Protein-loaded exosome as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biology, and particularly discloses a protein-loaded exosome as well as a preparation method and application thereof. The method for preparing the protein-loaded exosome comprises the following steps: (1) mixing a cholesterol-PEG-spy tag / probe tag with the exosome for reaction to obtain the spy tag / probe tag-loaded exosome; and (2) carrying out mixed reaction on the exosome loaded with the spy tag / probe tag and the fusion protein of the target loading protein and the spy catcher / probe, and forming an isopeptide bond through the spy tag / probe tag and the spy catcher / probe, so that the target loading protein is loaded on the exosome. The preparation method provided by the invention can avoid false positive, and the product is single and easy for mass production.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a protein-loaded exosome and a preparation method and application thereof. Background Art

[0002] Exosomes are vesicles containing a phospholipid bilayer in the range of 40-160 nm that are secreted from cells. Exosomes are derived from cells and have similar structures and properties to cells. Exosomes are composed of a phospholipid bilayer and cytoplasmic proteins, membrane proteins, RNA, DNA, and other components similar to those of maternal cells, but exosomes do not have organelles. Exosomes play a key role in various physiological and pathological conditions and are considered to be a new intercellular communication mechanism. Exogenous substances, including proteins, lipids, and genetic material, can be delivered to recipient cells. Therefore, exosomes can be used as biomarkers for disease diagnosis and prognosis. In addition, the large capacity and high delivery efficiency of exosomes promote the ability of exosomes to exchange proteins and genes with cells, giving them the potential for clinical application as gene and drug carriers. Compared with synthetic delivery carriers, exosomes have good biocompatibility. In addition, the surface of the exosome membrane contains various proteins that interact with integrins, giving exosomes the ability to cross various biological barriers in the body such as monocytes, cell adhesion, tissue penetration, diffusion into the blood, and even across the blood-brain barrier. Therefore, exosomes are considered to be natural carriers for delivering nucleic acids, proteins, etc.

[0003] Protein drugs are mainly obtained by connecting a variety of natural amino acids, and amino acids are important components of human growth. They promote or inhibit certain physiological processes in the human body, and then achieve therapeutic effects in physiological and biochemical processes. Compared with previous small molecule drugs, protein drugs have the characteristics of high activity, high specificity, low toxicity, clear biological functions, and are conducive to clinical application. In addition, the biological functions of protein drugs are clear. Among the current new drug research and development categories, protein drugs are one of the most active and fastest-progressing types. Through the permutation and combination ratio of N types of biological factors, countless protein drugs with various efficacy can be obtained. In fact, there are many protein drugs on the market that have significant effects in the treatment of AIDS, cancer, hepatitis, diabetes, and chronic pain. However, protein drugs are quite different from traditional small molecule chemical drugs, including large molecular weight, complex charge, and unstable structure. Protein drugs have disadvantages such as poor stability, poor membrane permeability, and short biological half-life, which limit the therapeutic potential and clinical application of protein drugs. Moreover, for protein drugs whose targets are inside cells, they need to be taken up by cells, escape from endosomal endosomes, and reach the target cytoplasm to exert their effects.

[0004] At present, methods for delivering proteins via exosomes include genetic engineering, chemical modification, electroporation, and extrusion. By integrating the expression plasmid of the desired protein into the donor cell through cell genetic engineering, and fusing the gene sequence of the guide protein or polypeptide with the gene sequence of the selected exosome membrane protein, specific guide peptides and proteins can be effectively displayed in the exosomes. The genetic engineering method is a convenient way to give exosomes new properties. However, this method is not suitable for exosomes from cells that are difficult to genetically engineer. Chemical modification is suitable for small molecules and polypeptides, but has problems such as inability to load large molecular proteins or low loading efficiency.

[0005] The prior art discloses a protein or polypeptide delivery carrier and its preparation method and application (Chinese patent application number: 202210540756.X), which uses self-assembly technology to construct a simpler and easier-to-operate exosome delivery system suitable for loading a variety of random proteins or polypeptides. However, the covalent coupling of DSPE-PEG and protein in this scheme is non-specific, which usually leads to uncertain modification sites and uneven products, thereby affecting the subsequent function of proteins / polypeptides. Therefore, how to better achieve protein loading in vitro with exosomes remains to be further studied. Summary of the invention

[0006] One of the purposes of the present invention is to provide a method for preparing protein-loaded exosomes that can avoid the occurrence of false positives and has a single product.

[0007] The present invention provides a method for preparing protein-loaded exosomes, comprising: (1) Mixing cholesterol-PEG-spytag / snooptag with exosomes to obtain exosomes loaded with spytag / snooptag; (2) The exosomes loaded with the spy tag / probe tag are mixed with a fusion protein of a target loading protein and a spycatcher / snoopcatcher to form an isopeptide bond between the spy tag / probe tag and the spycatcher / snoopcatcher, so that the target loading protein is loaded onto the exosomes.

[0008] The present invention utilizes membrane insertion technology to insert a spytag or snooptag of a molecular glue into the exosome membrane, and then specifically connects the target protein fusion protein containing the corresponding spycatcher or snoopcatcher through the molecular glue spytag / snooptag and spycatcher / snoopcatcher in the form of an isopeptide bond to load the target protein onto the exosome.

[0009] The present invention allows the surface of the exosomes to contain molecular glue polypeptides as a platform, which can be assembled with any protein containing the corresponding molecular glue protein to obtain protein-loaded exosomes.

[0010] During the research of the present invention, it was found that when inserting spy tags / probe tags on the exosome membrane, it is necessary to first connect them with a hydrophobic group, and insert the hydrophobic group into the membrane of the exosome to complete the loading of the spy tags / probe tags. The spy tags / probe tags themselves have specific hydrophilic and hydrophobic properties, and different hydrophobic groups have different hydrophilic and hydrophobic properties. If the physical length and physical properties of the combination of the two are not well designed, self-assembly will be caused, thereby forming micelles similar to the size of exosomes, resulting in the method of separating exosomes based on size being unable to effectively distinguish the two, causing false positives. Finally, it was found through exploration that when cholesterol is used as a hydrophobic group in the manner defined by the present invention and connected to the spy tag / probe tag through PEG, it will not be assembled to form micelles, thereby avoiding the occurrence of false positives. Moreover, the present invention realizes site-specific modification of proteins and reduces the impact on protein function by fusion connection of spycatcher / snoopcatcher and target loading protein. The obtained protein-loaded exosome product is single, does not affect the function of protein / polypeptide, and is conducive to the clinical transformation of exosomes. The overall preparation method is simple, the final product is single, and the separation is simple, so it has a wide range of applicability.

[0011] In the method of the present invention, the molecular weight of PEG in the cholesterol-PEG-spy tag / spy tag is 1000-5000, preferably the molecular weight of PEG is 2000; And / or, the exosomes are milk exosomes or stem cell exosomes.

[0012] In the method of the present invention, the amino acid sequence of the cholesterol-PEG-probe tag / probe tag is shown in SEQ ID No. 1; The amino acid sequence of the probe tag in the cholesterol-PEG-spy tag / probe tag is shown in SEQ ID No. 2; The amino acid sequence of the spy catcher is shown in SEQ ID No. 3; The amino acid sequence of the detector is shown in SEQ ID No. 4; The target loading protein and the spy catcher / detector in the fusion protein are directly connected via a connecting peptide, and the amino acid sequence of the connecting peptide is shown in SEQ ID No. 5 or 6.

[0013] In the method of the present invention, in the cholesterol-PEG-spy tag / probe tag, cholesterol-PEG and thespy tag / probe tag are linked by covalent reaction.

[0014] In step (1) of the method of the present invention, the cholesterol-PEG-spy tag / probe tag is mixed with exosomes, and the mixture is reacted at 37±1°C. The final concentration of the cholesterol-PEG-spy tag / probe tag in the mixture is 0.4mM-1.6mM, preferably 0.8mM.

[0015] In the method of the present invention, the preparation method of the fusion protein comprises: recombining the nucleotide sequences encoding the target loading protein and the spy catcher / detector by gene recombination, expressing them in a host cell and then purifying them; preferably, the host cell is Escherichia coli; And / or, in step (2), the exosomes loaded with spy tag / probe tag are mixed with the target loading protein and the fusion protein of spy catcher / probe catcher at 4-37°C.

[0016] In step (2), the fusion protein may be added in excess. Preferably, the number ratio of the exosomes loaded with spy tag / probe tag to the target loaded protein and the fusion protein of spy catcher / probe catcher in the present invention is (10 -9 ~10 -3 ):1.

[0017] The method of the present invention further comprises the steps of separating and purifying the product (removing free unreacted components) after the reaction of steps (1) and (2) is completed. The separation and purification method is ultrafiltration, tangential flow or size exclusion chromatography.

[0018] The present invention also provides a protein-loaded exosome, which is prepared by the above method.

[0019] The protein-loaded exosomes provided by the present invention can be used as a protein delivery system, have good biocompatibility, and utilize the biological function of the exosomes to penetrate the biological barrier to deliver the loaded protein to the target location.

[0020] The present invention also provides the use of the above method or exosomes in preparing drugs.

[0021] The present invention also provides a pharmaceutical composition, which comprises the above-mentioned exosomes and pharmaceutically acceptable excipients.

[0022] The beneficial effects of the present invention are at least: The present invention provides a method for simultaneously loading one or more proteins on exosomes, wherein each protein is connected to a spycatcher / snoopcatcher at a fixed position, and is connected to the spytag / snooptag loaded on the exosome by an isopeptide bond, thereby achieving point-specific connection of the protein with the exosome, without affecting the structure and function of the protein, and the obtained product is single and has a wide range of applicability. In addition, when preparing the exosomes loaded with proteins according to the present invention, self-assembly will not occur, thereby avoiding the occurrence of false positives.

[0023] The preparation method of the present invention is simple, does not require special modification of exosomes, is applicable to cells from various sources, and has a single product and is easy to mass produce. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of exosome loading with proteins.

[0025] Figure 2 The results of hydrophobic group screening for hydrophobic group-PEG-spytag.

[0026] Figure 3 The positive rate of exosomes loaded with Spytag mediated by different hydrophobic groups-PEG-spytag.

[0027] Figure 4 These are the positive rate test results at different Chol-PEG-Spytag feed concentrations when exosomes are loaded with spytag.

[0028] Figure 5 Nanoflow cytometry of exosomes loaded with GFP protein.

[0029] Figure 6 The figure shows the particle size of exosomes after loading with GFP protein. A is the particle size detection result of milk exosomes (EV) without protein loading, B is the particle size detection result of milk exosomes loaded with GFP (EV-GFP), and C is the statistical comparison result of the particle size of EV and EV-GFP.

[0030] Figure 7 These are transmission electron micrographs of exosomes before and after protein loading. Figure A is a transmission electron micrograph of milk exosomes (EV) without protein loading, B is an enlarged image of the area circled by the box in A, C is a transmission electron micrograph of exosomes loaded with GFP protein (EV-GFP), and D is an enlarged image of the area circled by the box in C. The scale bars in A and C are 200 nm; the scale bars in B and D are 50 nm.

[0031] Figure 8 The results of preparing the standard curve of GFP. A is the fluorescence detection results of GFP at different concentrations, and B is the standard curve of GFP.

[0032] Fig. 9 This is a confocal image of cells after exosomes were loaded with proteins. Representative 20x confocal microscope image, Representative images were taken with a 40x confocal microscope, and the scale bar represents 10 μm.

[0033] Fig.10 Nanoflow cytometry of stem cell exosomes loaded with GFP fluorescent marker.

[0034] Fig.11 Transmission electron microscopy image of stem cell exosomes loaded with BDNF.

[0035] Fig.12 This is the standard curve of stem cell exosome-BDNF.

[0036] Fig.13 These are the results of the stem cell exosome-BDNF migration experiment. DETAILED DESCRIPTION

[0037] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0038] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available or prepared according to conventional methods in the art unless otherwise specified.

[0039] Example 1 In this example, the experiment of loading Spytag on exosomes was carried out with different hydrophobic groups, as follows: 1. Preparation of Chol-PEG-Spytag, DSPE-PEG-Spytag, and Palmitic acid-PEG-Spytag: Chemical methods were used to synthesize PEGylated molecules with specific functions. Chol-PEG-maleamide, DSPE-PEG-maleamide and palmitic acid-PEG-maleamide were reacted with Spytag (amino acid sequence shown in SEQ ID No.1) containing cysteine ​​at the C-terminus in acetonitrile at room temperature overnight. This process utilized the specific reaction between maleamide and cysteine ​​to form a stable thioether bond.

[0040] Subsequently, the reaction product was freeze-dried to facilitate the subsequent purification step. Through dialysis purification technology, three PEGylated Spytag molecules were finally obtained: Chol-PEG-Spytag, DSPE-PEG-Spytag and palmitic acid-PEG-Spytag.

[0041] 2. Preparation of spycatcher-GFP fusion protein: The spycatcher-GFP was constructed into the vector pET-28a-sumo (subcloning site: BAMHI / XhoI) to obtain a plasmid.

[0042] Specifically, the amino acid sequence shown in SEQ ID No.7 is constructed into the vector. The amino acids 1-21 of the sequence shown in SEQ ID No.7 are the sequences of His tag and TEV protease, which are used for subsequent protein purification. The amino acids 22-377 are the sequences of spycatcher (SEQ ID No.3), connecting peptide (SEQ ID No.5) and GFP, respectively.

[0043] The above plasmid was introduced into E. coli BL21 (DE3), and spycatcher-GFP was expressed using the E. coli expression system. When the bacteria grew to OD 600 =0.8, 0.5 mM IPTG was used to induce expression at 16°C for 16 hours. The cells were then crushed and purified using a protein purification system (Ni column) to obtain spycatcher-GFP (which was formed by sequentially connecting the amino acid sequences of SEQ ID No. 3, SEQ ID No. 5 and GFP).

[0044] 3. Hydrophobic group screening experiment 1, the steps are as follows: (1) Take 100 μl of 1 mM Chol-PEG-Spytag, DSPE-PEG-Spytag and palmitic acid-PEG-Spytag, add 100 μl of 0.2 mg / ml spycatcher-GFP fusion protein, and react at 4°C overnight; (2) After cooling to 4°C, the tube was immediately centrifuged at 4,500 g for 3 min using a 100 kDa ultrafiltration tube to remove free spycatcher-GFP fusion protein; (3) Add 300 μl PBS (pH 7.4) to the ultrafiltration tube and centrifuge at 4500 g for 3 min to remove free spycatcher-GFP fusion protein; (4) Repeat step (3), collect the retained liquid in the upper layer of the ultrafiltration tube, store it at 4°C, and use it within 2 weeks.

[0045] (5) Sample positive rate determination: 1) Dilute the concentration standard 100 times with ultrapure water, perform quality control of the nanoflow cytometer according to the instructions, adjust the nanoflow cytometer to the optimal detection state (the signals of the scattering and fluorescence channels are both the strongest and uniform), and collect data on the diluted standard under the measurement parameters of the concentration standard (under the optimal state, the particle count value range of the quality control standard is usually 5000-7000).

[0046] 2) Detect the particle count of phosphate buffer under the Labelled Exo sample measurement parameters to determine the cleanliness of the phosphate buffer. Only phosphate buffer with a particle count of less than 200 can be used as a blank control for data collection and subsequent sample dilution. If the particle count is higher than 200, it needs to be filtered with a 0.22 μm filter membrane.

[0047] 3) Pre-dilute the sample 100 times with clean phosphate buffer, and collect data on the milk exosome sample under the Labelled Exo sample measurement parameters. The nanoflow cytometer particle count is accurate in the range of 4000 to 8000. If the sample particle count is too high or too low, the sample dilution multiple needs to be adjusted and then measured, and the final dilution multiple of the sample needs to be recorded. In addition, it should be noted that the baseline and automatic threshold of the scattering channel should be consistent with the blank control when the sample is tested on the machine. If the baseline rises during the sample test, a higher purity sample needs to be re-prepared.

[0048] In this example, nanoflow cytometry was performed on samples loaded with green fluorescent protein (GFP) to evaluate the particle size and positive rate of different PEGylated Spytag molecules. Figure 2 As shown in the figure, Chol-PEG-Spytag showed good solubility in aqueous solution, almost no nanoparticles were detected, and the positive rate was only 1.1%, indicating that it mainly existed in the form of single molecules in the solution without forming aggregates. In contrast, DSPE-PEG-Spytag and palmitic acid-PEG-Spytag had poor solubility in aqueous solution and tended to self-assemble to form nanoparticles. The nanoparticle size detected by DSPE-PEG-Spytag was about 65 nanometers, with a positive rate of up to 98.1%, while the nanoparticle size detected by palmitic acid-PEG-Spytag was about 67 nanometers, with a positive rate of 76.8%. These data indicate that these two PEG-Spytag molecules easily form nanoparticles with a size comparable to that of exosomes in aqueous solution, which may lead to false positive results in protein loading experiments.

[0049] Based on these findings, the present invention selects Chol-PEG-Spytag as the exosome protein loading tool for subsequent experiments. The good solubility and monodisperse state of Chol-PEG-Spytag make it an ideal choice because it reduces the risk of nonspecific aggregation and false positives, thereby improving the accuracy and reliability of the experiment.

[0050] 4. Hydrophobic group screening experiment 2, the steps are as follows: Exosomes loaded with Spytag: (1) Reagent dissolution: Prepare 1 mM Chol-PEG-Spytag, DSPE-PEG-Spytag and palmitic acid-PEG-Spytag respectively; take 1 bottle of milk exosome freeze-dried powder (3.3×10 11 / bottle), add 3.3ml secondary water to 10 11 Pieces / ml; (2) Take 100 μl 10 11 Milk exosomes of 100 g / ml were placed in a 1.5 ml EP tube, and 1 mM Chol-PEG-Spytag, DSPE-PEG-Spytag and palmitic acid-PEG-Spytag were added to a final concentration of 0.4 mM, respectively, and reacted at 37°C for 2 h; (3) After cooling to 4°C, the mixture was immediately centrifuged at 4,500 g for 3 min using a 100 kDa ultrafiltration tube to remove free Chol-PEG-Spytag, DSPE-PEG-Spytag, and palmitic acid-PEG-Spytag; (4) Add 300 μl PBS (pH 7.4) to the ultrafiltration tube and centrifuge at 4,500 g for 3 min to remove free Chol-PEG-Spytag, DSPE-PEG-Spytag, and palmitic acid-PEG-Spytag; (5) Repeat step (4) to collect the retained liquid in the upper layer of the ultrafiltration tube; (6) Add 150 μl of PBS to the entrapped liquid obtained at the end of the above exosome loading Spytag step to a total volume of 200 μl, and add 100 μl of 0.2 mg / ml spycatcher-GFP fusion protein prepared above (the ratio of the number of exosomes loaded with Spytag to the number of spycatcher-GFP fusion protein in the system is 3.78×10 -5 ), react at 4°C overnight; (7) After cooling to 4°C, the cells were immediately centrifuged at 4,500 g for 3 min using a 100 kDa ultrafiltration tube to remove free spycatcher-GFP fusion protein. (8) Add 300 μl PBS (pH 7.4) to the ultrafiltration tube and centrifuge at 4500 g for 3 min to remove free spycatcher-GFP fusion protein; (9) Repeat step (8), collect the retained liquid in the upper layer of the ultrafiltration tube, and detect the positive rate of exosomes loaded with Spytag.

[0051] Results Figure 3 The positive rates of Chol-PEG-Spytag, DSPE-PEG-Spytag and palmitic acid-PEG-Spytag loading Spytag were 98.4%, 50.5% and 39.8%, respectively. Therefore, Chol-PEG-Spytag was selected for loading protein.

[0052] Example 2 In this example, the concentration of Spytag used when exosomes are loaded with Spytag is studied, as follows: (1) Reagent dissolution: Weigh 1 mg of Chol-PEG-Spytag prepared in Example 1 and dissolve it in 227 μl of double distilled water to 1 mM; take 1 bottle of milk exosome freeze-dried powder (3.3×10 11 / bottle), add 3.3ml secondary water to 10 11 Pieces / ml; (2) Take 100 μl 10 11 Milk exosomes of 100 g / ml were placed in 1.5 ml EP tubes, and different volumes of 1 mM Chol-PEG-Spytag were added to the final concentrations of 0.1 mM, 0.2 mM, 0.4 mM, 0.8 mM, and 1.6 mM, respectively, and reacted at 37°C for 2 h. (3) After cooling to 4°C, the mixture was immediately centrifuged at 4,500 g for 3 min using a 100 kDa ultrafiltration tube to remove free Chol-PEG-Spytag. (4) Add 300 μl PBS (pH 7.4) to the ultrafiltration tube and centrifuge at 4,500 g for 3 min to remove free Chol-PEG-Spytag; (5) Repeat step (4) to collect the retained liquid in the upper layer of the ultrafiltration tube for loading spycatcher-GFP.

[0053] (6) Add 150 μl of PBS to the trapped liquid obtained at the end of the above exosome loading Spytag step to a total volume of 200 μl, and add 100 μl of 0.2 mg / ml spycatcher-GFP fusion protein prepared in Example 1 (the ratio of the number of exosomes loaded with Spytag to the number of spycatcher-GFP fusion protein in the system is 3.78×10-5 ), react at 4°C overnight; (7) After cooling to 4°C, the cells were immediately centrifuged at 4,500 g for 3 min using a 100 kDa ultrafiltration tube to remove free spycatcher-GFP fusion protein. (8) Add 300 μl PBS (pH 7.4) to the ultrafiltration tube and centrifuge at 4500 g for 3 min to remove free spycatcher-GFP fusion protein; (9) Repeat step (8), collect the retained liquid in the upper layer of the ultrafiltration tube, and detect the positive rate of exosomes loaded with Spytag (see Example 1 for the test method).

[0054] Results Figure 4 When the concentration of Chol-PEG-Spytag was 0.8mM, the positive rate of spytag loading was 85.6%, and when the concentration of Chol-PEG-Spytag reached 1.6mM, the positive rate was 100%. Therefore, 0.8mM, which has a higher positive rate of Chol-PEG-Spytag and a lower feed concentration, was selected as the optimal loading concentration.

[0055] Example 3 This embodiment provides a method for preparing exosomes loaded with protein. The schematic diagram of exosomes loaded with protein is shown in Figure 1 , including the following steps: 1. Exosomes were loaded with Spytag. For the specific method, see Example 2. During the preparation, the concentration of Chol-PEG-Spytag was 0.8 mM.

[0056] 2. Exosomes loaded with GFP protein (1) Add 150 μl of PBS to the above-obtained retentate to a total volume of 200 μl, and add 100 μl of 0.2 mg / ml spycatcher-GFP fusion protein prepared in Example 1 (the ratio of the number of exosomes loaded with Spytag to the number of spycatcher-GFP fusion protein in the system is 3.78×10 -5 ), react at 4°C overnight; (2) After cooling to 4°C, the tube was immediately centrifuged at 4,500 g for 3 min using a 100 kDa ultrafiltration tube to remove free spycatcher-GFP fusion protein; (3) Add 300 μl PBS (pH 7.4) to the ultrafiltration tube and centrifuge at 4500 g for 3 min to remove free spycatcher-GFP fusion protein; (4) Repeat step (3), collect the retained liquid in the upper layer of the ultrafiltration tube, store it at 4°C, and use it within 2 weeks.

[0057] 3. Identification of GFP-loaded exosome delivery system (1) Determination of exosome loading efficiency Take 10 μl of the GFP-loaded exosomes prepared in the above step and dilute 10-fold to a concentration of 10 by adding 90 μl of PBS. 10 / ml, vortex for 1min; Take 10 μl of the above solution and set the concentration to 10 10 10 / ml of GFP-loaded exosomes were added to 90 μl PBS and diluted 10-fold to a concentration of 10 9 / ml, vortex for 1 min; detect the positive rate of exosomes loaded with GFP protein (see Example 1 for the test method).

[0058] The results are as follows Figure 5 As shown, the positive rate of exosomes loaded with GFP protein was measured to be 92.1%, proving that most exosomes were loaded with GFP protein.

[0059] (2) Particle size characterization Take the concentration as 10 9 The sample (exosomes loaded with GFP protein) was analyzed by nanoflow cytometry. Figure 6 As shown, the particle size of milk exosomes (EV) without protein loading was 66±11 nm; the particle size of milk exosomes loaded with GFP (EV-GFP) was 79±19 nm, an increase of 13 nm.

[0060] (3) Morphological characterization Take 10 μl of the above solution and set the concentration to 10 10 10 μl of GFP-loaded exosomes were added to the copper mesh at 10 μg / ml and allowed to stand for 5 min to allow the sample to settle on the copper mesh. The excess sample was aspirated and 10 μl of uranyl acetate was added. The sample was allowed to stand for 1 min and then negatively stained and the morphology was observed using a transmission electron microscope.

[0061] The results are as follows Figure 7 As shown, milk exosomes (EV) without protein loading have a cup-shaped vesicle characteristic structure, and exosomes loaded with GFP protein (EV-GFP) also have a cup-shaped structure, with no obvious difference from exosomes (EV), indicating that loading protein does not affect the morphology of exosomes.

[0062] (4) Determination of encapsulation efficiency Sample determination: Take 10 μl with a concentration of 2×10 11 100 μl / ml exosome-GFP (exosomes loaded with GFP protein) were added into a cuvette containing 400 μl PBS, and the fluorescence intensity was measured at 512.6 nm.

[0063] Preparation of standard curve: Take 10 μl of 1.5 mg / ml GFP and add 90 μl PBS to dilute 10 times to 0.15 mg / ml.

[0064] Standard curve determination: Take 1μl, 3μl, 5μl, 7μl, 9μl, 11μl, 13μl, 15μl, 17μl GFP (concentration 0.15mg / ml) and add them to 400μl PBS. Use a fluorescence spectrophotometer to measure the fluorescence intensity at 512.6nm. Make a standard curve based on the fluorescence intensity and concentration (see the results). Figure 8 ), determine the concentration in the sample.

[0065] According to the measurement, the fluorescence intensity of the sample was 464.5. The GFP concentration of EV-GFP was calculated to be 48.7 μg / ml according to the standard curve, and the encapsulation efficiency (encapsulation amount / feeding amount × 100%) was 3.2%. The results showed that each exosome could load 3467 GFPs.

[0066] 4. GFP exosome uptake (1) Cell seeding: 293T cells were seeded into confocal culture dishes at 25,000 cells / well and incubated overnight in an incubator at 37°C and 5% CO2 in a liquid volume of 500 μl.

[0067] (2) Administration: 5 μl at a concentration of 10 11 The exosomes loaded with GFP protein (EV-GFP) prepared above were incubated for 6 hours at 100 μg / ml, then fixed with paraformaldehyde for 5 minutes, and washed 3 times with PBS. A blank control group and a control group with GFP added were also set up.

[0068] DAPI nuclear staining: DAPI (5 mg / ml) was diluted 1:500, added to the confocal dish containing DAPI in PBS and incubated for 5 min, and then washed three times with PBS.

[0069] The results are as follows Fig. 9 As shown, there is no obvious fluorescence after adding GFP confocal dish, while there is obvious fluorescence in the cytoplasm after adding exosome-loaded GFP protein, indicating that it can be taken up by cells and delivered into cells.

[0070] Example 4 Brain-derived neurotrophic factor (BDNF) is a protein with neurotrophic effects first discovered in pig brain by Barde et al. in 1982. The human BDNF gene has 9 promoters and 11 exons, 2 more exons than rats and mice, so human BDNF is more complex. These different transcripts may be cell-specific and activity-dependent, and can have unique effects on molecular and behavioral expression. BDNF mutant mice have thinner hippocampal spines, lower density, and reduced long-term potentiation (LTP). BDNF is most highly expressed in the hippocampus and cortex of the brain and is synthesized in the cell bodies of neurons and glial cells. In human neurons, BDNF transcripts are translated and cleaved into pro-BDNF precursors in neuronal cell bodies. Pro-BDNF is both converted intracellularly and secreted extracellularly, a process that helps regulate the different roles of pro-BDNF and mature BDNF in synaptic processes.

[0071] This embodiment provides a method for preparing exosomes loaded with BDNF, comprising the following steps: 1. Preparation of Spycatcher-BDNF The preparation method is as described in Example 1. Spycatcher-BDNF is expressed using an E. coli expression system. Its amino acid sequence is composed of the amino acid sequences of spycatcher (SEQ ID No. 3), a connecting peptide (SEQ ID No. 6) and BDNF (SEQ ID No. 8) connected in sequence.

[0072] 2. Exosomes are loaded with Spytag. The experimental steps are as follows: (1) Reagent dissolution: Weigh 1 mg of Chol-PEG-Spytag prepared in Example 1 and dissolve it in 227 μl of double distilled water to 1 mM; take 1 bottle of stem cell exosome freeze-dried powder (7.43×10 9 pcs / bottle), add 1ml of secondary water (double distilled water) to dissolve; (2) Take 100 μl 7.43×10 8 pcs / ml stem cell exosomes were placed in a 1.5ml EP tube, 60μl of 1mM Chol-PEG-Spytag was added, and the reaction was carried out at 37℃ for 2h; (3) After cooling to 4°C, the mixture was immediately centrifuged at 4,500 g for 3 min using a 100 kDa ultrafiltration tube to remove free Chol-PEG-Spytag. (4) Add 300 μl PBS (pH 7.4) to the ultrafiltration tube and centrifuge at 4,500 g for 3 min to remove free Chol-PEG-Spytag; (5) Repeat step (4) to collect the retained liquid in the upper layer of the ultrafiltration tube.

[0073] 3. Exosomes loaded with GFP protein (1) Add 150 μl of PBS to the entrapped liquid obtained at the end of the above exosome loading Spytag step to a total volume of 200 μl, and add 100 μl of 0.2 mg / ml spycatcher-GFP fusion protein prepared in Example 1 (the ratio of the number of exosomes loaded with Spytag to the number of spycatcher-GFP fusion protein in the system is 3.78×10 -5 ), react at 4°C overnight; (2) After cooling to 4°C, the tube was immediately centrifuged at 4,500 g for 3 min using a 100 kDa ultrafiltration tube to remove free spycatcher-GFP fusion protein; (3) Add 300 μl PBS (pH 7.4) to the ultrafiltration tube and centrifuge at 4,500 g for 3 min to remove free spycatcher-GFP fusion protein; (4) Repeat step (3) and add 400 μl PBS (pH 7.4). Collect the retained liquid in the upper layer of the ultrafiltration tube after centrifugation and store at 4°C. Use within 2 weeks.

[0074] It is convenient to analyze by nanoflow cytometry (see Example 1 for the method), and the results are shown in Fig.10 , stem cell exosomes loaded with green fluorescent protein (GFP) showed a positive rate of up to 60.1%. This data not only confirms the ability of exosomes to successfully load GFP, but also reflects the efficiency and accuracy of the experimental method of the present invention.

[0075] 4. Exosomes loaded with BDNF protein (1) Add 150 μl of PBS to the retentate obtained at the end of the above exosome loading Spytag step to a total volume of 200 μl, and add 36 μl of 0.87 mg / ml sc-BDNF (spycatcher-BDNF) fusion protein (the ratio of the number of exosomes loaded with Spytag to the number of spycatcher-BDNF fusion protein in the system is 3.78×10 -5 ), react at 4°C overnight; (2) After cooling to 4°C, the tube was immediately centrifuged at 4,500 g for 3 min using a 100 kDa ultrafiltration tube to remove free sc-BDNF fusion protein; (3) Add 300 μl PBS (pH 7.4) to the ultrafiltration tube and centrifuge at 4,500 g for 3 min to remove free sc-BDNF fusion protein; (4) Repeat step (3) and add 400 μl PBS (pH 7.4). Collect the retained liquid in the upper layer of the ultrafiltration tube after centrifugation and store at 4°C. Use within 2 weeks.

[0076] Electron microscopy images reveal the morphological characteristics of stem cell exosomes loaded with brain-derived neurotrophic factor (BDNF) (see Fig.11 ). The images clearly show the typical vesicle-like structure of exosomes, which is the hallmark morphology of exosomes. Importantly, the morphology of BDNF-loaded exosomes was not significantly different from that of unloaded exosomes, indicating that the BDNF loading process did not affect the natural morphology of exosomes.

[0077] 5. Identification of the exosome delivery system loaded with BDNF The BDNF ELISA kit was used to identify the loading amount of sc-BDNF. The specific experimental steps are as follows: (1) Reagent preparation: Prepare the diluted standard and the sample to be tested.

[0078] (2) Determination of ELISA strips: Calculate the ELISA strips required for the samples and standards to be tested, take the ELISA strips out of the aluminum foil bag, put the remaining ELISA strips back into the aluminum foil bag, seal the bag, and store at low temperature.

[0079] (3) Soaking the ELISA plate: Add 1× washing solution (350 μl / well) to soak the ELISA plate. After standing for 30 seconds, discard the liquid in the well and tap the ELISA plate dry. The amount of liquid has an important impact on the test results. Make sure that there is no washing solution left when tapping the plate for the last time.

[0080] (4) Sample addition and incubation: Add each gradient standard and diluted sample to be tested, 100 μl / well, ensure that the sample is added within 15 minutes, and incubate at room temperature for 2 hours.

[0081] (5) Washing the ELISA plate: discard the liquid in the wells, add 1× washing solution (350 μl / well) and wash the plate 5 times, then pat the plate dry.

[0082] (6) Detection antibody incubation: Add detection antibody prepared in advance to the working concentration to the ELISA plate, 100 μl / well, and incubate at room temperature for 2 hours.

[0083] (7) Washing the ELISA plate: discard the liquid in the wells, add 1× washing solution (350 μL / well) and wash the plate 5 times, and pat the ELISA plate dry.

[0084] (8) Enzyme conjugate incubation: Add the enzyme conjugate prepared to the working concentration into the ELISA plate, 100 μl / well, and incubate at room temperature for 20 minutes.

[0085] (9) Washing the ELISA plate: discard the liquid in the wells, add 1× washing solution (350 μl / well) and wash the plate 5 times, and pat the ELISA plate dry.

[0086] (10) Color development: Return the substrate solution to room temperature 10 minutes before use, add the substrate solution to the ELISA plate at 100 μl / well, and incubate at room temperature in the dark for 15 minutes.

[0087] (11) Stop: Add 50 μl / well of stop solution to the ELISA plate. The color will turn from blue to yellow. Gently shake the plate until the color is evenly distributed.

[0088] (12) Reading: Read the absorbance at 450 nm within 10 minutes.

[0089] Preparation of standard curve: Prepare the standard curve according to the absorbance and concentration. The absorbance test results of sc-BDNF with different concentrations are shown in Table 1. The standard curve of sc-BDNF is shown in Fig.12 The sc-BDNF loading amount was calculated to be 54.6 μg / ml based on the measured absorbance (results in Table 2).

[0090] Table 1 Table 2 6. Stem cell exosome-BDNF migration experiment: (1) Culture 200,000 cells / well in a 24-well plate and place in a culture chamber at 37°C and 5% CO2 for 24 h until the neurons form a monolayer covering the entire culture surface.

[0091] (2) Using a 200 μl pipette tip or a specialized scratching tool, make a straight scratch in the cell monolayer. Gently rinse the dish with PBS (phosphate-buffered saline) to remove loose cells and debris in the scratch.

[0092] (3) Replace the serum-containing Neurobasal™-A medium with one containing 5×10 9 The serum-free culture medium containing BDNF exosomes prepared as above was used to reduce the interference of cell proliferation on the experimental results.

[0093] (4) Immediately after scratching, take a photo of the scratched area using a microscope as the starting point of the experiment.

[0094] (5) Place the culture dish back into the incubator and allow the cells to culture under the above conditions for 48 hours.

[0095] (6) Take photos of the scratch area at different time points (0 h, 24 h, 48 h) during the culture process to monitor the dynamic process of cell migration.

[0096] The results are as follows Fig.13 As shown in the figure, compared with the control group without any treatment, the cells loaded with brain-derived neurotrophic factor (BDNF) showed a significant enhancement in migration ability. This result not only reveals the important role of BDNF in promoting cell migration, but also illustrates the effectiveness of the exosome delivery system of the present invention.

[0097] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing protein-loaded exosomes, characterized in that: include: (1) Mixing cholesterol-PEG-spy tag / probe tag with exosomes to obtain exosomes loaded with spy tag / probe tag; (2) The exosomes loaded with the spy tag / probe tag are mixed with the target loading protein and the fusion protein of the spy catcher / probe catcher to react, and the target loading protein is loaded onto the exosomes through the formation of an isopeptide bond between the spy tag / probe tag and the spy catcher / probe catcher.

2. The method according to claim 1, characterized in that The molecular weight of PEG in the cholesterol-PEG-spy tag / spy tag is 1000-5000, preferably the molecular weight of PEG is 2000; And / or, the exosomes are milk exosomes or stem cell exosomes.

3. The method according to claim 1 or 2, characterized in that: The amino acid sequence of the cholesterol-PEG-spy tag / spy tag is shown in SEQ ID No. 1; The amino acid sequence of the probe tag in the cholesterol-PEG-spy tag / probe tag is shown in SEQ ID No. 2; The amino acid sequence of the spy catcher is shown in SEQ ID No. 3; The amino acid sequence of the detector is shown in SEQ ID No. 4; The target loading protein and the spy catcher / detector in the fusion protein are directly connected via a connecting peptide, and the amino acid sequence of the connecting peptide is shown in SEQ ID No. 5 or 6.

4. The method according to any one of claims 1 to 3, characterized in that: In the cholesterol-PEG-spy tag / probe tag, cholesterol-PEG and thespy tag / probe tag are linked by covalent reaction.

5. The method according to any one of claims 1 to 4, characterized in that: In step (1), the cholesterol-PEG-spy tag / probe tag is mixed with exosomes, and the mixture is reacted at 37±1°C. The final concentration of the cholesterol-PEG-spy tag / probe tag in the mixture is 0.4 mM-1.6 mM, preferably 0.8 mM.

6. The method according to any one of claims 1 to 5, characterized in that: The preparation method of the fusion protein comprises: recombining the nucleotide sequences encoding the target loading protein and the spy catcher / detector by gene recombination, expressing them in a host cell, and then purifying them; preferably, the host cell is Escherichia coli; And / or, in step (2), the exosomes loaded with spy tag / probe tag are mixed with the target loading protein and the fusion protein of spy catcher / probe catcher at 4-37°C.

7. The method according to any one of claims 1 to 6, characterized in that: After the reaction of steps (1) and (2) is completed, the method further includes the steps of separating and purifying the products, wherein the separation and purification method is ultrafiltration, tangential flow or size exclusion chromatography.

8. A protein-loaded exosome, characterized in that: The method is prepared by any one of claims 1 to 7.

9. Use of the method according to any one of claims 1 to 7 or the exosomes according to claim 8 in the preparation of a drug.

10. A pharmaceutical composition, characterized in that Comprising the exosomes according to claim 8 and pharmaceutically acceptable excipients.

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