PH-responsive polyethylene glycol engineered small extracellular vesicle as well as preparation method and application thereof

Abstract

The invention relates to a pH-responsive polyethylene glycol engineered small extracellular vesicle as well as a preparation method and application thereof. 2, 5-dihydroxy-4-methyl-2, 5-dioxo-3-furanpropionic acid modified methoxy polyethylene glycol, which is called mPEG-CDM for short, is obtained by taking 2, 5-dihydroxy-4-methyl-2, 5-dioxo-3-furanpropionic acid as a raw material; a weakly alkaline buffer system solution of mPEG-CDM is obtained; a purified weakly alkaline buffer system dispersion liquid of sEVs is obtained; mixing the weakly alkaline buffer system solution of mPEG-CDM with the weakly alkaline buffer system dispersion liquid of purified sEVs to obtain a co-incubation system, and incubating; and after incubation, purifying and separating to obtain the pH-responsive PEG surface engineered sEVs, namely the pH-responsive polyethylene glycol engineered small extracellular vesicles. The engineered sEVs prepared by the method can be prevented from being quickly cleared by MPS in blood circulation, has excellent weak acid lesion site targeted enrichment capacity, and overcomes the core technical problem in application of PEG-coated sEVs. Meanwhile, the modification method is simple and convenient to operate and low in cost.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a pH-responsive polyethylene glycol-engineered small extracellular vesicle and a preparation method and application thereof. Background Art

[0002] Small extracellular vesicles (sEVs) are nanovesicles with a phospholipid bilayer membrane structure and a diameter of 30-150 nm secreted by cells. They contain substances derived from the parent cells and are responsible for intercellular material communication. In recent years, a large number of studies have confirmed that sEVs derived from specific cells have therapeutic functions for diseases. At the same time, by engineering these nanoscale vesicles, they can be used as efficient drug delivery carriers to achieve the treatment of diseases. At present, the application of sEVs has become a frontier research field in biomedicine, and it is expected to become the main medium for the next generation of cell therapy.

[0003] Systemic intravenous injection is the main administration method for sEVs to achieve therapeutic functions. However, different from traditional artificially prepared nanoparticles, the half-life of sEVs in the blood is only a few dozen minutes, and they will be rapidly cleared by the mononuclear phagocyte system (MPS) in the blood, making it difficult to efficiently accumulate at the lesion site. Their treatment often requires multiple injections at large doses, significantly increasing the cost of sEVs-based treatment. Therefore, how to overcome the rapid phagocytosis and clearance of sEVs by MPS after entering the blood and improve their enrichment ability at the lesion site is a key technical challenge for the clinical translation of sEVs-based treatment.

[0004] In recent years, researchers have developed a series of surface engineering modification methods for sEVs in order to overcome the problem of rapid clearance of sEVs by MPS. These methods include using genetic engineering techniques to fuse MPS escape proteins (such as CD47) on sEV membrane proteins and using chemical modification methods to prepare polyethylene glycol (PEG)-coated sEVs. Among these methods, the genetic engineering modification method has complex technical operations, is difficult to construct cell lines that stably express fusion proteins, and has low transformation potential. The chemical modification polyethylene glycol coating method has the advantages of simple operation, batch modification, and low cost, and is a highly potential surface engineering modification method for improving the blood circulation time of sEVs. However, although PEG coating can reduce the clearance efficiency of sEVs by MPS, the modification of PEG will also reduce the phagocytosis efficiency of sEVs by target cells, and does not effectively improve the enrichment ability of sEVs at the lesion site.

[0005] Chinese Patent CN114702621B discloses a pH-responsive random copolymer, which is obtained by copolymerizing a pH-responsive functional monomer and an exosome-specific recognition polymerizable monomer; the exosome-specific recognition polymerizable monomer is prepared from an exosome-specific recognition functionalized monomer as one of the raw materials, and the exosome-specific recognition functionalized monomer includes one or more of arginine, lysine, substituted phenylboronic acid, substituted polyhydroxy compounds, and substituted polyethylene glycol. The pH-responsive random copolymer provided by this solution can achieve the purification of exosomes by simply and rapidly adjusting the pH value during the separation and enrichment of exosomes, and further obtain purified exosomes by centrifugal separation.

[0006] This solution realizes the separation of exosomes by manufacturing a pH-responsive polymer, aiming to achieve the separation of exosomes, and does not involve the surface engineering modification of exosomes. Chinese Patent CN115804847B discloses a pH / hydrogen peroxide / MMP9 sequential response microsphere, a biological carrier carrying exosomes and applications. This solution discloses the protective effect of exosomes derived from regulatory T cells with high SPARC expression on cardiac function, as well as the levels of pH, H2O2, and MMP9 in the infarcted area, and synthesizes a conjugated peptide, a pH / H2O2 / MMP9 sequential response microsphere, and a biological carrier carrying exosomes. First, the acylhydrazone bond is broken under acidic conditions to release exosomes and hydrogel raw materials. Subsequently, H2O2 is used to oxidize Co 2+ to Co 3+ , forming a gel and fixing the released exosomes. Finally, under the hydrolysis of MMP9, the gel gradually degrades and releases exosomes, thereby exerting a continuous and long-term protective effect on the myocardium to improve cardiac function.

[0007] This solution is to manufacture a microsphere carrier with pH-responsive release performance to achieve the pH-responsive release of exosomes, and does not involve the surface engineering modification of exosomes. Summary of the Invention

[0008] In the prior art, the genetic engineering modification of sEVs is complex, it is difficult to construct a cell line with long-term expression of fusion proteins, and the transformation potential is low. The chemical modification of polyethylene glycol coating on sEVs will reduce the phagocytosis efficiency of sEVs by target cells and does not effectively improve the enrichment ability of sEVs at the lesion site. Based on the above technical problems, the present invention provides a pH-responsive polyethylene glycol-engineered small extracellular vesicle, its preparation method and application.

[0009] Combining the characteristics of a weakly acidic microenvironment of pH in some disease sites, the present invention constructs a pH-responsive polyethylene glycol-engineered small extracellular vesicle, its preparation method and application.

[0010] The method of the present invention can achieve the stable coating of sEVs with PEG in neutral blood circulation, avoid the massive clearance of sEVs by MPS, and at the same time remove the PEG coating of sEVs in the weakly acidic lesion microenvironment, thereby restoring the uptake ability of target site cells to sEVs and achieving the efficient enrichment of sEVs at the lesion site.

[0011] The object of the present invention can be achieved by the following technical solutions:

[0012] The present invention first provides a method for preparing pH-responsive polyethylene glycol-engineered small extracellular vesicles, comprising the following steps:

[0013] Obtain methoxypolyethylene glycol modified with 2,5-dihydroxy-4-methyl-2,5-dioxo-3-furanpropionic acid (CDM), abbreviated as mPEG-CDM;

[0014] Obtain a weakly basic buffer solution of mPEG-CDM;

[0015] Obtain a dispersion of purified sEVs in a weakly basic buffer system;

[0016] Mix the weakly basic buffer solution of mPEG-CDM with the dispersion of purified sEVs in a weakly basic buffer system to obtain a co-incubation system and perform incubation;

[0017] After incubation, purify and separate to obtain sEVs with pH-responsive PEG surface engineering modification, that is, pH-responsive polyethylene glycol-engineered small extracellular vesicles.

[0018] In an embodiment of the present invention, the structure of mPEG-CDM is shown in Formula I:

[0019]

[0020] In Formula I, the value range of n is 10 to 1000, preferably 10 to 400;

[0021] In an embodiment of the present invention, the synthesis method of mPEG-CDM is as follows:

[0022] Take 2,5-dihydroxy-4-methyl-2,5-dioxo-3-furanpropionic acid powder and dissolve it thoroughly in dichloromethane solution;

[0023] At 0 °C, add oxalyl chloride dropwise to the obtained solution;

[0024] After the dropwise addition is completed, add a catalytic amount of N,N-dimethylformamide to the reaction system;

[0025] React at 0 °C for 30 min and then at room temperature for 2 h;

[0026] After evaporating the reaction solvent, a dichloromethane solution dissolving mPEG-OH powder was added to the reaction system, and the mixture was stirred overnight at room temperature;

[0027] Finally, precipitation with diethyl ether and drying under vacuum were carried out to obtain mPEG-CDM powder.

[0028] In one embodiment of the present invention, the molar ratio of the 2,5-dihydroxy-4-methyl-2,5-dioxo-3-furanpropionic acid powder to the terminal hydroxyl group of polyethylene glycol added subsequently is 10-20:1;

[0029] In one embodiment of the present invention, the molar ratio of oxalyl chloride to 2,5-dihydroxy-4-methyl-2,5-dioxo-3-furanpropionic acid powder is 5-20:1; the concentration of the dichloromethane solution prepared is 0.1-0.2 g / mL.

[0030] In one embodiment of the present invention, in the weak alkaline buffer system solution of mPEG-CDM, the degree of polymerization of mPEG-CDM monomers ranges from 10 to 400, preferably 150 to 170; the pH range of the weak alkaline buffer system is 7.0 to 9.0, preferably 8.4.

[0031] In one embodiment of the present invention, the purified sEVs are obtained by methods such as ultracentrifugation, ultrafiltration, and tangential flow filtration.

[0032] In one embodiment of the present invention, the pH range of the weak alkaline buffer system dispersion of purified sEVs is 7.0 to 9.0, preferably 8.4.

[0033] In one embodiment of the present invention, the weak alkaline buffer system solution of mPEG-CDM and the weak alkaline buffer system dispersion of purified sEVs are mixed in equal volume.

[0034] In one embodiment of the present invention, in the co-incubation system, the concentration range of mPEG-CDM is 0.1 mg / mL to 50 mg / mL, preferably 2 mg / mL to 10 mg / mL; in the co-incubation system, the concentration range of sEVs is 1×10 9 ~1×10 12 per mL.

[0035] In one embodiment of the present invention, the conditions for incubation are: the incubation time is 30 min to 5 h, preferably 2 h; the incubation temperature range is 4°C to 40°C, preferably 25°C.

[0036] In one embodiment of the present invention, after incubation, the methods of purification and separation include ultracentrifugation, ultrafiltration, tangential flow filtration, etc.

[0037] The present invention further provides pH-responsive polyethylene glycol-engineered small extracellular vesicles prepared based on the above preparation method.

[0038] The present invention further provides the application of the pH-responsive polyethylene glycol-engineered small extracellular vesicles prepared based on the above preparation method, and the application of the pH-responsive polyethylene glycol-engineered small extracellular vesicles in the preparation of drugs.

[0039] In one embodiment of the present invention, the pH-responsive polyethylene glycol-engineered small extracellular vesicles are used as carriers to load anti-tumor drugs.

[0040] In one embodiment of the present invention, the tumor is glioma. At this time, the small extracellular vesicles are sEVs derived from glioma cells.

[0041] In one embodiment of the present invention, the pH-responsive polyethylene glycol-engineered small extracellular vesicles are used as carriers to load drugs for relieving stroke.

[0042] Using the scheme of the present invention, the prepared pH-responsive polyethylene glycol-engineered small extracellular vesicles can be used to prepare drugs for treating glioma and drugs for relieving stroke symptoms.

[0043] Under weakly basic conditions, the CDM group of mPEG-CDM can react with the free amino groups on the surface of sEVs membrane proteins to form an amide bond as shown in Structure II. This amide bond has pH responsiveness and can stably exist under physiological pH conditions, while it breaks under weakly acidic environments (pH < 6.8). Therefore, sEVs coated with mPEG-CDM (mPEG-CDM-sEVs) can achieve stable coating of PEG in the blood circulation, avoiding the rapid clearance of sEVs by MPS; when mPEG-CDM-sEVs enter the lesion tissue with a weakly acidic microenvironment, the amide bond is hydrolyzed by weak acid, the PEG modified on the surface of sEVs is removed, and sEVs recover the ability to be rapidly taken up by target cells, thereby realizing the enrichment of sEVs at the lesion site.

[0044]

[0045] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0046] The present invention firstly proposes a preparation method of pH-responsive polyethylene glycol (PEG) engineered small extracellular vesicles (sEVs with PEG surface engineering modification responsive to pH). The engineered sEVs prepared by this method can avoid being rapidly cleared by the mononuclear phagocyte system (MPS) in blood circulation, and at the same time have excellent targeting and enrichment ability at weakly acidic lesion sites, overcoming the core technical problems faced in the application of PEG-coated sEVs. Meanwhile, this modification method is simple to operate and low in cost.

[0047] This application uses mPEG-CDM to achieve PEGylation of the exosome surface, reducing the possibility of exosomes being phagocytosed by cells and enhancing their stability in blood circulation. When the exosomes modified with PEG-CDM enter an acidic pathological environment, weak acids in the acidic pathological environment can remove the modification of PEG-CDM and restore the phagocytosis ability of exosomes by cells. Therefore, the exosomes in the solution of this application are a kind of pH-responsive PEG-engineered small extracellular vesicles. The present invention constructs pH-responsive exosomes through the method of PEG modification, which can improve the delivery efficiency of exosomes to acidic microenvironments. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 : Uptake results of mPEG-CDM-modified sEVs by macrophages under different pH conditions;

[0049] Figure 2 : Distribution of mPEG-CDM-modified sEVs in glioma mice and stroke mice;

[0050] Figure 3 : Evaluation results of the effect of mPEG-CDM-modified GsEVs loaded with DOX in treating glioma;

[0051] Figure 4 : Evaluation results of the effect of mPEG-CDM-modified IsEVs in alleviating stroke symptoms. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0052] The present invention will be described in detail below with reference to the drawings and specific embodiments.

[0053] Example 1:

[0054] Synthesis of mPEG-CDM with Mn of 10KDa

[0055] Accurately weigh 0.28 g of CDM and 40 μL of N,N-dimethylformamide and dissolve them in 5 mL of dichloromethane. Subsequently, at 0 °C, add 0.38 g of oxalyl chloride to the reaction system, stir for 0.5 h, and then restore the reaction to room temperature and continue the reaction for 2 h. Rotate and dry the reaction solvent.

[0056] Subsequently, dichloromethane was added again to dissolve in the reaction system, 1 g (Mn = 10 KDa) mPEG-OH and a catalytic amount of pyridine were added to the reaction system, and the reaction continued for 12 h. Subsequently, the reaction solution was poured into 20 volumes of cold diethyl ether for precipitation, the precipitate was filtered, and the precipitate was washed repeatedly with diethyl ether 3 times. Vacuum drying was carried out to obtain 0.92 g of pale yellow solid powder. The structure of the product was identified by 1H NMR, and the results showed that the modification rate of CDM was 93%.

[0057] Example 2:

[0058] Preparation of Small Extracellular Vesicles with mPEG-CDM Surface-Modified Glioma Cells and Induced Pluripotent Stem Cells

[0059] The supernatants of murine glioma cells (GL261s) and induced pluripotent stem cells (iPSCs) were collected separately, and small extracellular vesicles (sEVs) in the supernatants were separated by a stepwise centrifugation procedure. The specific process was as follows: centrifugation at 400 g for 10 min to remove suspended cells and cell debris; centrifugation at 2000 g for 20 min to remove larger particles; centrifugation at 15,000 g for 30 min to remove cell debris and large particles; centrifugation at 100,000 g for 75 min to obtain purified sEVs. The precipitate was resuspended in normal saline and centrifuged again at 100,000 g for 75 min to obtain purified sEVs separately. The purified sEVs were suspended in 0.01 M sodium bicarbonate buffer (pH 8.4) for standby and prepared into a suspension of 2×10 10 / mL for subsequent mPEG-CDM modification reaction.

[0060] A certain amount of mPEG-CDM (prepared in Example 1) was weighed and dissolved in 0.01 M sodium bicarbonate buffer (pH 8.4) to make its final concentration reach 4 mg / mL. The mPEG-CDM solution was slowly added to the above two different sEVs suspensions separately, keeping the volume ratio at 1:1. The mixed solution was incubated at 25 °C for 2 h to ensure sufficient reaction between mPEG-CDM and the free amino groups on the sEVs membrane surface to form pH-responsive amide bonds.

[0061] After the reaction was completed, the modified sEVs solution was transferred to an ultracentrifugation tube and centrifuged at 100,000 g for 75 min to remove unreacted mPEG-CDM. Subsequently, it was resuspended with sterile PBS and centrifuged again to further purify the modified sEVs, and finally, sEVs from two cell sources modified with mPEG-CDM, namely mPEG-CDM-GsEVs and mPEG-CDM-IsEVs, were obtained.

[0062] Example 3:

[0063] pH-responsive cellular endocytosis of mPEG-CDM modified small extracellular vesicles

[0064] RAW 264.7 macrophages were seeded in 6-well plates at a density of 1×10 5 cells per well and cultured in a humidified environment at 37 °C and 5% CO2 until the cell confluence reached 80%. During the experiment, unmodified GsEVs (GL261-derived sEVs) and IsEVs (iPSCs-derived sEVs), as well as sEVs from two cell sources modified with mPEG-CDM (mPEG-CDM-GsEVs and mPEG-CDM-IsEVs), were added. sEVs labeled with the fluorescent dye DiD were added to the cell culture medium at a concentration of 1×10 9 / mL, and the cells were incubated at 37 °C for 12 hours. After incubation, the cells were washed 3 times with pre-cooled PBS to remove unuptaken sEVs, then fixed with 4% paraformaldehyde for 10 minutes and stained with DAPI for 10 minutes for the nucleus. As shown in Figure 1 Figure a, the macrophage uptake of mPEG-CDM modified sEVs was significantly lower than that of unmodified sEVs, indicating that mPEG-CDM modification significantly reduced the ability of sEVs to be phagocytosed by macrophages.

[0065] To simulate the acidic microenvironment, the prepared mPEG-CDM modified GsEVs (mPEG-CDM-GsEVs) and mPEG-CDM modified IsEVs (mPEG-CDM-IsEVs) were adjusted for pH. The specific steps were as follows: The suspension of mPEG-CDM modified sEVs (mPEG-CDM-GsEVs and mPEG-CDM-IsEVs respectively) was adjusted to pH 6.0 with 1 mM HCl, mixed evenly and incubated at room temperature for 10 min. After incubation, the solution was ultracentrifuged at 100,000 g for 75 min at 4 °C to remove unbound substances, and the sEVs precipitate was resuspended with PBS. The acid-treated sEVs were added to RAW 264.7 macrophages at a concentration of 1×10 9 / mL and incubated at 37 °C and 5% CO2 for 12 h. After incubation, the cells were washed 3 times with pre-cooled PBS, fixed with 4% paraformaldehyde for 20 minutes and stained with DAPI for 10 minutes for the nucleus. As shown in Figure 1As shown in Figure b, fluorescence microscopy revealed that the uptake of mPEG-CDM-modified sEVs without acid treatment was significantly reduced in RAW 264.7 macrophages; while after acid treatment, the uptake of sEVs by macrophages increased significantly, approaching the level of unmodified sEVs. This result indicates that mPEG-CDM-modified sEVs can remove the surface modification layer in an acidic environment, thereby restoring their ability to be taken up by cells, laying the foundation for targeting acidic lesion sites.

[0066] Example 4:

[0067] Enrichment ability of mPEG-CDM-modified small cell vesicles at lesion sites

[0068] In this example, the targeting and enrichment abilities of mPEG-CDM-modified small extracellular vesicles (sEVs) in two lesion models were evaluated. A nude mouse glioma model and a C57 mouse MCAO stroke model were used for the experiments respectively.

[0069] First, a nude mouse glioma model and a C57 mouse MCAO stroke model were established. For the nude mouse glioma model, 1×10 6 GL261 cells were suspended in 5 μL of sterile PBS and injected into the right striatum through a stereotaxic apparatus to ensure successful implantation of the tumor. For the C57 mouse MCAO stroke model, a transient middle cerebral artery occlusion (MCAO) surgery was performed, and reperfusion was carried out 1 hour later to induce local ischemic stroke in the mice.

[0070] After the models were successfully established, the GsEVs and IsEVs, as well as mPEG-CDM-modified GsEVs and IsEVs, were labeled with the fluorescent dye DiR. The labeled sEVs were diluted to a concentration of 1×1011 / mL, and a dose of 200 μL was injected via the tail vein into each experimental mouse. 12 h after administration, a small animal in vivo imaging system was used to collect the fluorescence signal to evaluate the distribution of sEVs in the body and the enrichment at the lesion sites.

[0071] Appendix Figure 2 As shown in Figure a, in the nude mouse glioma model, the unmodified GsEVs showed moderate fluorescence signals in the tumor area, while the fluorescence signals of the mPEG-CDM-modified GsEVs in the tumor area were significantly enhanced, and the fluorescence signals in the liver were significantly reduced, indicating that mPEG-CDM modification significantly avoided the rapid clearance of GsEVs and enhanced their enrichment at the tumor site. Similarly, as Figure 2As shown in b, in the C57 MCAO model, unmodified IsEVs showed weak fluorescence signals at the cerebral ischemia lesion sites, while IsEVs modified with mPEG-CDM showed significantly enhanced fluorescence signals in the cerebral ischemia area, and the fluorescence signals retained in the liver decreased significantly. This indicates that mPEG-CDM modification significantly improves the enrichment efficiency of sEVs in the weakly acidic lesion microenvironment.

[0072] In summary, this example verified that the targeting of small extracellular vesicles modified with mPEG-CDM was significantly improved in the nude mouse glioma model and the C57 MCAO stroke model, and significantly reduced the retention of sEVs in organs of the MPS system such as the liver, providing strong technical support for the clinical translation of sEVs in the treatment of tumors and strokes.

[0073] Example 5:

[0074] The ability of mPEG-CDM-modified glioma small extracellular vesicles to load doxorubicin for the treatment of glioma

[0075] To prepare doxorubicin (DOX)-loaded mPEG-CDM-modified sEVs, first, 1×10 10 mPEG-CDM-modified sEVs were suspended in PBS solution containing 0.2% (w / v) saponin, and 100 μM DOX (Selleck, USA) was added. Incubate at room temperature for 1 h to promote DOX loading. Subsequently, the DOX-loaded sEVs were purified by ultracentrifugation at 100,000 g and resuspended in PBS for later use.

[0076] In the experiment, C57BL / 6 mice were used to establish a glioma model. 1×10 6 GL261 cells were suspended in 5 μL of sterile PBS and precisely injected into the right striatum of the mice through a stereotaxic apparatus to ensure successful tumor implantation. After tumor implantation, the mice were randomly divided into four groups (n = 5 in each group): the mPEG-CDM-modified GsEVs loaded with DOX group (mPEG-CDM-GsEVs-DOX), the unmodified GsEVs loaded with DOX group (GsEVs-DOX), the free DOX group (2 μg / kg), and the PBS control group. According to the dosing protocol, starting from the 10th day after tumor implantation, intravenous injection was performed once every 3 days through the tail vein, 100 μL each time, for 6 consecutive doses. The DOX concentration in all DOX treatment groups was uniformly 2 μg / kg. After the dosing was completed, the mice were sacrificed, and the brain was taken for tumor volume measurement, and H&E staining was used to analyze the tumor size. The results showed (attached Figure 3) The group of GsEVs loaded with DOX modified by mPEG-CDM showed significant therapeutic effects in reducing tumor volume and had higher efficacy compared with the GsEVs-DOX group and the free DOX group. In summary, the results of this example indicate that DOX-loaded sEVs modified by mPEG-CDM can significantly enhance the therapeutic efficacy of drugs in glioblastoma.

[0077] As Figure 3 shown, this example confirmed the effect of doxorubicin (DOX)-loaded small extracellular vesicles (sEVs) modified by mPEG-CDM in the treatment of glioblastoma and was verified using a GL261 glioma C57 mouse model.

[0078] Example 6:

[0079] Ability of mPEG-CDM-modified induced pluripotent stem cell small extracellular vesicles to relieve stroke

[0080] In model establishment, local ischemic brain injury was induced by transient middle cerebral artery occlusion (MCAO). Reperfusion was performed within 1 hour after successful modeling to restore blood flow. The mice were then randomly divided into three experimental groups: a control group (PBS injection), an unmodified IsEVs group (IsEVs), and an mPEG-CDM-modified IsEVs group (mPEG-CDM-IsEVs). All treatments were administered by tail vein injection at a dose of 200 μL and a concentration of 1×10 11 particles / mL. The mice in the experimental groups received the first injection immediately after reperfusion and the second injection 24 h later to utilize the acidic microenvironment in the early stage of ischemia.

[0081] On the 3rd day after MCAO, the mouse brain tissue was taken for analysis of the infarct volume. The brain was sectioned and the surviving brain tissue area was labeled using MAP-2 staining. The experimental results are as shown in Figure 4 the appendix. Compared with the control group, the treatment with mPEG-CDM-modified IsEVs significantly reduced the infarct volume, indicating its targeted therapeutic effect on stroke lesions and neuroprotective effect.

[0082] This example confirmed that mPEG-CDM-modified IsEVs can effectively relieve the symptom manifestations of the C57 mouse MCAO stroke model.

[0083] The above description of the embodiments is to enable those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and all improvements and modifications made by those skilled in the art without departing from the scope of the present invention according to the disclosure of the present invention should be within the protection scope of the present invention.

Claims

1. A preparation method of pH-responsive polyethylene glycol-engineered small extracellular vesicles, characterized in that, It includes the following steps: Obtain methoxypolyethylene glycol modified with 2,5-dihydroxy-4-methyl-2,5-dioxo-3-furanpropionic acid, abbreviated as mPEG-CDM; Obtain a weakly basic buffer system solution of mPEG-CDM; Obtain a weakly basic buffer system dispersion of purified sEVs; Mix the weakly basic buffer system solution of mPEG-CDM with the weakly basic buffer system dispersion of purified sEVs to obtain a co-incubation system and conduct incubation; After incubation, purify and separate to obtain sEVs with pH-responsive PEG surface engineering modification, that is, pH-responsive polyethylene glycol-engineered small extracellular vesicles; The structure of the said mPEG-CDM is shown in Formula I: In Formula I, the value range of n is a positive integer between 10 and 400.

2. The preparation method of a pH-responsive polyethylene glycol-engineered small extracellular vesicle according to claim 1, wherein, The synthesis method of the said mPEG-CDM is as follows: Take 2,5-dihydroxy-4-methyl-2,5-dioxo-3-furanpropionic acid powder and fully dissolve it in dichloromethane solution; At 0 °C, add oxalyl chloride dropwise to the obtained solution; After the dropwise addition is completed, add a catalytic amount of N,N-dimethylformamide to the reaction system; React at 0 °C for 30 min and then at room temperature for 2 h; After evaporating the reaction solvent, add a dichloromethane solution dissolving mPEG-OH powder to the reaction system and stir at room temperature overnight; Finally, precipitate with ether and dry in vacuo to obtain mPEG-CDM powder.

3. The preparation method of a pH-responsive polyethylene glycol-engineered small extracellular vesicle according to claim 2, characterized in that, The molar ratio of the dosage of the said 2,5-dihydroxy-4-methyl-2,5-dioxo-3-furanpropionic acid to the terminal hydroxyl group of polyethylene glycol input subsequently is 10-20:1; the concentration of the dichloromethane solution prepared is 0.1-0.2 g / mL; The molar ratio of the said oxalyl chloride to 2,5-dihydroxy-4-methyl-2,5-dioxo-3-furanpropionic acid is 5-20:

1.

4. The preparation method of a pH-responsive polyethylene glycol-engineered small extracellular vesicle according to claim 2, characterized in that, In the weakly basic buffer system solution of mPEG-CDM, the polymerization degree range of mPEG-CDM monomers is 10-400, and the pH range of the weakly basic buffer system is 7.0-9.0; The pH range of the weakly basic buffer system dispersion of purified sEVs is 7.0-9.0; The weakly basic buffer system solution of mPEG-CDM and the weakly basic buffer system dispersion of purified sEVs are mixed in equal volume.

5. The preparation method of a pH-responsive polyethylene glycol-engineered small extracellular vesicle according to claim 2, characterized in that, In the co-incubation system, the concentration range of mPEG-CDM is 0.1 mg / mL to 50 mg / mL; in the co-incubation system, the concentration range of sEVs is 1×10 8 to 1×10 12 per mL; The conditions for incubation are: the incubation time is 30 min-5 h, and the incubation temperature range is 4 °C-40 °C.

6. A pH-responsive polyethylene glycol-engineered small extracellular vesicle prepared by the preparation method described in any one of claims 1-5.

7. Use of the pH-responsive polyethylene glycol-engineered small extracellular vesicles according to claim 6, characterized in that, Use of the said pH-responsive polyethylene glycol-engineered small extracellular vesicle in the preparation of drugs.

8. Use of the pH-responsive polyethylene glycol-engineered small extracellular vesicles according to claim 7, characterized in that, The said pH-responsive polyethylene glycol-engineered small extracellular vesicle is used as a carrier to load anti-tumor drugs.

9. Use of the pH-responsive polyethylene glycol-engineered small extracellular vesicles according to claim 8, characterized in that, The said tumor is glioma. At this time, the said small extracellular vesicle is sEVs derived from glioma cells.

10. Use of the pH-responsive polyethylene glycol-engineered small extracellular vesicles according to claim 7, characterized in that, The said pH-responsive polyethylene glycol-engineered small extracellular vesicle is used as a carrier to load drugs for relieving stroke.

Images