Large-scale preparation method and application of nano vesicles for targeted therapy of fibrosis

Abstract

The invention discloses a large-scale preparation method and application of nano vesicles for targeted therapy of fibrosis. The preparation method comprises the following steps: S1, extraction, purification and identification of mesenchymal stem cells; S2, construction and cell infection of an engineered lentiviral vector; S3, large-scale preparation and identification of the nano vesicles; according to the preparation method, the expression sequences of the TERT gene and the Lamp2b-iRGD fusion protein are introduced into the human umbilical cord-derived mesenchymal stem cells, so that efficient production and targeting enhancement of the nano-vesicles are realized; the MSR1-siRNA is loaded into the nano-vesicle by adopting an electroporation technology, and the expression of the MSR1 is inhibited through targeted delivery, so that the specific treatment capability of the nano-vesicle on fibrosis tissues is effectively improved.

Description

Technical Field

[0001] The present invention relates to the technical field of nanovesicle preparation, and in particular to a large-scale preparation method of nanovesicles for targeted treatment of fibrosis and application thereof. Background Art

[0002] Deafness, as a global health problem, has had a profound impact on my country's society and economy. According to the latest report of the World Health Organization (WHO) in 2021, approximately 466 million people worldwide are affected by hearing loss, accounting for 6.5% of the world's population. Cochlear implant surgery is currently the most effective means of hearing reconstruction for severe and above sensorineural hearing loss. However, although cochlear implants are widely believed to have good biocompatibility, the incidence of cochlear fibrosis caused by postoperative foreign body reaction is as high as about 90%. The newly formed fibrous tissue not only affects the patient's hearing reconstruction effect, but may also damage the fine structure of the cochlea and increase the risk of secondary surgery. Due to the special anatomical environment of the inner ear and the existence of the inner ear blood-labyrinth barrier, anti-fibrotic drugs are difficult to effectively reach the inner ear, thereby limiting their therapeutic effect. Therefore, a new treatment strategy for inner ear fibrosis is urgently needed.

[0003] Nanovesicles secreted by mesenchymal stem cells, as a natural and efficient drug carrier, have shown good prospects in the treatment of various diseases such as ischemic stroke. The nanovesicles are only 30-150 nanometers in diameter, rich in various functional components such as miRNA and protein, and have the ability to cross the blood-brain barrier, making them ideal inner ear drug delivery carriers. In addition, nanovesicles derived from mesenchymal stem cells have immunomodulatory effects and can effectively improve inflammatory responses. Compared with whole-cell therapy, mesenchymal stem cell nanovesicles also have the advantages of low immunogenicity, low risk of vascular occlusion, non-tumorigenicity, convenient storage, and high biosafety. Therefore, nanovesicles derived from mesenchymal stem cells are expected to inhibit foreign body reactions after cochlear implantation, improve the immune microenvironment of the inner ear, and thus inhibit cochlear fibrosis.

[0004] Nanovesicles have broad potential for application in the inner ear, but if they are not targeted and controlled, they may have adverse effects on the normal physiological functions of other cells in the cochlea, thereby limiting the accuracy and effectiveness of their clinical application. In order to improve the targeted therapeutic effect of nanovesicles, researchers have adopted a variety of modification methods, among which gene fusion technology is an effective method. By fusing the gene sequence of the target protein or peptide with the gene sequence of the cell membrane protein, specific proteins or peptides can be displayed on the surface of nanovesicles. If this method is adopted, the iRGD peptide targeting fibrotic tissue is modified on the surface of nanovesicles, which will greatly improve the therapeutic effect of cochlear fibrosis. However, in order to meet the needs of clinical treatment, a large number of nanovesicles are usually required, which requires the development of a large-scale preparation method. At present, the acquisition of primary mesenchymal stem cells is difficult, has a long cycle, and has a low success rate. In addition, during in vitro culture, with the increase of culture generations, the proliferation ability of mesenchymal stem cells gradually decreases, and replicative aging occurs, resulting in a decrease in the extraction efficiency of nanovesicles.

[0005] For this reason, macrophages play an important role in the formation of fibrotic pathology. Macrophages further aggravate the progression of fibrosis by secreting proinflammatory factors and stimulating fibroblast proliferation. Among them, Macrophage Scavenger Receptor 1 (MSR1) is a key molecule in fibrotic lesions. Studies have shown that overexpression of MSR1 significantly promotes fibroblast activation and excessive deposition of extracellular matrix, and is an important driving factor in the formation of fibrotic pathology. Therefore, genetic intervention strategies targeting MSR1, such as targeted inhibition of MSR1 expression by siRNA (small interfering RNA), can effectively block the process of fibrosis. Combining nanovesicles derived from mesenchymal stem cells and siRNA technology, MSR1-siRNA is efficiently loaded into nanovesicles by electroporation technology, making nanovesicles an ideal siRNA delivery carrier, which can not only accurately target fibrotic tissues, but also efficiently deliver siRNA to inhibit MSR1 expression. Through this strategy, it is expected to overcome the delivery challenges brought by the blood-labyrinth barrier of the inner ear and improve the accuracy and effectiveness of anti-fibrosis treatment.

[0006] For the above reasons, developing a method for large-scale preparation of nanovesicles for targeted fibrosis therapy has important clinical significance for the treatment of fibrotic diseases in the cochlea and other parts. Summary of the invention

[0007] In order to overcome the deficiencies of the prior art, one of the objectives of the present invention is to provide a large-scale preparation method of nanovesicles for targeted treatment of fibrosis to solve the above-mentioned traditional technical problems.

[0008] The second object of the present invention is to provide a nanovesicle prepared by the above preparation method.

[0009] The third purpose of the present invention is to provide an application, in which the nanovesicles obtained as above are used in the preparation of drugs for targeted treatment of fibrosis.

[0010] One of the purposes of the present invention is achieved by the following technical solution:

[0011] A large-scale preparation method of nanovesicles for targeted treatment of fibrosis, comprising the following preparation steps:

[0012] S1: Extraction, purification and identification of mesenchymal stem cells

[0013] Under sterile conditions, the tissue material to be extracted from mesenchymal stem cells is enzymatically treated with an enzymatic agent, filtered, and a single cell suspension is obtained, and the cell suspension is inoculated into a coated culture bottle, and cultured at 37°C and 5% CO2. After 72 hours of culture, the non-adherent cells are discarded, and fresh complete culture medium is added, and the culture is continued until the cell fusion rate reaches 80%-90%. The cells are washed with PBS and digested with trypsin for passage until high-purity mesenchymal stem cells are obtained. Finally, the specific surface markers of mesenchymal stem cells are detected by flow cytometry. At the same time, a three-line differentiation experiment of osteogenic, adipogenic and chondrogenic is performed to further verify the multidirectional differentiation potential of the cells, and mesenchymal stem cells within P5 and with good vitality are selected for the next experiment;

[0014] S2: Construction of engineered lentiviral vectors and cell infection

[0015] The three-plasmid co-transfection system is used to construct an engineered lentiviral vector. The engineered lentiviral vector is co-transfected with a packaging plasmid and an envelope plasmid into 293T cells. After culturing for 72 hours, the virus supernatant is collected, and ultracentrifugation or PEG precipitation is used for concentration and purification to obtain efficient and stable lentiviral particles. The engineered lentiviral vector used contains a CMV enhancer-driven iRGD-FLAG-Lamp2b fusion protein expression sequence, an EF1a core promoter-regulated TERT gene, a puromycin resistance gene for screening, and a WPRE enhancement element and a PPT / CTS region for improving the expression efficiency and infection ability of the virus.

[0016] Subsequently, the lentiviral particles are used to infect the mesenchymal stem cells obtained in step S1, and the specific steps are as follows:

[0017] The mesenchymal stem cells were cultured to the logarithmic growth phase. When the cell confluence reached 30%-50%, an appropriate amount of virus particles was added to each well, and the cells were gently shaken and incubated in a 37°C, 5% CO2 incubator. Then, after 8-16 hours of infection, the old culture medium was discarded, and fresh complete culture medium was added. The culture was continued for 48-72 hours, and then 1 μg / mL of puromycin was added to start resistance screening. After 7-14 days of screening, the cells that survived the resistance screening were diluted and plated by the single-cell cloning method, and the stable strains were further purified by monoclonal culture. The screened cell lines were further identified, and the integration and expression of the target gene were verified by qPCR and Western Blot.

[0018] In this step, a three-plasmid co-transfection system is first used to construct a lentiviral vector. After plasmid co-transfection, the lentiviral supernatant is collected, concentrated and purified to obtain efficient and stable lentiviral particles for subsequent cell transfection and functional verification. Next, the purified lentiviral particles are constructed and concentrated to infect the mesenchymal stem cells, and the screened cell lines are further identified.

[0019] S3: Large-scale preparation and characterization of nanovesicles

[0020] A mesenchymal stem cell line that has been screened by puromycin and stably expresses TERT and Lamp2b-Flag-iRGD fusion protein is used for large-scale culture. When the cells reach 80%-90% confluence, the medium is replaced with a serum-free medium without nanovesicles. The culture is continued for 48 hours, and the culture supernatant is collected and centrifuged at low speed to remove suspended cells and debris, followed by medium-speed centrifugation to further remove cell debris and larger organelles.

[0021] The pretreated supernatant is pre-concentrated, and then separated by size exclusion chromatography. According to the length of the separation column and the properties of the filler, the eluate containing the nanovesicles is collected, and the collected eluate is concentrated using an ultrafiltration centrifuge tube or a dialysis membrane. Finally, the nanovesicles are resuspended in an appropriate amount of PBS to obtain a high-concentration nanovesicle sample, and the extracted nanovesicle sample is stored at 4°C for a short term, or at -80°C for a long term;

[0022] S4: Loading and release of MSR1-siRNA

[0023] The purified nanovesicles were mixed with the target MSR1-siRNA in an optimized ratio and placed in a special electroporation buffer. The electroporator was used to set the appropriate electric field strength and pulse parameters (such as voltage, pulse time and number) to temporarily electroporate the membrane of the nanovesicles, so that MSR1-siRNA successfully entered the interior of the nanovesicles. The treated nanovesicle samples were immediately transferred to ice, and the unloaded free siRNA was removed by low-speed centrifugation. Subsequently, the particle size, zeta potential and dispersibility of the nanovesicles were characterized using nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS) techniques to ensure the structural integrity of the nanovesicles during the loading process to detect the loading efficiency and encapsulation amount of MSR1-siRNA. In a simulated in vivo release environment, the release characteristics of MSR1-siRNA were evaluated by measuring the release curve to confirm its targeted delivery and effective release ability in fibrotic tissues. Finally, the loaded nanovesicles can be stored at 4°C for a short period of time or at -80°C for a long period of time.

[0024] Further, in step S1, the tissue material to be extracted for mesenchymal stem cells is umbilical cord or other tissue material cut into small pieces of 1-2 mm.

[0025] Furthermore, in step S1, the enzymatic agent is a mixture containing collagenase Type I, hyaluronidase and trypsin, the content of collagenase Type I is 1-2 mg / mL, the content of hyaluronidase is 0.1-0.5 mg / mL, and the content of trypsin is 0.125-0.25%.

[0026] Furthermore, in steps S1 and S2, the coated culture flask is an externally purchased culture flask suitable for culturing mesenchymal stem cells, and the complete culture medium is an externally purchased culture medium suitable for culturing mesenchymal stem cells, which will not be described in detail herein.

[0027] Further, in step S1, the specific surface markers include high expression of CD73, CD90 and CD105, and low expression or negative expression of CD34, CD45 and HLA-DR.

[0028] Further, in step S2, the specific steps of constructing the engineered lentiviral vector using the three-plasmid co-transfection system are:

[0029] Firstly, the TERT gene and Lamp2b-Flag-iRGD fusion gene were cloned into the multiple cloning site of the lentiviral vector, respectively. A dual promoter system was designed, which contained the EF1α promoter and the CMV promoter in the same lentiviral vector to drive the expression of TERT and Lamp2b-Flag-iRGD, respectively. At the same time, T2A peptide was used to connect the TERT gene and the puro resistance gene to achieve their simultaneous expression. After plasmid co-transfection, the lentiviral supernatant was collected, concentrated and purified to obtain efficient and stable lentiviral particles for subsequent cell transfection and functional verification.

[0030] Furthermore, in step S2, the engineered lentiviral vector further includes a helper plasmid and a packaging plasmid.

[0031] Furthermore, in step S2, before the step of gently shaking and placing in a 37° C., 5% CO 2 incubator for incubation, it also includes adding polybrene at a concentration of 4-8 μg / mL.

[0032] Furthermore, in step S2, during the resistance screening process, fresh complete culture medium is replaced every 2-3 days, and screening drugs are continuously added.

[0033] Furthermore, in step S3, the rotation speed of the low-speed centrifugation is 300-500×g, and the centrifugation time is 10 min; the rotation speed of the medium-speed centrifugation is 2000-3000×g, and the centrifugation time is 15-20 min.

[0034] Further, in step S3, the size exclusion chromatography separation step is:

[0035] First, select a size exclusion chromatography filler suitable for nanovesicle separation, ensure that the filler pore size is suitable for separating nanovesicles of 30-150nm in size, and can exclude larger protein aggregates and small particles. After filling the size exclusion chromatography column to an appropriate height, balance the column with equilibration buffer until the column reaches a stable flow rate and pressure; resuspend the pretreated nanovesicle sample with an appropriate amount of buffer and load it to the top of the size exclusion chromatography column; start elution separation at an appropriate flow rate, and collect the eluate containing nanovesicles according to the length of the separation column and the properties of the filler.

[0036] Furthermore, the size exclusion chromatography filler is Sepharose CL-2B or a similar hydrophilic gel material.

[0037] Furthermore, the equilibrium buffer is PBS or HBS; the appropriate buffer is PBS; and the appropriate flow rate is 0.5-1 mL / min.

[0038] Further, in step S4, the mass ratio of the purified nanovesicles to the target MSR1-siRNA is 10:1-100:1, and the electroporation buffer is a solution containing 10 mM HEPES, 5 mM KCl, 1 mM MgCl2, and 270 mM sucrose, and the pH is adjusted to 7.4.

[0039] Further, in step S4, the specific electric field strength ranges from 100 to 500 V / cm, the pulse parameters are pulse time 100 to 500 μs, number of pulses: 1 to 10 times, and time interval 0.5 to 5 seconds.

[0040] The second object of the present invention is achieved by adopting the following technical solution:

[0041] One application is to use nanovesicles loaded with MSR1-siRNA to prepare a drug for inhibiting MSR1 expression, wherein the drug can be delivered to fibrotic tissue in a targeted manner and effectively release MSR1-siRNA in the tissue, thereby achieving specific treatment of fibrotic tissue, wherein the nanovesicles loaded with MSR1-siRNA are the nanovesicles obtained by the preparation method described above.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] 1. The large-scale preparation method of nanovesicles of the present invention achieves efficient production and enhanced targeting of nanovesicles by introducing the expression sequences of TERT gene and Lamp2b-iRGD fusion protein into human umbilical cord-derived mesenchymal stem cells; and electroporation technology is used to load MSR1-siRNA into nanovesicles, and MSR1 expression is inhibited by targeted delivery, thereby effectively improving the specific therapeutic ability of nanovesicles for fibrotic tissue.

[0044] 2. The large-scale preparation method of nanovesicles of the present invention also has the following advantages:

[0045] (1) Efficient production: Compared with the traditional nanovesicle preparation method, the present invention achieves long life and efficient proliferation of cell lines by utilizing human umbilical cord mesenchymal stem cells (hUC-MSCs) and combining them with efficient expression of the TERT gene, significantly improving the yield and stability of nanovesicles, and is suitable for large-scale production.

[0046] (2) Strong stability: By introducing the telomerase (TERT) gene, the cell division ability is prolonged, the impact of cell aging is reduced, the quality of the nanovesicles is ensured to be stable, and long-term production and application are facilitated.

[0047] (3) Precise targeting: By displaying iRGD peptide on the surface of nanovesicles, nanovesicles are endowed with the ability to specifically target fibrotic tissues. Compared with existing non-specific nanovesicles, this invention can deliver therapeutic substances more accurately, reduce the impact on surrounding healthy tissues, and improve the therapeutic effect.

[0048] (4) Efficient system for multi-gene expression: The lentiviral vector designed in the present invention contains both Lamp2b-Flag-iRGD fusion protein and puro resistance gene, and also utilizes T2A peptide to achieve efficient expression of multiple genes, thereby simplifying the vector construction and operation process and ensuring the versatility and efficiency of targeted therapy.

[0049] (5) High-purity extraction: Compared with the traditional ultracentrifugation method, the present invention extracts exosomes through size exclusion chromatography (SEC), which not only improves the purity of exosomes, but also effectively removes large molecular proteins and other impurities, ensuring that the extracted nanovesicles have higher purity and consistency.

[0050] (6) Mild separation conditions: Size exclusion chromatography uses mild separation conditions, which avoids the damage to the exosome structure caused by high shear force and high centrifugal force, helps to preserve the functionality and biological activity of nanovesicles, and improves the quality of nanovesicles.

[0051] (7) High safety: Compared with whole-cell therapy, the present invention uses nanovesicles derived from mesenchymal stem cells, which have low immunogenicity and no tumorigenicity, reducing the safety risks in clinical applications. At the same time, the introduction of the puromycin resistance gene ensures the high efficiency of cell screening, further improving the safety of treatment.

[0052] (8) Precision treatment: Through targeted nanovesicles, the accumulation of drugs in non-target tissues can be reduced, potential side effects can be reduced, and the effectiveness of treatment and patient compliance can be improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 This is a construction map of the engineered lentiviral vector of Example 1;

[0054] Figure 2 is a transmission electron microscope (TEM) morphology image of the nanovesicles of Example 1;

[0055] Figure 3 The particle size distribution and concentration diagram of the nanovesicles of Example 1;

[0056] Figure 4 This is a diagram of the characteristic proteins of the nanovesicles of Example 1. DETAILED DESCRIPTION

[0057] A large-scale preparation method of nanovesicles of the present invention comprises the following steps:

[0058] S1: Extraction, purification and identification of human umbilical cord mesenchymal stem cells

[0059] The umbilical cord is placed under sterile conditions, the blood vessels are removed and cut into small pieces of 1-2 mm. Enzymatic hydrolysis is performed on the umbilical cord tissue using an enzymatic hydrolyzer (enzymatic hydrolyzer is a mixed solution containing collagenase Type I, hyaluronidase and trypsin, the content of collagenase Type I is 1-2mg / mL, the content of hyaluronidase is 0.1-0.5mg / mL, and the content of trypsin is 0.125-0.25wt%), and then filtered through a cell sieve to obtain a single cell suspension, and inoculated in a coated culture bottle, cultured under the culture conditions of 37°C and 5% CO2. After 72 hours, the non-adherent cells are discarded and fresh culture medium is added. Continue to culture until the cell fusion rate reaches 80%-90%, wash the cells with PBS and perform trypsin digestion and passage. Repeat this process to obtain highly purified umbilical cord mesenchymal stem cells. Flow cytometry was used to detect specific surface markers of umbilical cord mesenchymal stem cells, including high expression of CD73, CD90 and CD105, and low or negative expression of CD34, CD45 and HLA-DR. At the same time, osteogenic, adipogenic and chondrogenic differentiation experiments were performed to further verify the multidirectional differentiation potential of the cells. Mesenchymal stem cells with good vitality within P5 were selected for the next experiment.

[0060] S2: Construction of engineered lentiviral vectors and cell infection

[0061] The three-plasmid co-transfection system was used to construct the engineered lentiviral vector. First, the TERT gene and the Lamp2b-Flag-iRGD fusion gene were cloned into the multiple cloning site (MCS) of the lentiviral vector. To ensure the simultaneous and efficient expression of the two genes, a dual promoter system was designed, that is, the EF1α promoter and the CMV promoter were included in the same lentiviral vector to drive the expression of TERT and Lamp2b-Flag-iRGD, respectively. At the same time, the T2A peptide was used to connect the TERT gene and the puro resistance gene to achieve their simultaneous expression, so that after infecting the cells, the cells can both efficiently express TERT and be screened by the puromycin resistance gene. The vector also includes an auxiliary plasmid (pMD2.G) and a packaging plasmid (psPAX2) to ensure the packaging and production of lentiviral particles. After plasmid co-transfection, the lentiviral supernatant was collected, concentrated and purified to obtain efficient and stable lentiviral particles for subsequent cell transfection and functional verification.

[0062] Next, the engineered lentiviral vector was co-transfected with the packaging plasmid and the envelope plasmid into 293T cells, and after culturing for 72 hours, the viral supernatant was collected, and concentrated and purified by ultracentrifugation or PEG precipitation to obtain efficient and stable lentiviral particles; wherein the engineered lentiviral vector used contained the iRGD-FLAG-Lamp2b fusion protein expression sequence driven by the CMV enhancer, the TERT gene regulated by the EF1a core promoter, the puromycin resistance gene for screening, and the WPRE enhancement element and PPT / CTS region for improving the expression efficiency and infection ability of the virus;

[0063] Subsequently, the lentiviral particles are used to infect the mesenchymal stem cells obtained in step S1, and the specific steps are as follows:

[0064] First, culture mesenchymal stem cells to the logarithmic growth phase. When the cell confluence reaches 30%-50%, add an appropriate amount of virus particles (MOI, Multiplicity of Infection, usually 5-100) to each well (6-well plate). To improve the infection efficiency, add an appropriate amount of polybrene (such as Polybrene, final concentration of 4-8μg / mL), shake gently, and incubate in a 37°C, 5% CO2 incubator.

[0065] 8-16 hours after infection, discard the old culture medium, add fresh complete culture medium, continue to culture for 48-72 hours, and then add 1μg / mL of puromycin to start resistance screening. During the resistance screening process, replace fresh complete culture medium every 2-3 days, and continue to add screening drugs. After 7-14 days of screening, the cells that survived the resistance screening were diluted and plated by single-cell cloning method, and monoclonal culture was used to further purify the stable strain. The screened cell lines were further identified, and the integration and expression of the target genes (TERT and Lamp2b-Flag-iRGD fusion genes) were verified by qPCR and Western Blot.

[0066] S3: Large-scale preparation and characterization of nanovesicles

[0067] Use mesenchymal stem cell lines that have been screened by puromycin and stably express TERT and Lamp2b-Flag-iRGD fusion proteins for large-scale culture. When the cells reach 80%-90% confluence, replace them with serum-free medium without nanovesicles and continue to culture for 48 hours. Collect the culture supernatant and remove suspended cells and debris by low-speed centrifugation (300-500×g, 10 minutes). Then centrifuge at medium speed (2000-3000×g, 15-20 minutes) to further remove cell debris and larger organelles. The pretreated supernatant is pre-concentrated and then separated by size exclusion chromatography. First, select a size exclusion chromatography filler suitable for nanovesicle separation, such as Sepharose CL-2B or similar hydrophilic gel materials. Make sure that the filler pore size is suitable for separating nanovesicles of 30-150nm in size, while being able to exclude larger protein aggregates and small particles. After filling the size exclusion chromatography column to an appropriate height, equilibrate the column with an equilibrium buffer (usually PBS or HBS) until the column reaches a stable flow rate and pressure. Resuspend the pretreated nanovesicle sample with an appropriate amount of buffer (such as PBS) and load it to the top of the size exclusion chromatography column. Start elution separation at an appropriate flow rate (usually 0.5-1mL / min). Depending on the length of the separation column and the properties of the filler, collect the eluate containing the nanovesicles. Use an ultrafiltration centrifuge tube or a dialysis membrane to concentrate the collected eluate, and finally resuspend the nanovesicles in an appropriate amount of PBS to obtain a high-concentration nanovesicle sample. Store the extracted nanovesicle sample at 4°C for a short term or at -80°C for a long term.

[0068] S4: Loading and release of MSR1-siRNA

[0069] The purified nanovesicles are mixed with the target MSR1-siRNA according to the optimized ratio and placed in a special electroporation buffer, wherein the mass ratio of the purified nanovesicles to the target MSR1-siRNA is 10:1-100:1, and the special electroporation buffer is a solution containing 10mM HEPES, 5mM KCl, 1mM MgCl2, and 270mM sucrose, and the pH is adjusted to 7.4. The electroporator is used to set the appropriate electric field strength and pulse parameters (the range of the specific electric field strength is 100-500V / cm, the pulse parameters are pulse time 100-500μs, pulse number: 1-10 times, and time interval 0.5-5 seconds) to temporarily electroporate the membrane of the nanovesicle, so that MSR1-siRNA successfully enters the interior of the nanovesicle. The treated nanovesicle sample is immediately transferred to ice, and the unloaded free siRNA is removed by low-speed centrifugation.

[0070] Subsequently, nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS) techniques were used to characterize the particle size, zeta potential, and dispersibility of the nanovesicles to ensure the structural integrity of the nanovesicles during the loading process to detect the loading efficiency and encapsulation amount of MSR1-siRNA. In a simulated in vivo release environment (pH 7.4, with the addition of 10U / mL nuclease or 2mg / mL proteinase K), the release characteristics of MSR1-siRNA were evaluated by measuring the release curve to confirm its targeted delivery and effective release ability in fibrotic tissue. Finally, the loaded nanovesicles can be stored at 4°C for a short period of time or at -80°C for a long period of time.

[0071] The present invention is further described below in conjunction with specific embodiments. It should be noted that, under the premise of no conflict, the embodiments described below or the technical features can be arbitrarily combined to form new embodiments. In the following embodiments, the coated culture bottle is a purchased culture bottle suitable for mesenchymal stem cell culture, the complete medium is a purchased medium suitable for mesenchymal stem cell culture, and the serum-free medium is a purchased medium suitable for mesenchymal stem cell culture. The specific manufacturer is not described here.

[0072] Example 1

[0073] A large-scale preparation method of nanovesicles comprises the following steps:

[0074] S1: Extraction, purification and identification of human umbilical cord mesenchymal stem cells

[0075] The umbilical cord is placed under sterile conditions, the blood vessels are removed and cut into small pieces of 1-2 mm. Enzymatic hydrolysis is performed on the umbilical cord tissue using an enzymatic hydrolyzer (enzymatic hydrolyzer is a mixed solution containing collagenase Type I, hyaluronidase and trypsin, and the content of collagenase Type I is 1.5mg / mL, the content of hyaluronidase is 0.3mg / mL, and the content of trypsin is 0.2wt%), and then filtered through a cell sieve to obtain a single cell suspension, and inoculated in a coated culture bottle, and cultured under the culture conditions of 37°C and 5% CO2. After 72 hours, the non-adherent cells are discarded and fresh culture medium is added. Continue to culture until the cell fusion rate reaches 80%-90%, wash the cells with PBS and trypsinize and pass them on. Repeat this process to obtain highly purified umbilical cord mesenchymal stem cells. Flow cytometry was used to detect specific surface markers of umbilical cord mesenchymal stem cells, including high expression of CD73, CD90 and CD105, and low or negative expression of CD34, CD45 and HLA-DR. At the same time, osteogenic, adipogenic and chondrogenic differentiation experiments were performed to further verify the multidirectional differentiation potential of the cells. Mesenchymal stem cells with good vitality within P5 were selected for the next experiment.

[0076] S2: Construction of engineered lentiviral vectors

[0077] A three-plasmid co-transfection system was used to construct an engineered lentiviral vector. First, the TERT gene and the Lamp2b-Flag-iRGD fusion gene were cloned into the multiple cloning site (MCS) of the lentiviral vector, respectively. In order to ensure the simultaneous and efficient expression of the two genes, a dual promoter system was designed, that is, the EF1α promoter and the CMV promoter were included in the same lentiviral vector to drive the expression of TERT and Lamp2b-Flag-iRGD, respectively. At the same time, the T2A peptide was used to connect the TERT gene and the puro resistance gene to achieve their simultaneous expression, so that after infecting the cells, the cells can not only efficiently express TERT, but also be screened by the puromycin resistance gene. The vector also includes an auxiliary plasmid (pMD2.G) and a packaging plasmid (psPAX2) to ensure the packaging and production of lentiviral particles. After plasmid co-transfection, the lentiviral supernatant was collected, concentrated and purified to obtain efficient and stable lentiviral particles for subsequent cell transfection and functional verification. Figure 1 Construction map of the engineered lentiviral vector shown.

[0078] Next, the engineered lentiviral vector was co-transfected with the packaging plasmid and the envelope plasmid into 293T cells. After culturing for 72 hours, the viral supernatant was collected and concentrated and purified by ultracentrifugation or PEG precipitation to obtain efficient and stable lentiviral particles. The engineered lentiviral vector used contained the iRGD-FLAG-Lamp2b fusion protein expression sequence driven by the CMV enhancer, the TERT gene regulated by the EF1a core promoter, the puromycin resistance gene for screening, and the WPRE enhancing element and PPT / CTS region for improving the expression efficiency and infection ability of the virus.

[0079] S3: Infection and identification of engineered lentivirus

[0080] The lentiviral particles constructed, concentrated and purified in the previous step are used to infect human umbilical cord mesenchymal stem cells. First, the mesenchymal stem cells are cultured to the logarithmic growth phase, and when the cell confluence reaches 30%-50%, 80um of viral particles (MOI, Multiplicity of Infection) are added to each well (6-well plate).

[0081] After 10 hours of infection, discard the old culture medium, add fresh complete culture medium, continue to culture for 48 hours, and then add 1μg / mL puromycin to start resistance screening. During the resistance screening process, replace fresh complete culture medium every 2-3 days, and continue to add screening drugs. After 14 days of screening, the cells that survived the resistance screening were diluted and plated by single cell cloning method, and monoclonal culture was used to further purify the stable strain. The screened cell lines were further identified, and the integration and expression of the target gene were verified by qPCR and Western Blot.

[0082] S4: Large-scale preparation and characterization of nanovesicles

[0083] A mesenchymal stem cell line that has been screened by puromycin and stably expresses TERT and Lamp2b-Flag-iRGD fusion proteins was used for large-scale culture. When the cells reached a confluence of 80%-90%, they were replaced with a serum-free medium without nanovesicles and cultured for 48 hours. The culture supernatant was collected and suspended cells and debris were removed by low-speed centrifugation (300-500×g, 10 minutes). Subsequently, medium-speed centrifugation (2000-3000×g, 15 minutes) was used to further remove cell debris and larger organelles. The pretreated supernatant was pre-concentrated and then separated by size exclusion chromatography. First, the size exclusion chromatography filler for Sepharose CL-2B nanovesicle separation was selected to ensure that the filler pore size was suitable for separating nanovesicles of 30-150nm in size, while being able to exclude larger protein aggregates and small particles. After the size exclusion chromatography column was filled to an appropriate height, the column was equilibrated with PBS equilibration buffer until the column reached a stable flow rate and pressure. The pretreated nanovesicle sample is resuspended in an appropriate amount of PBS buffer and loaded onto the top of the size exclusion chromatography column. The elution separation is started at a flow rate of 0.5 mL / min. According to the length of the separation column and the properties of the filler, the eluate containing the nanovesicles is collected. The collected eluate is concentrated using an ultrafiltration centrifuge tube or a dialysis membrane, and the nanovesicles are finally resuspended in an appropriate amount of PBS to obtain a high-concentration nanovesicle sample. The extracted nanovesicle sample is stored at 4°C for a short term, or at -80°C for a long term.

[0084] S4: Loading and release of MSR1-siRNA

[0085] Electroporation technology is used to efficiently load MSR1-siRNA into nanovesicles. The specific method is: the purified nanovesicles are mixed with the target MSR1-siRNA in an optimized ratio and placed in a special electroporation buffer, wherein the mass ratio of the purified nanovesicles to the target MSR1-siRNA is 20:1, and the special electroporation buffer is a solution containing 10mM HEPES, 5mM KCl, 1mM MgCl2, and 270mM sucrose, and the pH is adjusted to 7.4. The electroporator is used to set the appropriate electric field strength and pulse parameters (the range of specific electric field strength is 100-500V / cm, the pulse parameters are pulse time 100-500μs, pulse number: 5 times, and time interval 1 second) to temporarily electroporate the membrane of the nanovesicle, so that MSR1-siRNA successfully enters the interior of the nanovesicle. The treated nanovesicle sample is immediately transferred to ice, and the unloaded free siRNA is removed by low-speed centrifugation. Subsequently, nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS) techniques were used to characterize the particle size, zeta potential, and dispersity of the nanovesicles to ensure the structural integrity of the nanovesicles during the loading process.

[0086] To verify the loading effect, fluorescently labeled MSR1-siRNA was used for fluorescence quantitative analysis to evaluate the encapsulation efficiency and loading amount of siRNA. After loading, the nanovesicles can be directly used in cell experiments or animal experiments to study their targeted delivery and the effect of inhibiting MSR1 expression. To ensure functionality, the release characteristics of MSR1-siRNA were evaluated by measuring the release curve in a simulated in vivo release environment to confirm its targeted delivery and effective release ability in fibrotic tissue. Finally, the loaded nanovesicles can be stored at 4°C for a short period of time, or stored at -80°C for a long period of time.

[0087] After testing, such as Figure 2 As shown, the morphology of the nanovesicles was observed using a transmission electron microscope (TEM). It can be seen from the figure that MSR1-siRNA is efficiently loaded onto the surface of the nanovesicles.

[0088] like Figure 3 As shown in the figure, the particle size distribution and concentration of nanovesicles were measured using a nanoparticle size analyzer. It can be seen from the figure that the maximum concentration range of nanovesicles is 5.0×10 7 -5.3×10 7 between.

[0089] The characteristic proteins of nanovesicles were analyzed by Western Blot. Figure 4 shown.

[0090] Example 2

[0091] A large-scale preparation method of nanovesicles comprises the following steps:

[0092] S1: Extraction, purification and identification of human umbilical cord mesenchymal stem cells

[0093] The umbilical cord is placed under sterile conditions, the blood vessels are removed and cut into small pieces of 1-2 mm. Enzymatic hydrolysis is performed on the umbilical cord tissue using an enzymatic hydrolyzer (enzymatic hydrolyzer is a mixed solution containing collagenase Type I, hyaluronidase and trypsin, the content of collagenase Type I is 2mg / mL, the content of hyaluronidase is 0.3mg / mL, and the content of trypsin is 0.125wt%), and then filtered through a cell sieve to obtain a single cell suspension, and inoculated in a coated culture bottle, cultured under the culture conditions of 37°C and 5% CO2. After 72 hours, the non-adherent cells are discarded and fresh culture medium is added. Continue to culture until the cell fusion rate reaches 80%-90%, wash the cells with PBS and trypsinize and pass on. Repeat this process to obtain highly purified umbilical cord mesenchymal stem cells. Flow cytometry was used to detect specific surface markers of umbilical cord mesenchymal stem cells, including high expression of CD73, CD90 and CD105, and low or negative expression of CD34, CD45 and HLA-DR. At the same time, osteogenic, adipogenic and chondrogenic differentiation experiments were performed to further verify the multidirectional differentiation potential of the cells. Mesenchymal stem cells with good vitality within P5 were selected for the next experiment.

[0094] S2: Construction of engineered lentiviral vectors

[0095] A three-plasmid co-transfection system was used to construct an engineered lentiviral vector. First, the TERT gene and the Lamp2b-Flag-iRGD fusion gene were cloned into the multiple cloning site (MCS) of the lentiviral vector, respectively. In order to ensure the simultaneous and efficient expression of the two genes, a dual promoter system was designed, that is, the EF1α promoter and the CMV promoter were included in the same lentiviral vector to drive the expression of TERT and Lamp2b-Flag-iRGD, respectively. At the same time, the T2A peptide was used to connect the TERT gene and the puro resistance gene to achieve their simultaneous expression, so that after infecting the cells, the cells can not only efficiently express TERT, but also be screened by the puromycin resistance gene. The vector also includes an auxiliary plasmid (pMD2.G) and a packaging plasmid (psPAX2) to ensure the packaging and production of lentiviral particles. After plasmid co-transfection, the lentiviral supernatant was collected, concentrated and purified to obtain efficient and stable lentiviral particles for subsequent cell transfection and functional verification. Figure 1 Construction map of the engineered lentiviral vector shown.

[0096] Next, the engineered lentiviral vector was co-transfected with the packaging plasmid and the envelope plasmid into 293T cells. After culturing for 72 hours, the viral supernatant was collected and concentrated and purified by ultracentrifugation or PEG precipitation to obtain efficient and stable lentiviral particles. The engineered lentiviral vector used contained the iRGD-FLAG-Lamp2b fusion protein expression sequence driven by the CMV enhancer, the TERT gene regulated by the EF1a core promoter, the puromycin resistance gene for screening, and the WPRE enhancing element and PPT / CTS region for improving the expression efficiency and infection ability of the virus.

[0097] S3: Infection and identification of engineered lentivirus

[0098] The lentiviral particles constructed, concentrated and purified in the previous step are used to infect human umbilical cord mesenchymal stem cells. First, the mesenchymal stem cells are cultured to the logarithmic growth phase. When the cell confluence reaches 30%-50%, 80um of viral particles (MOI, Multiplicity of Infection) are added to each well (6-well plate). To improve the infection efficiency, an appropriate amount of polybrene (such as Polybrene, with a final concentration of 4μg / mL) can be added, gently shaken and placed in a 37°C, 5% CO2 incubator for incubation.

[0099] After 10 hours of infection, discard the old culture medium, add fresh complete culture medium, continue to culture for 48 hours, and then add 1μg / mL puromycin to start resistance screening. During the resistance screening process, replace fresh complete culture medium every 2-3 days, and continue to add screening drugs. After 14 days of screening, the cells that survived the resistance screening were diluted and plated by single cell cloning method, and monoclonal culture was used to further purify the stable strain. The screened cell lines were further identified, and the integration and expression of the target gene were verified by qPCR and Western Blot.

[0100] S4: Large-scale preparation and characterization of nanovesicles

[0101] A mesenchymal stem cell line that has been screened by puromycin and stably expresses TERT and Lamp2b-Flag-iRGD fusion proteins was used for large-scale culture. When the cells reached a confluence of 80%-90%, they were replaced with a serum-free medium without nanovesicles and cultured for 48 hours. The culture supernatant was collected and suspended cells and debris were removed by low-speed centrifugation (300-500×g, 10 minutes). Subsequently, medium-speed centrifugation (2000-3000×g, 15 minutes) was used to further remove cell debris and larger organelles. The pretreated supernatant was pre-concentrated and then separated by size exclusion chromatography. First, the size exclusion chromatography filler for Sepharose CL-2B nanovesicle separation was selected to ensure that the filler pore size was suitable for separating nanovesicles of 30-150nm in size, while being able to exclude larger protein aggregates and small particles. After the size exclusion chromatography column was filled to an appropriate height, the column was equilibrated with PBS equilibration buffer until the column reached a stable flow rate and pressure. The pretreated nanovesicle sample is resuspended in an appropriate amount of PBS buffer and loaded onto the top of the size exclusion chromatography column. The elution separation is started at a flow rate of 0.5 mL / min. According to the length of the separation column and the properties of the filler, the eluate containing the nanovesicles is collected. The collected eluate is concentrated using an ultrafiltration centrifuge tube or a dialysis membrane, and the nanovesicles are finally resuspended in an appropriate amount of PBS to obtain a high-concentration nanovesicle sample. The extracted nanovesicle sample is stored at 4°C for a short term, or at -80°C for a long term.

[0102] S4: Loading and release of MSR1-siRNA

[0103] Electroporation technology is used to efficiently load MSR1-siRNA into nanovesicles. The specific method is: the purified nanovesicles are mixed with the target MSR1-siRNA in an optimized ratio and placed in a special electroporation buffer, wherein the mass ratio of the purified nanovesicles to the target MSR1-siRNA is 50:1, and the special electroporation buffer is a solution containing 10mM HEPES, 5mM KCl, 1mM MgCl2, and 270mM sucrose, and the pH is adjusted to 7.4. The electroporator is used to set the appropriate electric field strength and pulse parameters (the range of specific electric field strength is 100-500V / cm, the pulse parameters are pulse time 100-500μs, pulse number: 5 times, and time interval 1 second) to temporarily electroporate the membrane of the nanovesicle, so that MSR1-siRNA successfully enters the interior of the nanovesicle. The treated nanovesicle sample is immediately transferred to ice, and the unloaded free siRNA is removed by low-speed centrifugation. Subsequently, nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS) techniques were used to characterize the particle size, zeta potential, and dispersity of the nanovesicles to ensure the structural integrity of the nanovesicles during the loading process.

[0104] Subsequently, nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS) techniques were used to characterize the particle size, zeta potential, and dispersibility of the nanovesicles to ensure the structural integrity of the nanovesicles during the loading process to detect the loading efficiency and encapsulation amount of MSR1-siRNA. In a simulated in vivo release environment (pH 7.4, with the addition of 10U / mL nuclease or 2mg / mL proteinase K), the release characteristics of MSR1-siRNA were evaluated by measuring the release curve to confirm its targeted delivery and effective release ability in fibrotic tissue. Finally, the loaded nanovesicles can be stored at 4°C for a short period of time or at -80°C for a long period of time.

[0105] After testing, the performance of the nanovesicles of Example 2 is the same as that of the nanovesicles of Example 1.

[0106] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A large-scale preparation method of nanovesicles for targeted treatment of fibrosis, characterized in that: The method comprises the following preparation steps: S1: Extraction, purification and identification of mesenchymal stem cells Under sterile conditions, the tissue material to be extracted from mesenchymal stem cells is enzymatically treated with an enzymatic agent, filtered, and a single cell suspension is obtained, and the cell suspension is inoculated into a coated culture bottle, and cultured at 37°C and 5% CO2. After 72 hours of culture, the non-adherent cells are discarded, and fresh complete culture medium is added, and the culture is continued until the cell fusion rate reaches 80%-90%. The cells are washed with PBS and digested with trypsin for passage until high-purity mesenchymal stem cells are obtained. Finally, the specific surface markers of mesenchymal stem cells are detected by flow cytometry. At the same time, a three-line differentiation experiment of osteogenic, adipogenic and chondrogenic is performed to further verify the multidirectional differentiation potential of the cells, and mesenchymal stem cells within P5 and with good vitality are selected for the next experiment; S2: Construction of engineered lentiviral vectors and cell infection The three-plasmid co-transfection system is used to construct an engineered lentiviral vector. The engineered lentiviral vector is co-transfected with a packaging plasmid and an envelope plasmid into 293T cells. After culturing for 72 hours, the virus supernatant is collected, and ultracentrifugation or PEG precipitation is used for concentration and purification to obtain efficient and stable lentiviral particles. The engineered lentiviral vector used contains a CMV enhancer-driven iRGD-FLAG-Lamp2b fusion protein expression sequence, an EF1a core promoter-regulated TERT gene, a puromycin resistance gene for screening, and a WPRE enhancement element and a PPT / CTS region for improving the expression efficiency and infection ability of the virus. Subsequently, the lentiviral particles are used to infect the mesenchymal stem cells obtained in step S1, and the specific steps are as follows: First, the mesenchymal stem cells were cultured to the logarithmic growth phase. When the cell confluence reached 30%-50%, an appropriate amount of virus particles was added to each well, and the cells were gently shaken and incubated in a 37°C, 5% CO2 incubator. Then, after 8-16 hours of infection, the old culture medium was discarded, and fresh complete culture medium was added. The culture was continued for 48-72 hours, and then 1 μg / mL of puromycin was added to start resistance screening. After 7-14 days of screening, the cells that survived the resistance screening were diluted and plated by the single-cell cloning method, and the stable strains were further purified by monoclonal culture. The screened cell lines were further identified, and the integration and expression of the target gene were verified by qPCR and Western Blot. S3: Large-scale preparation and characterization of nanovesicles A mesenchymal stem cell line that has been screened by puromycin and stably expresses TERT and Lamp2b-Flag-iRGD fusion protein is used for large-scale culture. When the cells reach 80%-90% confluence, the medium is replaced with a serum-free medium without nanovesicles. The culture is continued for 48 hours, and the culture supernatant is collected and centrifuged at low speed to remove suspended cells and debris, followed by medium-speed centrifugation to further remove cell debris and larger organelles. The pretreated supernatant is pre-concentrated, and then separated by size exclusion chromatography. According to the length of the separation column and the properties of the filler, the eluate containing the nanovesicles is collected, and the collected eluate is concentrated using an ultrafiltration centrifuge tube or a dialysis membrane. Finally, the nanovesicles are resuspended in an appropriate amount of PBS to obtain a high-concentration nanovesicle sample, and the extracted nanovesicle sample is stored at 4°C for a short term, or at -80°C for a long term; S4: Loading and release of MSR1-siRNA The purified nanovesicles were mixed with the target MSR1-siRNA in an optimized ratio and placed in a special electroporation buffer. The electroporator was used to set the appropriate electric field strength and pulse parameters to temporarily electroporate the membrane of the nanovesicles, so that MSR1-siRNA successfully entered the interior of the nanovesicles. The treated nanovesicle samples were immediately transferred to ice and the unloaded free siRNA was removed by low-speed centrifugation. Subsequently, the particle size, zeta potential and dispersibility of the nanovesicles were characterized using nanoparticle tracking analysis and dynamic light scattering technology to ensure the structural integrity of the nanovesicles during the loading process. In a simulated in vivo release environment, the release characteristics of MSR1-siRNA were evaluated by measuring the release curve to confirm its targeted delivery and effective release ability in fibrotic tissues. Finally, the loaded nanovesicles can be stored at 4°C for a short period of time, or stored at -80°C for a long period of time.

2. The large-scale preparation method of nanovesicles for targeted treatment of fibrosis according to claim 1, characterized in that: In step S1, the tissue material to be extracted for mesenchymal stem cells is umbilical cord cut into small pieces of 1-2 mm.

3. The large-scale preparation method of nanovesicles for targeted treatment of fibrosis according to claim 1, characterized in that: In step S1, the enzymatic agent is a digestive enzyme mixture containing collagenase, hyaluronidase and DNase I, wherein the content of collagenase is 0.5-2 mg / mL, the content of hyaluronidase is 0.2-1 mg / mL, and the content of DNase I is 0.1-0.2 mg / mL.

4. The large-scale preparation method of nanovesicles for targeted treatment of fibrosis according to claim 1, characterized in that: In step S1, specific surface markers include high expression of CD73, CD90 and CD105, and low or negative expression of CD34, CD45 and HLA-DR.

5. The large-scale preparation method of nanovesicles for targeted treatment of fibrosis according to claim 1, characterized in that: In step S2, the specific steps of constructing a lentiviral vector using a three-plasmid co-transfection system are as follows: Firstly, the TERT gene and Lamp2b-Flag-iRGD fusion gene were cloned into the multiple cloning site of the lentiviral vector, respectively. A dual promoter system was designed, which contained the EF1α promoter and the CMV promoter in the same lentiviral vector to drive the expression of TERT and Lamp2b-Flag-iRGD, respectively. At the same time, T2A peptide was used to connect the TERT gene and the puro resistance gene to achieve their simultaneous expression. After plasmid co-transfection, the lentiviral supernatant was collected, concentrated and purified to obtain efficient and stable lentiviral particles for subsequent cell transfection and functional verification.

6. The large-scale preparation method of nanovesicles for targeted treatment of fibrosis according to claim 1, characterized in that: In step S2, the lentiviral vector further includes a helper plasmid and a packaging plasmid.

7. The large-scale preparation method of nanovesicles for targeted treatment of fibrosis according to claim 1, characterized in that: In step S2, before gently shaking and placing in a 37°C, 5% CO2 incubator for incubation, it also includes adding polybrene at a concentration of 4-8 μg / mL; during the resistance screening process, fresh complete culture medium is replaced every 2-3 days, and screening drugs are continuously added.

8. The large-scale preparation method of nanovesicles for targeted treatment of fibrosis according to claim 1, characterized in that: In step S3, the steps of size exclusion chromatography separation are: First, select a size exclusion chromatography filler suitable for nanovesicle separation, ensure that the filler pore size is suitable for separating nanovesicles of 30-150nm in size, and can exclude larger protein aggregates and small particles. After filling the size exclusion chromatography column to an appropriate height, balance the column with equilibration buffer until the column reaches a stable flow rate and pressure; resuspend the pretreated nanovesicle sample with an appropriate amount of buffer and load it to the top of the size exclusion chromatography column; start elution separation at an appropriate flow rate, and collect the eluate containing nanovesicles according to the length of the separation column and the properties of the filler.

9. The large-scale preparation method of nanovesicles for targeted treatment of fibrosis according to claim 1, characterized in that: In step S4, the specific electric field strength ranges from 100 to 500 V / cm, the pulse parameters are pulse time 100 to 500 μs, pulse number: 1 to 10 times, and time interval 0.5 to 5 seconds.

10. An application, using nanovesicles loaded with MSR1-siRNA to prepare a drug for inhibiting the expression of MSR1, wherein the drug can be delivered to fibrotic tissue by targeting and effectively releasing MSR1-siRNA in the tissue, thereby achieving specific treatment of fibrotic tissue, wherein: The nanovesicle loaded with MSR1-siRNA is the nanovesicle obtained by the preparation method according to any one of claims 1 to 9.

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