preparation prepared by the above-mentioned bionic vesicle preparation or preparation method in the preparation of drugs for treating pulmonary fibrosis. As an implementation manner, the pulmonary fibrosis includes specific fibrosis.

[0038] To further illustrate the present invention, the following describes in detail a bionic vesicle preparation provided by the present invention with reference to the accompanying drawings and embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0039] Example 1 Preparation method of specific pulmonary fibrosis bionic vesicle preparation

[0040] Mesenchymal stem cells (MSCs) were cultured in a mesenchymal-specific medium (purchased from Tianjin Simpson International Life Science Co., Ltd.). The mesenchymal-specific medium contained penicillin-streptomycin at a volume concentration of 1%. The mesenchymal cells were cultured at 37°C and 5% CO2 until the cell confluence reached 90%. Then, the mesenchymal medium was removed, and the cells were washed. The cells were added to a serum-free stem cell medium, and the culture conditions remained unchanged. The cells were cultured for another 48 h, and the culture fluid was collected.

[0041] The culture fluid was centrifuged at 500 g for 10 min to remove cell debris; then, it was centrifuged at 23,000 g for 10 min to remove larger particles in the culture fluid; finally, it was centrifuged at 100,000 g for 70 min to precipitate extracellular vesicles (denoted as MSC EVs). The precipitated MSC EVs were collected, resuspended in PBS or a serum-free medium, and stored at -80°C for later use. The MSC EVs were observed using a transmission electron microscope, and the results are as Figure 1 shown in a of [Figure]. It can be seen that the extracellular vesicles are nearly circular cup-shaped, and the particle size of the extracellular vesicles is within 200 nm.

[0042] The BCA protein quantification kit was used to quantify the MSC EVs. The concentration of the MSC EVs was 100 μg / mL of MSCEVs. The MSC EVs were mixed with 10 μg / mL of DPSE-PEG-HA labeled with fluorescein isothiocyanate (FITC) at a volume ratio of 1:10 and incubated overnight at 4°C for membrane insertion. After incubation, a 100 KD ultrafiltration tube was used, and centrifugation was performed at 4°C and 2,000 g for 15 min to remove the MSC EVs that did not insert DPSE-PEG-HA, obtaining HA-MSC EVs.

[0043] Nintedanib was loaded into HA-MSC EVs using the electroporation technique. The mass ratio of nintedanib to HA-MSC EVs was 1:4, that is, the loading rate of nintedanib in N@HA-MSC EVs was 20%, obtaining N@HA-MSC EVs.

[0044] The average particle size of N@HA-MSC EVs was detected using a Malvern particle size analyzer, and the results are as Figure 1 shown in b of [Figure]. As can be seen from Figure 1 b of [Figure], the average particle size of N@HA-MSC EVs is about 200 nm.

[0045] The specific operation steps of the electroporation technique are as follows:

[0046] Sample mixing: Mix the small molecule drug solution and the vesicle suspension, mix well according to the set ratio, and avoid the instability of vesicles caused by high-concentration drugs.

[0047] Transfer to the electroporation device: Transfer the mixed liquid between the electroporation electrodes, ensuring that the sample volume fills the electrode gap and avoiding interference from bubbles.

[0048] Set the electroporation parameters: The electric field strength is 500 V / cm; Pulse time: The pulse width is 10 ms and the interval is 0.1 s. Pulse number: 5 times, avoiding over-treatment to damage the vesicles.

[0049] Perform electroporation: Start the electroporator and apply the electric field. After the pulse, let it stand for 3 minutes to allow the small molecule drug to diffuse into the interior of the vesicles.

[0050] Cooling treatment: After the pulse is completed, immediately place the sample in an ice bath and cool for 30 min to stabilize the vesicle membrane structure.

[0051] Experiment on exploring the maximum membrane insertion amount of extracellular vesicles (MSC EVs) and DPSE-PEG-HA in Example 2

[0052] In this example, fluorescence imaging and flow cytometry are used to determine the maximum membrane insertion amount.

[0053] Prepare DPSE-PEG-HA with concentrations of 1 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL, and the above concentrations are all the concentrations of DPSE-PEG-HA in PBS solution. Mix the above different concentrations of DPSE-PEG-HA with 100 μg / mL of MSC EVs respectively, and the mixing ratio is DPSE-PEG-HA:MSC EVs = 500 μL:50 μL. After incubation for 1 h, HA-MSC EVs samples are obtained. They are named HA-MSC EVs samples 1 to 4 in sequence.

[0054] Fluorescence imaging experiment: Image HA-MSC EVs samples 1 to 4 using a confocal microscope or a fluorescence microscope respectively. During fluorescence imaging, the fluorescently labeled DPSE-PEG-HA will label the outer vesicle membrane after membrane insertion, so the degree of membrane insertion at different concentrations can be judged by observing the fluorescence intensity and fluorescence distribution of the samples. The fluorescence imaging results are as Figure 2 shown, and it can be seen from Figure 2 that increasing the concentration of DPSE-PEG-HA will lead to an increase in the fluorescence signal, and then it is judged that the maximum membrane insertion amount is 10 μg / mL of DPSE-PEG-HA per 500 μL inserted into 50 μL of 100 μg / mL of MSC EVs.

[0055] Flow cytometry detection test: After fluorescence staining, samples 1-4 of HA-MSC EVs were analyzed by flow cytometry. Flow cytometry can accurately detect the fluorescence intensity and distribution of each extracellular vesicle. Through the fluorescence channel with an excitation wavelength of 405 nm and an emission wavelength of 610 nm, the fluorescence intensity of each extracellular vesicle can be quantified, so as to evaluate the membrane insertion effect after treatment with different concentrations of DPSE-PEG-HA. The detection results of the flow cytometry detection of samples 1-4 in the present invention are as Figure 3 shown, as Figure 3 can be seen, it can be seen that increasing the concentration of DPSE-PEG-HA will lead to an increase in the fluorescence signal, and then it is judged that the maximum membrane insertion amount is: the mass ratio of extracellular vesicles to DSPE-PEG-HA is 1:1.

[0056] Example 3 Experiment on the optimal loading rate of nintedanib in N@HA-MSC EVs

[0057] In this example, high-performance liquid technology was used to detect N@HA-MSC EVs as follows to determine the optimal loading rate of nintedanib in N@HA-MSC EVs. The loading rate is the mass percentage of nintedanib in N@HA-MSC EVs. In this example, HA-MSC EVs with a mass ratio of MSC EVs to DSPE-PEG-HA of 1:1 were used for the experiment.

[0058] Sample 1: N@HA-MSC EVs were prepared according to the mass ratio of nintedanib:HA-MSC EVs of 1:1 using the preparation method described in Example 1; the loading ratio of nintedanib in Sample 1 was 50%.

[0059] Sample 2: N@HA-MSC EVs were prepared according to the mass ratio of nintedanib:HA-MSC EVs of 1:4 using the preparation method described in Example 1; the loading ratio of nintedanib in Sample 2 was 20%.

[0060] Sample 3: N@HA-MSC EVs were prepared according to the mass ratio of nintedanib:HA-MSC EVs of 1:10 using the preparation method described in Example 1; the loading ratio of nintedanib in Sample 3 was 10%.

[0061] Sample 4: N@HA-MSC EVs were prepared according to the mass ratio of nintedanib:HA-MSC EVs of 1:20 using the preparation method described in Example 1; the loading ratio of nintedanib in Sample 4 was 5%.

[0062] The above samples were respectively experimented with an Agilent 1260 high performance liquid chromatograph to explore the optimal loading rate of nintedanib in N@HA-MSC EVs. The experimental process was as follows: Using an XBridge RP-C18 chromatographic column (250 mm × 4.6 mm, 3.5 μm), gradient elution was performed with methanol (A) - 0.1% trifluoroacetic acid (B) as the mobile phase, and the flow rate was 1 mL·min -1 , the detection wavelength was 287 nm, the column temperature was 35 °C, and the injection volume was 10 μl. The group with the best loading effect was shown, that is, the high performance liquid chromatography detection result of sample 2, as shown in Figure 1 c shown in

[0063] As can be seen from Figure 1 c, the optimal loading rate of nintedanib in N@HA-MSC EVs was 20%.

[0064] Example 4 Construction of an in vitro pulmonary fibrosis model and evaluation of the in vitro intervention effect of N@HA-MSC EVs

[0065] 1) Construction of an in vitro pulmonary fibrosis model

[0066] L-929 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) by volume and 1% penicillin / streptomycin by volume. The culture conditions were in a constant temperature incubator at 37 °C and 5% CO2. When the cell density reached 80%, passage was carried out using a 0.25% trypsin-EDTA solution, and the cells were cultured to a density of 1×10 5 cells / mL.

[0067] After the L-929 cells adhered to the wall, 1 mL of RPMI-1640 containing 5 ng / mL TGF-β factor was added to a 24-well cell culture plate. After induction for 24 hours, in vitro induction culture was carried out to obtain a pulmonary fibrosis model.

[0068] 2) Evaluation of the in vitro intervention effect of N@HA-MSC EVs

[0069] N@HA-MSC EVs were added to the in vitro pulmonary fibrosis model, and the cells were cultured for another 24 hours. The cells were fixed with paraformaldehyde, and then the expression of proteins related to cell fibrosis such as α-smooth muscle actin (α-SMA) and fibronectin (Fn) was examined using immunofluorescence staining technology to evaluate the fibrosis reversal effect of the drug delivery system. The examination results are as shown in Figure 4 shown.

[0070] As can be seen from Figure 4It can be seen that in in vitro cell experiments, the present invention uses TGF-β to construct a cell fibrosis model, co-cultures N@HA-MSC EVs with L-929 fibroblasts, and then uses immunofluorescence technology to examine the expression of fibroblast-related proteins α-SMA and Fn in L929 cells. It can be observed that after treatment with N@HA-MSC EVs, the fluorescence signals corresponding to α-SMA and Fn proteins are significantly reduced. This further proves that after the intervention, the signal intensity is significantly weakened, and a good intervention effect can be achieved. The above preliminary experiment results lay a foundation for the subsequent formal experiments.

[0071] In summary, the biomimetic vesicle preparation provided by the present invention can be directly delivered to the pulmonary fibrosis injury site through inhaled administration, act on the lesion site at a higher local concentration, while significantly reducing the exposure of the drug in the systemic circulation and reducing the occurrence of side effects, so as to achieve specific targeted intervention and regulation of fibroblasts. Moreover, the biomimetic vesicle preparation designed by the present invention can reduce the influence of the lung clearance mechanism on the drug deposition rate, improve the drug deposition rate, and thus improve the drug effect.

[0072] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments without creative efforts as in this embodiment, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A bionic vesicle preparation, characterized in that, The raw materials include mesenchymal stem cell extracellular vesicles, nintedanib, and DSPE-PEG-HA; The DSPE-PEG-HA is conjugated to the surface of mesenchymal stem cell extracellular vesicles to obtain mesenchymal stem cell extracellular vesicles conjugated with DSPE-PEG-HA; the nintedanib is loaded into the mesenchymal stem cell extracellular vesicles conjugated with DSPE-PEG-HA.

2. The biomimetic vesicle preparation according to claim 1, wherein The mass ratio of the DSPE-PEG-HA conjugated to the mesenchymal stem cell extracellular vesicles is 1-2:1-10.

3. The bionic vesicle preparation according to claim 1, wherein The mass ratio of the nintedanib to the mesenchymal stem cell extracellular vesicles conjugated with DSPE-PEG-HA is 1-2:1-10.

4. The biomimetic vesicle preparation according to claim 1, wherein The particle size of the bionic vesicle preparation is 100 nm to 200 nm.

5. The biomimetic vesicle preparation according to claim 1, wherein The dosage form of the bionic vesicle preparation includes any one of aerosol, dry powder inhalant, or nebulized inhalant.

6. The preparation method of the bionic vesicle preparation according to any one of claims 1 to 5, characterized in that, It includes the following steps: Conjugate DSPE-PEG-HA to the surface of mesenchymal stem cell extracellular vesicles and incubate for 24 h to obtain mesenchymal stem cell extracellular vesicles conjugated with DSPE-PEG-HA; Load nintedanib into the mesenchymal stem cell extracellular vesicles conjugated with DSPE-PEG-HA to obtain the bionic vesicle preparation.

7. The preparation method according to claim 6, characterized in that, Before quantifying the mesenchymal stem cell extracellular vesicles, it also includes the steps of culturing mesenchymal stem cells and extracting mesenchymal stem cell extracellular vesicles.

8. The preparation method according to claim 6, characterized in that, Use electroporation technology to load nintedanib into the mesenchymal stem cell extracellular vesicles conjugated with DSPE-PEG-HA.

9. The preparation method according to claim 6, characterized in that, The conditions for the incubation are: overnight incubation at 4°C.

10. Use of the bionic vesicle preparation according to any one of claims 1-5 or the bionic vesicle preparation prepared by the preparation method according to any one of claims 6-9 in the preparation of a drug for treating pulmonary fibrosis.

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