Preparation method and application of an engineered exosome

Abstract

The application discloses a preparation method and application of an engineered exosome, and relates to the technical field of biology.The application provides a preparation method of an engineered exosome, and a carrier construction step is provided for constructing CD63-VEGF into a FUGW carrier, and replacing an Ubc promoter in the FUGW carrier with EF1a to obtain FUGW(EF1a)-CD63-VEGF, wherein the sequence of the FUGW(EF1a)-CD63-VEGF is shown as SEQ ID NO:5.The application successfully develops the engineered exosome capable of carrying VEGF by using synthetic biology technology, and the application provides a train of thought for exosome drugs and drug development with the exosome as a delivery carrier.The engineered exosome carrying VEGF has a remarkable effect on promoting hair growth.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, in particular to a preparation method and application of an engineered exosome. BACKGROUND

[0002] Exosomes are extracellular vesicles with a diameter of 30-150nm and a phospholipid bilayer, which are membrane vesicles released into the extracellular space after fusion of intracellular multivesicular bodies with the cell membrane. Exosomes contain many components, including proteins, lipids, mRNAs, mitochondrial DNA, miRNAs and many other non-coding RNAs. According to proteomic studies, exosomes not only have specific proteins dependent on the type of secretory cells, but also have specific subpopulations of cell proteins found in exosomes regardless of cell type. Exosome components such as lipids, metabolites, functional proteins and nucleic acids play an important role in intercellular and intracellular communication and show great potential for using exosomes as drug delivery carriers. With the in-depth study of exosomes, it is found that exosomes can not only mediate the transmission of intercellular signals, but also play an important role in disease screening and diagnosis, disease treatment and as drug delivery carriers.

[0003] All cells can produce exosomes, but different types of exosomes have different functions, such as exosomes secreted by cancer cells can promote the spread and metastasis of cancer, exosomes produced by stem cells can be used for medical beauty, immune regulation and can play a repair function and can be developed as drugs alone.

[0004] Exosomes are composed of lipid membrane bilayers, which express surface ligands and receptors for donor cells, and are surrounded by and contain hydrophilic cores. As a natural carrier, exosomes can target specific organs, protect drugs and prolong the circulation time of drugs, and mediate long-distance intercellular communication. Exosomes have good biocompatibility, small size and low immunogenicity, and can be used as natural carriers for small molecule drugs, transmembrane proteins and nucleic acid drugs. The method of loading drugs into exosomes can be divided into two types: endogenous loading and exogenous loading. Endogenous loading is to load drug molecules into parent cells before isolating exosomes, so that the exosomes secreted by the parent cells carry drug molecules. Exogenous loading is to isolate exosomes first, and then load drug molecules directly into the membrane surface or cavity of the exosomes by passive or active methods. Endogenous loading does not damage the exosome membrane, and the most commonly used method is to incubate the engineered exosome drug loading with the donor cells, but how to make the exosomes carry specific gene expression products to further increase their functional effects, and how to introduce drugs into the exosomes are still problems to be solved. SUMMARY

[0005] The purpose of the present application is to provide a preparation method and application of an engineered exosome.

[0006] To achieve the above-mentioned purposes of the application, the technical solutions of the application are as follows.

[0007] In one aspect, the application provides a preparation method of an engineered exosome, comprising a vector construction step, for constructing CD63-VEGF into FUGW vector, and replacing Ubc promoter in FUGW vector with EF1a to obtain FUGW(EF1a)-CD63-VEGF, wherein the sequence of the FUGW(EF1a)-CD63-VEGF is shown as SEQ ID NO: 5.

[0008] Specifically, the preparation method of the engineered exosome comprises the following steps.

[0009] S1, vector construction;

[0010] S2, lentivirus packaging;

[0011] S3, lentivirus transfection of stem cells;

[0012] S4, exosome purification: collecting supernatant in a culture dish, collecting supernatant by first centrifugation, collecting supernatant by second centrifugation, filtering, adding a purification reagent to the filtrate, and collecting exosomes by centrifugation after mixing.

[0013] Specifically, the lentivirus packaging of step S2 comprises the following steps: cell resuscitation, cell passage, cell plating, and lentivirus plasmid packaging.

[0014] Further, the cells are selected from one or more of 293T cells, A549 cells, MCF-7 cells, HEL cells, HUVEC cells, and NIH3T3 cells.

[0015] Still further, the cells are 293T cells.

[0016] According to some embodiments of the application, the cell resuscitation step comprises the following steps: placing the cryopreserved cells in a 37℃ water bath, after thawing, aspirating the cells into a centrifuge tube containing cell culture medium, centrifuging at 800rpm for 5min, discarding the supernatant, adding culture medium to resuspend the cells, inoculating the cells into a cell culture bottle containing culture medium, and culturing in a 37℃, 5% CO2 incubator.

[0017] According to some embodiments of the application, the cell passage step comprises the following steps: when the cell density reaches more than 80%, adding trypsin for digestion, adding cell culture medium to terminate the digestion, centrifuging at 800rpm for 5min at room temperature, discarding the supernatant, adding cell culture medium to resuspend the cells, inoculating 1x10 6 cells into a culture bottle containing culture medium, and culturing in a 37℃, 5% CO2 cell incubator.

[0018] According to some embodiments of the present application, the cell plating step is: when the cell density is greater than 80%, trypsin is added, cell culture medium is added to stop the digestion, centrifugation is performed, the supernatant is discarded, cell culture medium is added to resuspend the cells, 4x10 6 cells are inoculated into a culture bottle containing the culture medium, and culture is performed in a 37°C, 5% CO2 cell incubator.

[0019] According to some embodiments of the present application, the lentivirus plasmid package includes plasmid FUGW(EF1a)-CD63-VEGF, packaging plasmid 1, packaging plasmid 2, and envelope plasmid.

[0020] Further, the mass ratio of the FUGW(EF1a)-CD63-VEGF, packaging plasmid 1, packaging plasmid 2, and envelope plasmid is (1-5):(1-3):(1-3):1.

[0021] Further, the mass ratio of the FUGW(EF1a)-CD63-VEGF, packaging plasmid 1, packaging plasmid 2, and envelope plasmid is 2:1:1:1.

[0022] According to some embodiments of the present application, the packaging plasmid 1 is pLP1-KANA, the packaging plasmid 2 is pLP2-KANA, and the envelope plasmid is PVSVG-KANA.

[0023] Specifically, the lentivirus package includes the following steps:

[0024] (1) FUGW(EF1a)-CD63-VEGF, packaging plasmid 1, packaging plasmid 2, and envelope plasmid are mixed according to the ratio, and the mixture is added to the culture medium, denoted as A;

[0025] (2) The transfection reagent is added to the culture medium, denoted as B;

[0026] (3) A and B are mixed and added to the culture dish for culture.

[0027] Further, the mass ratio of the transfection reagent to the total plasmid in step (2) is 1:(1-3).

[0028] Further, the mass ratio of the transfection reagent to the total plasmid in step (2) is 1:2.

[0029] Further, the culture medium in step (2) is selected from any one of OptiMEM medium, DMEM medium, and RPMI medium.

[0030] Further, the culture medium in step (2) is OptiMEM medium.

[0031] Further, the transfection reagent in step (2) is PEI.

[0032] Further, the culture condition in step (3) is 35-38℃, 4-8% CO2.

[0033] Still further, the culture condition in step (3) is 37℃, 5% CO2. 2。

[0034] Specifically, the supernatant in the culture dish is collected after culturing for 36-48h in step (3).

[0035] Further, the supernatant is collected after culturing for 48h in step (3) and centrifuged at 2000g for 10min at 4℃, and then filtered through a 0.45μm filter membrane and centrifuged at 20000g for 2h at 4℃ for lentivirus concentration.

[0036] Specifically, the lentivirus transfection of stem cells in step S3 comprises the following steps:

[0037] 1) Lentivirus concentrated solution infects stem cells, and a promoting infection reagent is added for culture in an incubator;

[0038] 2) Puromycin is added to the culture medium for culture in an incubator, and subculture is performed.

[0039] Further, 1ml of lentivirus concentrated solution infects 6*10 6 stem cells in step 1).

[0040] Further, the final concentration of the promoting infection reagent in step 1) is 5-10μg / ml; still further, the final concentration of the promoting infection reagent in step 1) is 8μg / ml.

[0041] Further, the culture condition in step 1) is 37℃, 5% CO2.

[0042] Further, the culturing time in step 1) is 24-48h, and the cell confluence is 80%.

[0043] Further, the stem cells in step 1) are selected from any one of human umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells and adipose mesenchymal stem cells;

[0044] Still further, the stem cells in step 1) are human umbilical cord mesenchymal stem cells.

[0045] Further, the final concentration of puromycin in step 2) is 1-5μg / ml; still further, the final concentration of puromycin in step 2) is 2μg / ml.

[0046] Further, the culture condition in step 2) is 37℃, 5% CO2, and the cell confluence is 50%.

[0047] Further, the first centrifugation condition in step S4 is 3-8℃, 1000-3000g, 10-30min.

[0048] Further, the first centrifugation condition in step S4 is 4℃, 2000g, 20min.

[0049] Further, the second centrifugation condition in step S4 is 3-8℃, 8000-12000g, 50-70min.

[0050] Further, the second centrifugation condition in step S4 is 4℃, 10000g, 60min.

[0051] Further, the purification reagent in step S4 is PEG8000.

[0052] Further, the final concentration of the purification reagent is 8-12%; further, the final concentration of the purification reagent is 10%.

[0053] Specifically, the purification reagent and the final concentration of 8-12% are mass / volume percentages.

[0054] In another aspect, the present application provides the engineered exosome prepared by the above preparation method.

[0055] In another aspect, the present application provides the use of the above engineered exosome in the preparation of a product for promoting hair growth.

[0056] Specifically, the product includes but is not limited to a drug.

[0057] Further, the engineered exosome is used as an active ingredient in the drug.

[0058] Further, the drug includes a pharmaceutically acceptable carrier.

[0059] Further, the pharmaceutically acceptable carrier includes but is not limited to excipients, buffers, emulsifiers, stabilizers, diluents, binders, preservatives, lubricants, pH regulators, cryoprotectants, flavorings, fillers.

[0060] Further, the dosage form of the drug includes but is not limited to gastrointestinal administration and non-gastrointestinal administration according to the administration method.

[0061] Further, the gastrointestinal administration dosage form includes, but is not limited to, tablets, powders, granules, solutions, capsules, emulsions, suspensions, oils.

[0062] Further, the non-gastrointestinal administration dosage form includes, but is not limited to, injection administration dosage form, respiratory administration dosage form, skin administration dosage form, mucosal administration dosage form, and cavity administration dosage form.

[0063] Further, the injection administration dosage form includes, but is not limited to, intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intracavity injection.

[0064] Further, the respiratory administration dosage form includes, but is not limited to, spray, aerosol, and powder mist.

[0065] Further, the skin administration dosage form includes, but is not limited to, lotion, ointment, external solution, plaster, paste, patch.

[0066] Further, the mucosal administration dosage form includes, but is not limited to, eye drops, nose drops, eye ointment, sublingual tablets, film patches.

[0067] Further, the cavity administration dosage form includes, but is not limited to, suppositories, aerosols, effervescent tablets, drops, and dripping pills.

[0068] In another aspect, the present application provides a product for promoting hair growth, wherein the product comprises the engineered exosome described above.

[0069] Specifically, the product includes, but is not limited to, a drug.

[0070] Further, the engineered exosome is used as an active ingredient in the drug.

[0071] Further, the drug comprises a pharmaceutically acceptable carrier.

[0072] Further, the pharmaceutically acceptable carrier includes, but is not limited to, excipients, buffers, emulsifiers, stabilizers, diluents, binders, preservatives, lubricants, pH adjusters, cryoprotectants, flavoring agents, fillers.

[0073] Further, the dosage form of the drug includes, but is not limited to, gastrointestinal administration dosage form and non-gastrointestinal administration dosage form according to the administration method.

[0074] Further, the gastrointestinal administration dosage form includes, but is not limited to, tablets, powders, granules, solutions, capsules, emulsions, suspensions, oils.

[0075] Further, the non-gastrointestinal administration forms include, but are not limited to, injection administration forms, respiratory administration forms, skin administration forms, mucosa administration forms, and cavity administration forms.

[0076] According to some embodiments of the present application, the present application provides a preparation method of the engineered exosome, comprising a vector construction step, for constructing CD63-GFP into FUGW vector, and replacing Ubc promoter in FUGW vector with EF1a to obtain FUGW(EF1a)-CD63-GFP, wherein the sequence of the FUGW(EF1a)-CD63-GFP is shown as SEQ ID NO: 4.

[0077] Specifically, the preparation method of the engineered exosome comprises the following steps:

[0078] (1) Vector construction;

[0079] (2) Transfecting cells: adding FUGW(EF1a)-CD63-GFP into a culture medium, denoted as A; adding transfection reagent into the culture medium, denoted as B; mixing A and B and then adding into a culture dish for culture;

[0080] (3) Exosome purification: collecting supernatant in the culture dish, collecting the supernatant by first centrifugation, collecting the supernatant by second centrifugation, filtering, adding purification reagent into the filtrate, mixing and then collecting exosomes by centrifugation.

[0081] Further, the mass ratio of FUGW(EF1a)-CD63-GFP to the transfection reagent in step (2) is 1:(1-3).

[0082] Further, the mass ratio of FUGW(EF1a)-CD63-GFP to the transfection reagent in step (2) is 1:2.5.

[0083] Further, the culture medium in step (2) is selected from any one of OptiMEM culture medium, DMEM culture medium, and RPMI culture medium.

[0084] Further, the culture medium in step (2) is OptiMEM culture medium.

[0085] Further, the transfection reagent in step (2) is PEIpro.

[0086] Further, the culture condition in step (2) is 35-38℃, 4-8% CO2.

[0087] Further, the culture condition in step (2) is 37℃, 5% CO 2。

[0088] Specifically, the supernatant in the culture dish is collected after culturing for 36-48 hours in step (3).

[0089] Further, the first centrifugation in step (3) is performed at 3-8 DEG C for 10-30 min at 1000-3000 g.

[0090] Further, the first centrifugation in step (3) is performed at 4 DEG C for 20 min at 2000 g.

[0091] Further, the second centrifugation in step (3) is performed at 3-8 DEG C for 50-70 min at 8000-12000 g.

[0092] Further, the second centrifugation in step (3) is performed at 4 DEG C for 60 min at 10000 g.

[0093] Further, the purification reagent in step (3) is PEG8000.

[0094] Further, the final concentration of the purification reagent is 8-12%; and more preferably, the final concentration of the purification reagent is 10%.

[0095] The present application has the following advantages:

[0096] The present application successfully develops the engineered exosome carrying specific gene expression products by using synthetic biology technology, which provides a new idea for exosome drug and drug development using exosome as a delivery carrier. BRIEF DESCRIPTION OF DRAWINGS

[0097] Figure 1 The figure is the transcription result of the mRNA level of the target gene, wherein the control group is 293T cells, Ubc-GFP is 293T cells into which FUGW(Ubc)-GFP plasmid is introduced, EF1a-GFP is 293T cells into which FUGW(EF1a)-GFP plasmid is introduced, Ubc-CD63-GFP is 293T cells into which FUGW(Ubc)-CD63-GFP plasmid is introduced, and EF1a-CD63-GFP is 293T cells into which FUGW(EF1a)-CD63-GFP plasmid is introduced.

[0098] Figure 2 The figure is the fluorescence observation result of 293T cells into which FUGW(EF1a)-GFP and FUGW(EF1a)-CD63-GFP plasmids are introduced.

[0099] Figure 3Figure for WB detection of different exosome protein expression graphs. M is marker, control group is 293T cell produced exosome, EF1a-GFP is 293T cell produced exosome introduced FUGW(EF1a)-GFP plasmid, Ubc-CD63-GFP is 293T cell produced exosome introduced FUGW(Ubc)-CD63-GFP plasmid, EF1a-CD63-GFP is 293T cell produced exosome introduced FUGW(EF1a)-CD63-GFP plasmid.

[0100] Figure 4 Figure for WB detection of engineered exosome VEGF expression.

[0101] Figure 5 Comparison figure for animal level verification of the effect of engineered exosome germination. DETAILED DESCRIPTION

[0102] In order to make the technical means, creative features, purposes and effects realized by the present application easy to understand, the following specific embodiments are further illustrated, but the following embodiments are only preferred embodiments of the present application, not all. Based on the embodiments in the embodiments, other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application. In the following examples, if not otherwise specified, the operation method used is a conventional operation method, the equipment used is a conventional equipment, and the equipment and materials used in each example are the same.

[0103] Example 1

[0104] 1 Vector construction

[0105] GFP and CD63-GFP were constructed into FUGW vector by genetic engineering technology, and the Ubc promoter in FUGW vector was replaced by EF1a, and four kinds of vectors were constructed, namely FUGW(Ubc)-GFP (SEQ ID NO: 1), FUGW(Ubc)-CD63-GFP (SEQ ID NO: 2), FUGW(EF1a)-GFP (SEQ ID NO: 3), FUGW(EF1a)-CD63-GFP (SEQ ID NO: 4).

[0106] 2 Obtaining of engineered exosome carrying GFP

[0107] (1) 293T cell recovery: One week in advance, take one vial of frozen 293T cells, quickly put into 37°C water bath, shake quickly until the cells melt. After disinfecting the outer surface of the frozen tube with 75% alcohol, transfer to the biosafety cabinet, use a 1 mL pipette to transfer the cells to a 15 mL centrifuge tube containing 5 mL of pre-added adherent 293T cell culture medium (DMEM basic containing 10% FBS). After centrifugation at room temperature at 800 rpm for 5 min, discard the supernatant, add 1 mL of adherent 293T cell culture medium to resuspend the cells, take 300 μL of cells and inoculate into a T25 cell culture bottle containing 5 mL of pre-added adherent 293T cell culture medium, and transfer to a 37°C, 5% CO2 cell incubator for culture.

[0108] (2) 293T cell passage: After 2 days of culture, when the cell density is greater than 80%, discard the culture medium, wash with 1 mL of PBS, repeat once, add 1 mL of trypsin and incubate at room temperature for 15 s, add 2 mL of adherent 293T cell culture medium to terminate digestion, transfer 3 mL of cell suspension to a 15 mL centrifuge tube. After centrifugation at room temperature at 800 rpm for 5 min, discard the supernatant, add 1 mL of adherent 293T cell culture medium to resuspend the cells, and take 100 μL of cell suspension for counting. According to the counting result, inoculate 1*106 cells into a T182 cell culture bottle containing 30 mL of pre-added adherent 293T cell culture medium, and transfer to a 37°C, 5% CO2 cell incubator for culture.

[0109] (3) 293T cell plating: After 2 days of culture, when the cell density is greater than 80%, discard the culture medium, wash with PBS, add trypsin for digestion, add culture medium to terminate digestion, centrifuge and discard the supernatant, add culture medium to resuspend the cells, take 100 μL of cell suspension, count. According to the counting result, inoculate 2*10 6 cells into a 10 cm dish containing 10 mL of pre-added adherent 293T cell culture medium, and transfer to a 37°C, 5% CO2 cell incubator for culture.

[0110] (4) Transfection: After 1 day of culture, when the cell density was 80%, the supernatant was discarded, and 15 mL of adherent 293T cell culture medium was added. When transfecting, 8 μg of the constructed plasmid was taken and added to 500 μL of OptiMEM, labeled as tube A; 20 μg of PEIpro (1:2.5 ratio of plasmid mass, i.e., 20 μL of 1 mg / mL stock solution) was added to 500 μL of OptiMEM, labeled as tube B. After 5 min of room temperature standing of tube A and tube B, the contents of tube A and tube B were mixed gently, the mixed solution was allowed to stand at room temperature for 15 min, then it was evenly dropped into a 10 cm culture dish, and the culture dish was moved crosswise to mix the solution with the culture medium. The culture was carried out in a 37°C, 5% CO2 cell incubator. From the transfection to the recovery of the culture supernatant, the culture medium was not changed.

[0111] (5) Exosome purification (note: operation on ice): After 36-48 h of culture, the supernatant of each dish was collected into a 50 mL centrifuge tube (the supernatants of two dishes can be combined, i.e., 30 mL), placed on ice, and transferred to a cell-free place, centrifuged at 2000 g for 20 min at 4°C, and then the supernatant was collected into a new 50 mL centrifuge tube. After centrifugation at 10000 g for 60 min at 4°C, the supernatant was collected, filtered through a 0.22 μm filter membrane into a new 50 mL centrifuge tube. 50% PEG 8000 was added to the filtrate to a final concentration of 10%, mixed well, and allowed to stand at 4°C overnight. The next day, the treatment solution was centrifuged at 10000 g for 60 min at 4°C to collect the exosomes. After centrifugation was completed, a yellowish-white precipitate was visible on the wall of the centrifuge tube. The supernatant was removed, and after the residual supernatant was removed using a pipette, 200 μL of PBS was added to resuspend the precipitate, labeled with the name and date, and stored at -80°C. After resuspension, green fluorescence can be seen under a fluorescence microscope; 40 μL of the sample can also be taken, and the RFU value should be higher than that of the PBS control group when detected at an excitation wavelength of 485 nm and an emission wavelength of 528 nm on a multifunctional enzyme labeler.

[0112] 3. Results verification of exosomes carrying GFP

[0113] 3.1 mRNA level verification of expression of the target gene and effect of different promoters on expression yield of the target gene

[0114] Different plasmids were transfected into 293T cells, and after 36 h of culture, the total RNA of the cells was extracted using a FastPure® Cell / Tissue Total RNA Isolation Kit V2 (Vazyme, Catalog No.: RC112-01) kit. The total RNA extraction steps were as follows: the 293T cells were digested with trypsin, centrifuged at 1000 rpm for 5 min at room temperature, and the cells were collected; 3 x 10 6Cells were treated with 500 μl of Buffer RL and vortexed until no obvious cell clumps were observed. Subsequent steps were performed according to the "08-2 / RNA Extraction" procedure in the instruction manual. Finally, 30-50 μl of RNase-free ddH2O was added as needed. The RNA concentration was measured to be 777.7 - 1454.7 ng / μl; OD260 / OD280: 2.12 - 2.14. The cells were stored at -80℃.

[0115] The transcriptional amount of the target gene relative to different plasmids was detected by qPCR using primers for detecting the CD63 gene. The experimental steps are as follows: Kit: 2×RealStar Fast SYBR qPCR Mix (UNG) (Genstar, catalog number: A302);

[0116] Procedure: Refer to the instruction manual and add the corresponding component in a 20 μl system. Make 3 replicates for each sample. When adding the Mix, be careful to avoid light.

[0117] qPCR reaction procedure:

[0118] Table 1 qPCR reaction procedure

[0119]

[0120] Table 2 Related Primers

[0121]

[0122] Specific results are as follows Figure 1 As shown, from Figure 1 It can be seen that the introduced target gene was successfully transcribed, and the EF1a promoter was stronger than the Ubc promoter.

[0123] 3.2 Fluorescence microscopy observation of CD63-GFP fusion protein expression in 293T cells

[0124] CD63 is a marker protein on the surface of exosomes, belonging to the tetraspanic membrane protein superfamily. If CD63-GFP is successfully expressed in 293T cells, fluorescence will be observed around the cell membrane under a fluorescence microscope, while the cytoplasm will not show fluorescence. By comparing the fluorescence results of 293T cells containing FUGW(EF1a)-GFP and FUGW(EF1a)-CD63-GFP plasmids respectively, it was found that the CD63-GFP fusion protein was successfully expressed on the surface of the 293T cell membrane. Figure 2 ).

[0125] 3.3 Western blotting validation of engineered exosomes

[0126] CD63 is a marker protein of exosomes. If the exosomes successfully contain the GFP target protein, the corresponding band can be observed by WB. The WB experiment results show that the obtained exosomes contain the GFP target protein. Figure 3 As can be seen from the figure, exosomes containing GFP are successfully obtained, and the EF1a promoter is stronger than the Ubc promoter.

[0127] The specific process of WB detection is as follows:

[0128] Glue preparation: clamp two clean glass plates, fix them in the support, prepare 10% separation glue solution, add 5-7.5 ml of glue solution between the two glass plates, press the glue with anhydrous ethanol, after 30 min, pour off the ethanol, and use filter paper to absorb the residual ethanol between the glass plates; then add 2-3 ml of concentrated glue solution, then insert the 10-hole comb between the glass plates, wait for 30-60 min until the protein gel is solidified, take out the glass plates, soak them in pure water, and store them at 4°C.

[0129] Table 3 Composition of concentrated glue and separation glue

[0130]

[0131] Take 200 μl of exosomes, add RIPA (containing a final concentration of 1 mM phenylmethylsulfonyl fluoride (PMSF)) at a ratio of 1:1, lyse on ice for 20 min, mix 3 times during the period; centrifuge (13000 g, 5 min, 4°C), take the supernatant, and determine the total protein concentration by BCA;

[0132] WB sample preparation: take 80 μl of supernatant after RIPA lysis, add 20 μl of 5x loading buffer (without 2-mercaptoethanol), then 100°C, 10 min;

[0133] Loading: load 15 μl of marker. After BCA quantification, the total protein amount is 200-300 μg, and the volume is 30-35 μl; if multiple samples are run together, equal amounts and equal volumes should also be noted;

[0134] For example, sample B is 5 mg / ml, and sample C is 3 mg / ml. After mixing the sample with 5x loading buffer and heating at 100°C for 10 min, take 3.75 μl of sample B, mix with 6.25 μl of 1x loading buffer; take 6.25 μl of sample C, mix with 3.75 μl of 1x loading buffer, which can meet the requirements of "total protein amount of 15 μg, volume of 10 μl";

[0135] Running: Place the glass plate containing the protein gel into the electrophoresis tank, pour 1x running buffer into the tank, set 100 V constant voltage for 100-120 min. When the bromophenol blue migrates to the bottom of the gel, stop the electrophoresis;

[0136] Transferring: Open the mold, separate the black and white sides, place the filter paper on the negative side (black) of the mold, and place it in the transfer solution. Take out the protein gel, cut off the concentrated gel and excess separation gel, and place it on the filter paper on the negative side (black) of the mold. Remove the bubbles. Soak the PVDF membrane in methanol for 15 seconds, then take it out and place it above the gel. Remove the bubbles, place the filter paper on top, remove the bubbles again, clamp the mold, and apply a constant current of 300 mA for 120 min.

[0137] Blocking and washing: Soak the transferred PVDF membrane in 5% blocking solution at room temperature on a shaker at 60 rpm for 60 min. Then wash the membrane with TBST at room temperature on a shaker at 100 rpm for 5 min.

[0138] Primary antibody (CD63 Antibody (MX-49.129.5) (mouse origin)) incubation and washing: Place the PVDF membrane in a container, add 10 ml of diluted antibody, and incubate overnight at 4°C on a shaker at 60 rpm. The next day, recover the primary antibody and wash the membrane with TBST at room temperature on a shaker at 100 rpm for 10 min.

[0139] Secondary antibody (HRP-labeled goat anti-mouse IgG (H+L)) incubation and washing: Place the PVDF membrane in a container, add 10 ml of diluted antibody, and incubate at room temperature on a shaker at 60 rpm for 60 min. Recover the secondary antibody and wash the membrane with TBST at room temperature on a shaker at 100 rpm for 10 min.

[0140] Development: Mix the two liquids in the development reagent kit according to the 1:1 ratio (500 μl:500 μl), evenly drop them onto the membrane, and place it in a multifunctional imager for development and photography.

[0141] 4. Obtaining of engineered exosomes carrying VEGF

[0142] According to the method described in step 1, use genetic engineering technology to construct the VEGF gene into the determined expression vector, i.e. FUGW (EF1a)-CD63-VEGF (SEQ ID NO: 5). Perform lentivirus packaging according to the following steps:

[0143] 4.1 Lentivirus packaging

[0144] 4.1.1 293T cell recovery: Take one vial of 293T cells, quickly put into 37 °C water bath, shake quickly until the cells melt. After disinfecting the outside of the cryopreservation tube with 75% alcohol, transfer to the biosafety cabinet, and use a 1 ml pipette to transfer the cells to a 15 ml centrifuge tube containing 5 ml of pre-added adherent 293T cell culture medium (DMEM medium containing 10% FBS). After centrifugation at room temperature at 800 rpm for 5 min, discard the supernatant, add 1 ml of adherent 293T cell culture medium to resuspend the cells, and take 500 μl of cells to inoculate a T75 cell culture bottle containing 12 ml of pre-added adherent 293T cell culture medium, and transfer to a 37 °C, 5% CO2 cell incubator for culture.

[0145] 4.1.2 293T cell passage: When the cell confluence is greater than 80% (24-48 h), discard the culture medium, wash with 3 ml of PBS, repeat once, add 2 ml of trypsin for 15 s of room temperature digestion, add 2 ml of adherent 293T cell culture medium to terminate digestion, and transfer 4 ml of cell suspension to a 15 ml centrifuge tube. After centrifugation at room temperature at 800 rpm for 5 min, discard the supernatant, add 1 ml of adherent 293T cell culture medium to resuspend the cells, and take 100 μl of cell suspension for counting. According to the counting result, inoculate 1*10 6 cells into a T182 cell culture bottle containing 25 ml of pre-added adherent 293T cell culture medium, and transfer to a 37 °C, 5% CO2 cell incubator for culture.

[0146] 4.1.3 293T cell plating: When the cell confluence is greater than 80% (24-48 h), discard the culture medium, refer to 4.1.2, add PBS for washing, add trypsin for digestion, add medium to terminate digestion, centrifuge and discard the supernatant, add medium to resuspend the cells, take 100 μl of cell suspension, and count. According to the counting result, inoculate 4*10 6 cells into a 10 cm dish containing 10 ml of pre-added adherent 293T cell culture medium, and prepare the same amount for 4 dishes, and transfer to a 37 °C, 5% CO2 cell incubator for culture.

[0147] 4.1.4 Packaging of lentiviral plasmid: After 24 h of plating, when the cell confluence is greater than 80%, perform the following operations for each dish.

[0148] The transfection operation is performed according to the plasmid ratio of FUGW-EFla-CD63-VEGF: pLP1-KANA: pLP2-KANA: PVSVG-KANA = 2:1:1:1 (w / w / w / w). The total amount of plasmid required for each 10 cm culture dish is 25 μg (the specific volume of each plasmid added to each dish is calculated according to the plasmid ratio and concentration), added to 500 μl of OptiMEM, labeled as tube A; PEI 50 μg (1:2 ratio with the total amount of plasmid required, i.e. 50 μl of 1 mg / ml stock solution), added to 500 μl of OptiMEM, labeled as tube B. After 5 minutes of incubation at room temperature, the solution in tube A is added to tube B and mixed gently, the mixture is incubated at room temperature for 15 minutes, then it is added dropwise to the 10 cm culture dish, and the dish is moved crosswise to mix the solution with the culture medium, and then incubated at 37°C in a 5% CO2 cell incubator. The culture medium can not be changed from plating to recovery of the culture supernatant, and the specific determination is based on the cell state (if the medium needs to be changed, it is changed after 24 hours of plating and before the transfection operation).

[0149] 4.1.5.1 Virus preservation: The concentrated virus solution can be stored at 4°C for 3 days or at -80°C.

[0150] 4.1.5.1 Virus preservation: The concentrated virus solution can be stored at 4°C for 3 days or at -80°C.

[0151] 4.1.6 Human umbilical cord mesenchymal stem cells (hUC-MSC) lentivirus infection, culture supernatant collection (P3 recovery, P4 infection, P5 screening verification, P6-P10 supernatant collection).

[0152] 4.1.6.1 Puromycin concentration: The final concentration of puromycin is 2 μg / ml.

[0153] 4.1.6.2 Lentivirus infection: 1 ml of virus concentrated solution is used to infect 6*10 6hUC-MSC cells, as follows. Referring to 4.1.1, thaw and passage hUC-MSCs, and plate 3*10 6 cells / 10 ml / 10 cm dish (2 dishes total), and passage the cells into 10 cm dishes with 10 ml of 3D FloTrix Mesenchymal Stem Cell Serum-Free Medium added in advance, and, for each dish, 500 μΐ of concentrated virus solution, and a final concentration of 8 μg / ml of the infection-promoting agent Polybrene (10 mg / ml stock to 8 μΐ per dish). After mixing evenly by crosswise movement, incubate at 37°C in a 5% CO2cell culture incubator for 24-48 h until the cells reach 80% confluence.

[0154] 4.1.6.3 Puromycin selection: Referring to 4.1.2, collect the cells from the 2 dishes and combine and passage into 1 T182 flask, and, at the time of passage, add to the medium a final concentration of 2 μg / ml of puromycin (e.g., 25 ml of medium, plus 50 μΐ of 1 mg / ml puromycin), and transfer to a 37°C, 5% CO2cell culture incubator for 48 h until the cells reach approximately 50% confluence, at which time the hUC-MSCs should be at or below passage 6, and discard the supernatant from this round of culture.

[0155] 4.1.6.4 Passage and collection of culture supernatant: Passage the cells from the previous step of puro selection into 1 new T182 flask, without adding puromycin, and passage at a ratio of 1:2, and incubate each passage for 48-72 h, and collect the culture supernatant from passage 6 (20 ml), passage 7 (40 ml), passage 8 (80 ml), passage 9 (160 ml), and passage 10 (320 ml), and centrifuge at 4°C, 3000 g, for 20 min, and retain the supernatant, and store at -20°C.

[0156] 4.1.7 Exosome purification (note: perform on ice): After 36-48 h of incubation, collect the supernatant from each dish into a 50 mL centrifuge tube, place on ice, and transfer to an extracellular location, and centrifuge at 4°C, 2000 g, for 20 min, and collect the supernatant into a new 50 mL centrifuge tube. Centrifuge at 4°C, 10,000 g, for 60 min, and collect the supernatant, and filter the supernatant through a 0.22 μιη filter into a new 50 mL centrifuge tube. Add 50% PEG 8000 to the filtrate to a final concentration of 10%, and mix, and let stand at 4°C overnight. The next day, centrifuge the treated solution at 4°C, 10,000 g, for 60 min to collect the exosomes. After the centrifugation is complete, a yellow-white precipitate will be visible on the wall of the centrifuge tube, remove the supernatant, and, using a pipette, remove any remaining supernatant, and add 200 μΐ^of PBS to resuspend the precipitate, label with name and date, and store at -80°C.

[0157] The obtained engineered exosomes carrying VEGF were verified in the manner described in step 3, and the results are shown in Figure 4

[0158] 5. Verification of the function of engineered exosomes carrying VEGF

[0159] After obtaining the engineered exosomes carrying VEGF, animal experiments were carried out to verify whether they have better hair growth effect, and the verification was carried out according to the following experimental steps:

[0160] 5.1 Experimental grouping

[0161] C57BL6 mice (6 weeks old) were randomly grouped after 3 days of adaptation, and photographed (before hair removal). The specific grouping is shown in Table 4 below:

[0162] Table 4 Experimental grouping

[0163]

[0164] 5.2 Hair removal: After the back was shaved after anesthesia, hair removal cream was used for 4 minutes, then the hair was removed, and hair removal cream was used for another 2 minutes, then the back hair was completely removed. The hair removal area was 0.5 cm behind the ear to 1 cm in front of the tail, about 4 cm long and 3 cm wide. Photographed (after hair removal);

[0165] 5.3 Drug administration once: 50 μl of exosomes was injected subcutaneously at 4 points on the back with a 1 ml syringe, a total of 200 μl, 1.2 × 10 9 particles;

[0166] 5.4 Observation: Regular observation before and after exosome administration. When taking pictures each time, the white background, the height of the camera, the focused light source, the direction of the light source and the brightness were the same. The experimental results are shown in Figure 5 From the figure, it can be seen that the engineered exosomes carrying VEGF can significantly promote hair growth.

[0167] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.​

Claims

1. The application of engineered exosomes in the preparation of products that promote hair growth, characterized in that, The method for preparing engineered exosomes includes a vector construction step, in which CD63-VEGF is constructed into the FUGW vector and the Ubc promoter in the FUGW vector is replaced with EF1a to obtain FUGW(EF1a)-CD63-VEGF. The sequence of FUGW(EF1a)-CD63-VEGF is shown in SEQ ID NO:5.

Images