A silk-affinity engineered exosome and its construction method and functional application

Abstract

The present invention provides a silk fibroin affinity engineered exosome, its construction method, and functional application, belonging to the field of biomedical materials and cell molecular therapy technology. The present invention obtains a silk fibroin affinity peptide with strong affinity for silk fibroin through multiple rounds of screening. Fusion expression of the silk fibroin affinity peptide in the exosome membrane protein is beneficial for enhancing the binding performance of the exosome and the affinity protein. At the same time, the exosome membrane protein can efficiently capture a large number of specific miRNAs by fusion expression of the MS2 domain, thereby exerting a specific functional role. After the silk fibroin protein is combined with the silk fibroin affinity engineered exosome, the stability of the exosome and its internalization effect in the receptor cells are improved, providing a research foundation for the application of biomolecular active materials in the field of tissue repair and regenerative medicine, and providing theoretical and technical support for the efficient and precise treatment of tissue damage repair in clinical practice.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials and cell molecule therapy, and specifically relates to a silk fibroin affinity engineered exosome, a construction method thereof, and functional applications. Background Art

[0002] With the rapid development of cell-based molecular therapy technologies, exosome therapy has become a hot topic in biomedical research and has garnered widespread attention. Exosomes, a class of extracellular vesicles, serve as transport vehicles, carrying specific intracellular components such as proteins and RNA. By fusing to the target cell membrane or internalizing them through endocytosis, they deliver active factors directly into the target cell, exerting effective regulatory effects on recipient tissues. Compared to stem cell therapy, stem cell-derived exosomes not only contain multiple active ingredients secreted by stem cells but also avoid transient rejection reactions caused by xenogeneic cell surface antigens, making them safer for clinical use. Currently, the use of engineered exosomes as carriers for active ingredients such as specific genes and active drugs to create functional exosomes with specific regulatory effects has become a hot topic and a development direction for exosome therapy. However, functional exosomes require an effective sustained-release storage model to maintain their regulatory effects.

[0003] In clinical treatment, the route of administration is directly linked to therapeutic efficacy. If a drug fails to effectively and sustainably act on the body, the therapeutic effect is often significantly compromised. Exosomes have a short half-life, making them difficult to maintain in vivo. However, biomaterials, as carriers of active molecules, offer unlimited possibilities for exosome-based therapeutic approaches. The combination of functional exosomes and biomaterials for the sustained delivery of targeted regulatory molecules holds promise for precision therapy in various wound repair applications. Exosome-incorporated bioactive materials have already been applied in the repair and treatment of injuries to skin, cardiovascular, bone, and cartilage tissues. Among tissue repair and regeneration biomaterials, silk fibroin, as a natural protein component, possesses antimicrobial properties, preventing pathogen invasion and proliferation, thereby reducing the risk of infection. Its excellent biocompatibility, degradability, and mechanical properties make it a promising biomaterial for tissue repair and regeneration applications. Notably, the exosome loading capacity of silk fibroin materials may be closely related to the exosome binding efficiency of the biomaterial. Enhancing efficient exosome-silk binding and sustained release is a key technical challenge in the construction of silk-affinity engineered exosomes. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a silk fibroin affinity engineered exosome and a construction method thereof, so that the constructed silk fibroin affinity engineered exosome has efficient binding and sustained release properties in silk fibroin protein.

[0005] The present invention also aims to provide a functional exosome combined with silk fibroin, which has a sustained internalization effect and good storage stability and in vivo stability, thereby effectively improving the therapeutic effect of the active ingredients of the exosomes.

[0006] The present invention provides a silk fibroin affinity engineered exosome, wherein the membrane protein of the exosome is a membrane protein Lactadherin fused with a silk fibroin binding peptide;

[0007] The silk fibroin binding peptide is inserted between the SP and C1C2 domains of the membrane protein Lactadherin;

[0008] The amino acid sequence of the silk fibroin binding peptide is shown in SEQ ID NO: 1.

[0009] Preferably, the amino acid sequence of the membrane protein fused with the silk fibroin binding peptide is as shown in SEQ ID NO: 3.

[0010] Preferably, the exosomes are loaded with miR146a; the membrane protein of the exosomes is also fused to express the MS2 domain;

[0011] The MS2 domain is expressed at the C-terminus of the C1C2 domain in the membrane protein Lactadherin;

[0012] The amino acid sequence of the MS2 domain is shown in SEQ ID NO: 2.

[0013] The present invention provides a method for constructing the silk fibroin affinity engineered exosomes, comprising the following steps:

[0014] 1) constructing a lentiviral plasmid containing a coding sequence for a membrane protein fused with a silk fibroin binding peptide;

[0015] 2) transfecting cells with the lentiviral plasmid containing the coding sequence of the membrane protein fused with the silk fibroin binding peptide described in step 1), and collecting the packaged lentiviral particles;

[0016] 3) Infecting cells with the packaged lentiviral particles described in step 2), culturing the positive cells obtained after screening, collecting the supernatant, and isolating the silk fibroin affinity engineered exosomes.

[0017] Preferably, the nucleotide sequence of the coding sequence of the membrane protein fused with the silk fibroin binding peptide in step 1) is as shown in SEQ ID NO: 4.

[0018] Preferably, the transfection method in step 2) is to mix preparation 1 and preparation 2, add them to cells incubated in serum-free medium, replace them with serum medium after 4 to 6 hours, and continue to culture for 48 to 72 hours;

[0019] The preparation 1 is Opti-MEM culture medium containing Lipo2000; the volume ratio of Opti-MEM to Lipo2000 is 250:10;

[0020] The preparation 2 is an Opti-MEM culture medium containing miR146a plasmid and MS2 plasmid; the mass ratio of the Opti-MEM, miR146a plasmid and MS2 plasmid is 250:2:2.

[0021] Preferably, the nucleotide sequence of the coding sequence of the membrane protein fused with the silk fibroin binding peptide and the MS2 domain is shown in SEQ ID NO: 5;

[0022] When the exosomes are loaded with miR146a, the lentiviral plasmid containing the coding sequence of the membrane protein fused with the silk fibroin binding peptide and the lentiviral plasmid containing the miR146a precursor sequence are co-transfected into cells.

[0023] The present invention provides a functional exosome combined with silk fibroin, which is a complex formed by combining the silk fibroin affinity engineered exosome or the silk fibroin affinity engineered exosome obtained by the construction method and silk fibroin;

[0024] The mass ratio of the silk fibroin affinity engineered exosomes to silk fibroin protein is approximately 1:10.

[0025] The present invention provides the use of the silk fibroin affinity engineered exosomes or the functional exosomes bound to silk fibroin in tissue repair and regeneration.

[0026] Preferably, the tissue repair and regeneration includes one or more of the following tissue damage repairs: skin tissue damage repair and regeneration, cardiovascular tissue repair and regeneration, bone tissue repair and regeneration, and cartilage tissue repair and regeneration.

[0027] The present invention provides a silk fibroin affinity engineered exosome (SGM-Exo). The present invention obtains a silk fibroin affinity peptide with strong affinity to silk fibroin through multiple screening. Silk fibroin binding peptide is designed based on the silk fibroin affinity peptide, and the silk fibroin binding peptide is fused and expressed in exosome membrane protein, thereby enhancing the binding efficiency and sustained release of exosomes and silk fibroin, providing a research basis for the preparation of functional drugs using exosomes as active molecule carriers.

[0028] Furthermore, the C-terminus of the membrane protein of the exosomes of the present invention is also fused with the MS2 domain, which can efficiently capture a large amount of miR146a through the pac sites at both ends of miR146a. MiR146a acts as an active ingredient in the drug and plays a role in inhibiting inflammatory response.

[0029] The present invention provides a functional exosome bound to silk fibroin (SGM-Exo@SFP), which is a complex formed by the combination of the silk fibroin affinity engineered exosomes or the silk fibroin affinity engineered exosomes obtained by the construction method and silk fibroin. In the experiment of the present invention, the silk fibroin patch extract bound to functional exosomes was subjected to HaCat cells. The results showed that the functional exosomes can be continuously internalized and act on the recipient cells. At the same time, the stability of SGM-Exo@SFP was evaluated from three aspects: in vitro storage, in vivo application, and miRNA. The results showed that the storage stability of SGM-Exo@SFP was significantly improved regardless of whether it was stored at room temperature or low temperature; in vivo application experiments showed that the sustained release and activity stability of the silk fibroin affinity engineered exosomes after binding to silk fibroin were improved; at the same time, the expression level of the basic miR146a contained in the silk fibroin affinity engineered exosomes was more stable, thereby improving the application effect of the functional exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram for the construction of silk fibroin affinity-engineered exosomes;

[0031] Figure 2 The results are the internalization and stability of the binding rate of silk fibroin-affinity exosomes to silk fibroin;

[0032] Figure 3 This is the result of silk affinity engineered exosomes being released from SFP and then internalized into HaCaT cells;

[0033] Figure 4 Figure 1 shows the stability evaluation results of the SGM-Exo@SFP prepared in this invention. A shows that SFP enhanced the stability of SGM-Exos at 37°C; B shows that SFP enhanced the stability of SGM-Exos at 4°C; C shows that SFP-loaded SGM-Exos retained longer in vivo than the SGM-Exo group alone. Signal activity is expressed as photons / s / cm2 / steradian (sr); data represent the mean ± SD of six different experiments (**p < 0.01, ***p < 0.001).

[0034] Figure 5 The changes in miR146a expression in SGM-Exo@SFP. DETAILED DESCRIPTION

[0035] The present invention provides a silk fibroin affinity engineered exosome, wherein the membrane protein of the exosome is the membrane protein Lactadherin fused with a silk fibroin binding peptide; the silk fibroin affinity peptide is inserted between the SP and C1C2 domains of the membrane protein Lactadherin, and the amino acid sequence of the silk fibroin binding peptide is shown in SEQ ID NO: 1 (LSLSPGHFSFVDLSLSPGHFSFVDLSLSPGHFSFVD).

[0036] In the present invention, the silk fibroin binding peptide is obtained by three-fold repetition in series on the basis of a silk fibroin affinity peptide. The silk fibroin affinity peptide is preferably obtained by screening a 12-peptide library phage and verifying it by ELISA. The amino acid sequence of the silk fibroin affinity peptide is preferably SEQ ID NO: 11. The silk fibroin affinity peptide has a significant silk fibroin binding activity compared to other screened silk fibroin affinity peptides (amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10). The amino acid sequence of the membrane protein fused with the silk affinity peptide is preferably as shown in SEQ ID NO: 3 (MPRPRLLAALCGALLCAPSLLVALSLSPGHFSFVDLSL SPGHFSFVDLSLSPGHFSFVDTKCVEPLGLENGNIANSQIAASSVRVTFLGLQHWVPELARLNRAGMVNAWTPSSNDDNPWIQVNLLRRMWVTGVVTQGASRLASHEYLKAFKLAYSLNGHEFDFIHDVNKKHKEFVGNWNKNAVHVNLFETPVEAQYVRLYPTSCHTACTLRFELLGCELNGCANPLGLKNNSIPDKQITASSSYKTWGLHLFSWNPSYARLDKQGNFNAWVAGSYGNDQWLQVDLGSSKEVTGIITQGAPNFGSVQFVASYKVAYSNDSANWTEYQDPRTGSSKIFPGNWDNHSHKKNLFETPILARYVRILPVAWHNRIALRLELLGC).

[0037] In the present invention, the exosomes contain miR146a. The exosome membrane protein also expresses a fused MS2 domain. The MS2 domain is expressed at the C-terminus of the C1C2 domain of the membrane protein lactadherin. The amino acid sequence of the MS2 domain is shown in SEQ ID NO: 2 (ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSV RQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNS DCELIVKAMQGLLKDGNPIPSAIAANSGIY). The fusion expression of the bacteriophage MS2 capsid protein and the membrane protein allows for expression on the surface of the engineered exosome membrane. Since a specific miRNA (miR146a) has two Pac sites upstream and downstream, the MS2 domain can recognize the Pac sites and efficiently bind and capture miR146a, thereby encapsulating a large amount of miR146a in the expressed exosomes and endowing the engineered exosomes with specific anti-inflammatory regulatory functions.

[0038] In the present invention, in order to detect the expression and cellular internalization of the subsequently constructed engineered exosomes, when the membrane protein is fused and expressed, a reporter gene is preferably also fused and expressed. In an embodiment of the present invention, the reporter gene is preferably Gaussia luciferase (Gluc) luciferase.

[0039] The present invention provides a method for constructing the silk fibroin affinity engineered exosomes, comprising the following steps:

[0040] 1) Constructing a lentiviral plasmid containing the coding sequence of a membrane protein fused with a silk fibroin binding peptide and an MS2 domain;

[0041] 2) co-transfecting cells with the lentiviral plasmid containing the coding sequence of the membrane protein fused with the silk fibroin binding peptide and the MS2 domain and the lentiviral plasmid containing the miR146a precursor sequence described in step 1), and collecting the packaged lentiviral particles;

[0042] 3) Infecting cells with the packaged lentiviral particles described in step 2), culturing the positive cells obtained after screening, collecting the supernatant, and isolating the silk fibroin affinity engineered exosomes.

[0043] The invention constructs a lentiviral plasmid containing a coding sequence for expressing a membrane protein fused with a silk fibroin binding peptide and an MS2 structural domain.

[0044]

[0045]

[0046] In the present invention, the method for constructing a lentiviral plasmid containing the coding sequence of a membrane protein fused to a silk fibroin binding peptide (SFBP) and an MS2 domain preferably comprises the following steps: constructing a recombinant vector containing an exogenous gene based on the target SFBP and MS2 coding sequences; verifying the correctness of the recombinant plasmid by sequencing; and further extracting and purifying high-quality, endotoxin-free SFBP and MS2 recombinant plasmids. After obtaining the lentiviral plasmid containing the coding sequence of a membrane protein fused to a silk fibroin affinity peptide and an MS2 domain, the present invention co-transfects cells with the lentiviral plasmid containing the coding sequence of a membrane protein fused to a silk fibroin affinity peptide and an MS2 domain and a lentiviral plasmid containing a miR146a precursor sequence, and collecting the packaged lentiviral particles.

[0047] After obtaining the lentiviral plasmid containing the coding sequence of the membrane protein fused with the silk affinity peptide, the present invention transfects cells with the lentiviral plasmid containing the coding sequence of the membrane protein fused with the silk affinity peptide, and collects the packaged lentiviral particles.

[0048] The present invention has no particular limitation on the transfected cells and transfection method, and cells and conventional lentiviral transfection methods well known in the art can be used. In the embodiment of the present invention, the cells are 293T cells.

[0049] In the present invention, when exosomes are loaded with a large amount of miR146a, it is preferred to construct a lentiviral plasmid containing the miR146a precursor sequence. The method for constructing the lentiviral plasmid containing the miR146a precursor sequence preferably comprises constructing a recombinant vector containing pre-miRNA146a based on the target gene sequence (pre-miRNA146a sequence); sequencing to verify the correct recombinant plasmid, and further extracting and purifying a high-quality endotoxin-free pre-miR146a recombinant plasmid.

[0050] In the present invention, the lentiviral plasmid containing the coding sequence of the membrane protein fused with the silk affinity peptide and the MS2 domain and the lentiviral plasmid containing the miR146a precursor sequence are preferably co-transfected into cells, and the packaged lentiviral particles are collected.

[0051] The present invention has no particular limitation on the transfected cells and transfection method, and cells and conventional lentiviral transfection methods well known in the art can be used. In the embodiment of the present invention, the cells are 293T cells.

[0052] After obtaining the packaged lentiviral particles, the present invention infects cells with the packaged lentiviral particles, and the positive cells obtained after screening are cultured, and the supernatant is collected to separate the silk fibroin affinity engineered exosomes.

[0053] In the present invention, the cells are preferably human placenta-derived mesenchymal stem cells (PMSCs). After infection, the infected human placenta-derived mesenchymal stem cells are screened. The method for screening PMSCs preferably comprises replacing the culture medium with fresh medium 6 hours after lentiviral infection, and then replacing the culture medium containing the highest concentration of puromycin 48 hours later. The PMSCs were purchased from Beijing Han's United Biotechnology Co., Ltd.

[0054] In the present invention, the screening medium preferably contains 1 μg / mL puromycin (purchased from Sigma, MO, USA) and is maintained for 14 days, with the resistance medium replaced every other day. Surviving cells are designated as positive cells for subsequent culture. The preferred conditions for culturing the positive cells are: screened engineered PMSCs positive cells are digested and transferred to a new culture dish, where they are continued to be cultured in a medium containing 1 μg / mL puromycin at 37°C, 5% CO2, and passaged.

[0055] In the present invention, the method for collecting the supernatant is preferably centrifugation. The centrifugation speed is preferably 1000 rpm. The centrifugation time is preferably 5 minutes. The method for isolating the silk fibroin affinity-engineered exosomes is preferably using an ultracentrifuge at 100,000 rpm, centrifuging three times for 30 minutes each, and finally mixing with a small amount of PBS to obtain a silk fibroin affinity-engineered exosome solution.

[0056] In the present invention, the binding and release properties of the prepared silk fibroin affinity engineered exosomes with silk fibroin protein were verified. The results showed that the binding rate of the silk fibroin affinity engineered exosomes prepared by the present invention with SFP was 3 times that of the control group (G-Exos). It can be seen that SFBP improves the binding rate and efficiency of SGM-Exos with SFP.

[0057] The present invention provides a functional exosome combined with silk fibroin, which is a complex formed by combining the silk fibroin affinity engineered exosome or the silk fibroin affinity engineered exosome obtained by the construction method and silk fibroin.

[0058] In the present invention, the method for constructing the functional exosomes bound to silk fibroin preferably loads 100 μL of exosome solution containing 100 μg of exosomes onto a silk fibroin patch with a diameter of 10 mm to form the functional exosomes bound to silk fibroin.

[0059] The present invention provides the use of the functional exosomes combined with silk fibroin in the preparation of medicines for tissue repair and regeneration.

[0060] In the present invention, the tissue repair and regeneration preferably includes one or more of the following tissue damage repairs: skin damage repair and regeneration, cardiovascular repair and regeneration, bone tissue repair and regeneration, and cartilage tissue repair and regeneration.

[0061] The following examples provide a detailed description of the silk fibroin affinity engineered exosomes, their construction method, and applications provided by the present invention, but they should not be construed as limiting the scope of protection of the present invention.

[0062] Example 1

[0063] A screening method and verification method for silk fibroin affinity peptides with high affinity to silk fibroin

[0064] 1 Biopanning

[0065] (1) A 10 mg / mL silk fibroin solution was added to a plate as an antigen (Ag) and air-dried at 60°C in a sterile environment to form a film.

[0066] (2) Add 1×10 11 Phages of the 12-peptide library of PDU (NEB commercial peptide library: Ph.D.-12TM PhageDisplay Peptide Library Kit) were incubated at 37°C for 1.5 h.

[0067] (3) Rinse with 0.05% PBST 4 times and PBS twice to remove unbound phages.

[0068] (4) The antigen-bound phages were eluted with glycine-HCl (pH to 2.2) and then neutralized with tris-HCl (pH 9.2) until the pH reached 7.0.

[0069] 2. Detect the titer of eluted phage

[0070] (1) Cultivate Escherichia coli TG1 until the log phase.

[0071] (2) Dilute the eluted phage and mix 10 μL of the dilution with 80 μL of E. coli TG11.

[0072] (3) Incubate at 37°C for 15 minutes, then pour into two YT-IPTG / Xgal plates and incubate the plates upside down at 37°C overnight.

[0073] 3. Amplification of eluted phage

[0074] (1) Add 100 μL of E. coli TG1 to 40 mL of 2YT and shake at 37°C until the OD 600 =0.6.

[0075] (2) Add the eluted product and shake at 37°C for 5 h.

[0076] (3) Centrifuge the supernatant and transfer it to a sterile tube. Add 1 / 5 (v / v) PEG / NaCl to the supernatant. After mixing, place the mixture on ice for 2 hours.

[0077] (4) Centrifuge the pellet with 1 ml of PBS and transfer the supernatant to a sterile tube. This will yield the amplified phage for the next biological screening. Perform titer testing as in step 2.

[0078] Table 1 Screening conditions for each round

[0079]

[0080] Table 2 Biopanning results

[0081]

[0082] After three rounds of bio-panning, phage supernatants were used for phage polyclonal ELISA detection.

[0083] 4. Polyclonal phage ELISA

[0084] The plate was coated with 2 mg / mL antigen and incubated at 60°C to form a film.

[0085] 100 μL of diluted anti-M13-HRP antibody (1:5000, purchased from abcam) was added to each well and incubated at 37°C for 1 hour.

[0086] At room temperature, 100 μL of TMB was added to each well, followed by 100 μL of 2M HCl solution. The plate was read at 450 nm using a microplate reader.

[0087] Table 3 Polyclonal phage ELISA results

[0088]

[0089] According to the results, the second round has the characteristics of fewer rounds of panning and lower enrichment, while too many rounds of panning have high enrichment but reduced phage diversity. The output phages of the second round are selected for single clone screening.

[0090] 5. Monoclonal phage ELISA

[0091] (1) 96 clones were selected from the second round of eluted phage dilution and plated plates and cultured at 37°C with shaking at 220 rpm for 6 hours.

[0092] (2) After centrifugation, the supernatant was collected for ELISA detection.

[0093] Table 4 ELISA test results of antigen group R2P1 output phage

[0094]

[0095] Table 5 ELISA background value detection

[0096]

[0097] Table 6 Monoclonal phage (second round elution phage) ELISA test results (after removing background value)

[0098]

[0099] Clones with ELISA values ​​greater than 0.3 after background removal were selected for sequencing and secondary verification by phage ELISA. After sequencing, eight different correct sequences were obtained, and these clones were re-amplified and secondary verified.

[0100] 6 positive phage clones were verified for the second time.

[0101] The 10 mg / mL antigen-coated plates (test group) were incubated at 60°C to form a film.

[0102] Then, the positive clones were detected again by ELISA.

[0103] 7. Sequence analysis of the selected phage clones.

[0104] Table 7 Results of secondary ELISA verification of positive clones

[0105]

[0106] Table 8 ELISA background value detection

[0107]

[0108] Table 9 Secondary validation results of positive phage ELISA (after removing background value)

[0109]

[0110]

[0111] The amino acid sequences and nucleotide sequences of the six positive clones are shown in Table 10 below.

[0112] Table 106 Sequence information of positive clones

[0113]

[0114] According to the antigen binding results, among the 6 positive clones, 20000063F-R2P1-G8 still showed a positive result (i.e., a positive value) in the second verification after removing the background value. 20000063F-R2P1-G8 was selected as the preferred silk affinity peptide.

[0115] Example 2

[0116] A method for constructing engineered exosomes with silk affinity comprises the following steps:

[0117] 1. Use the screened silk fibroin affinity peptide segment (LSLSPGHFSFVD) to design and synthesize silk fibroin binding peptide (SFBP) fusion protein, thereby constructing a silk fibroin affinity exosome.

[0118] Table 11 Silk fibroin binding peptide (SFBP) sequence

[0119]

[0120] SFBP was inserted between the SP and C1C2 domains of the exosomal membrane protein Lactadherin, and the bioluminescent reporter system Gaussia luciferase (Gluc) luciferase (SEQ ID NO: 20, KPTENNEDFNIVAVASNFATTDLDADRGKLPGKKLPLEVLKEL EANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESAQGGIGE AIVDIPEIPGFKDLEPLEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQRCATFASKIQGQVDKIKGAGGDAS, coding sequence of SEQ ID NO: 21, AAGCCCACCGAGAACAACGAAGACTTCAACATCGGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGA GCTGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACCGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTC The MS2 domain (SEQ ID NO: 2, encoding sequence SEQ ID NO: 19) was fused to the C-terminus of the exosomal membrane protein Lactadherin. There are two pac sites upstream and downstream of the specific miRNA (miR146a). MS2 can efficiently capture miR146a through pac, thereby giving exosomes specific anti-inflammatory regulatory function (see Figure 1 ).

[0121] The recombinant SFBP-Gluc-MS2 lentiviral plasmid (SGM-pLv) and pac-pre-miR146a-pac lentiviral plasmid (miR146a-pLv) were transfected into 293T cells, and the SGM-miR146a-Lv lentiviral particles were collected after packaging. PMSCs were infected with SGM-miR46a-Lv, and then the transduced positive cells (SGM-miR146a-PMSCs) were selected with culture medium containing 1 μg / mL puromycin (Sigma, MO, USA). Silk fibroin affinity engineered exosomes (SGM-miR146a-Exo) were extracted by culturing SGM-miR146a-PMSCs and collecting the supernatant ( Figure 1 ).

[0122] Comparative Example 1

[0123] The Gluc fusion protein was transferred into PMSC cells, the cell supernatant was collected, and the exosomes in the supernatant were extracted to obtain G-Exos.

[0124] Example 3

[0125] Binding efficiency of silk fibroin affinity engineered exosomes (SGM-Exo) in silk fibroin.

[0126] The protein activity of exosomes was monitored by Gaussia luciferase (Gluc) activity.

[0127] Solutions with different SGM-Exo contents (1-32 μg) were added to a silk fibroin patch with a diameter of 10 mm and allowed to interact at 37°C for 10 minutes in a 48-well plate. Unbound exosomes were washed away. After incubation in a 37°C incubator for different periods of time, the exosomes bound to the silk material were released and the supernatant PBS was collected. After the exosomes were fully released into the PBS solution, they were moved to another 48-well plate and analyzed by BLI detection using the Gluc signal. The binding rates of SGM-Exos and G-Exos to the silk fibroin patch (SFP) were compared. The results showed that as the concentration of exosomes increased, the binding rate of SGM-Exos to SFP increased, and the binding rate of G-Exos to SFP also increased. At the same exosome concentration, the binding rate of SGM-Exos to SFP was 3 times that of G-Exos ( Figure 2 ).

[0128] Example 4

[0129] Internalization experiment of silk affinity-engineered exosomes

[0130] The silk protein extract of exosomes with silk affinity (SGM-Exo@SFP) was applied to HaCat cells to study the internalization of exosomes in recipient cells. The internalization of G-Exos was analyzed in HaCat cells. HaCat cells were seeded at 5×10 4 SGM-Exos (100 μg / mL) and SGM-Exo@SFP were added to cells / 24-well plates one day before culture for 1, 3, 6, 9, 12, 18, 24, and 36 h. The cells were washed twice with PBS, and the internalized fluid was analyzed by a luminescence imaging system.

[0131] The results confirmed that SGM-Exo@SFP can be continuously internalized into the recipient cells, providing SGM-Exo( Figure 3 ).

[0132] Example 5

[0133] Stability evaluation of silk fibroin affinity engineered exosomes (SGM-Exo@SFP)

[0134] 1. Active storage stability test

[0135] The stability of the two storage temperatures (room temperature RT and 4 ° C) was examined and it was found that the Gluc signal of the SGM-Exo@SFP group lasted for a longer time than that of the SGM-Exo group at either RT or 4 ° C, which indicated that SFP could improve the stability of SGM-Exos. Figure 4 A and B).

[0136] 2. Evaluating the Stability of SGM-Exos Combined with Silk Fibroin in Vivo

[0137] 100 μg of SGM-Exos loaded with SFP were then implanted subcutaneously in the backs of nude mice. A control group received 100 μg of SGM-Exos without SFP. Images were taken using the IVIS Lumina imaging system 0, 12, 24, 36, 48, and 72 hours after administration.

[0138] BLI data showed that the Gluc signal of both groups of patients was detected within 12 hours. Compared with the rapid decline of the Gluc signal in the SGM-Exo group, the Gluc signal in the SGM-Exo@SFP group remained at a certain level for more than 72 hours ( Figure 4 Middle C).

[0139] SFBP increased the binding rate of SGM-Exos to SFP, and SFP could improve the sustained release and activity stability of silk-affinity engineered exosomes.

[0140] 3. Stability of miRNA in Silk Fibroin-Affinity Engineered Exosomes (SGM-Exo@SFP)

[0141] By measuring the basal expression of miRNAs in silk-affinity engineered exosomes, the stability of miRNAs in silk-affinity engineered exosomes before and after binding to silk protein was compared. MiRNAs were isolated from exosomes and further synthesized into cDNA as templates for quantitative PCR (qPCR) detection. The detection primers and internal reference primer, GAPDH, were commercialized primers from Tiangen and Shanghai Bioengineering (UGAGAACUGAAUUCCAUGGGUU, SEQ ID NO: 22), respectively. The reaction system and procedures were based on the manufacturer's instructions. On a Stratagene MX3005P qPCR system, a total reaction volume of 20 μL was set. miR146a was selected as a representative miRNA for PCR assays, with three replicates per reaction. Three replicates were set up using primers designed for GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (TIANGEN commercialized primers) with the same amount of template as the internal reference gene for normalization. For all qPCR assays, the efficiency of different primer sets was tested by combining samples of all templates used in each experiment and creating a standard curve by serially diluting cDNA. -ΔΔCT The target genes were quantified and analyzed by t test.

[0142] The results showed that the basal expression level of miR146a in the SGM-Exo@SFP group was higher than that in the SGM-Exo group, indicating the protective effect of SGM-Exo@SFP on miRNA ( Figure 5 ).

[0143] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A silk fibroin affinity engineered exosome, characterized in that: The membrane protein of the exosome is Lactadherin, a membrane protein fused with a silk fibroin binding peptide; The silk fibroin binding peptide is inserted between the SP and C1C2 domains of the membrane protein Lactadherin; The amino acid sequence of the silk fibroin binding peptide is shown in SEQ ID NO: 1; The amino acid sequence of the membrane protein fused with the silk fibroin binding peptide is shown in SEQ ID NO:

3.

2. The silk fibroin affinity engineered exosomes according to claim 1, characterized in that The exosomes are loaded with miR146a; the C-terminus of the C1C2 domain in the membrane protein of the exosomes is further fused to express the MS2 domain; The amino acid sequence of the MS2 domain is shown in SEQ ID NO:

2.

3. A method for constructing the silk fibroin affinity engineered exosomes according to claim 1 or 2, characterized in that: The following steps are involved: 1) Constructing a lentiviral plasmid containing the coding sequence of a membrane protein fused with a silk fibroin binding peptide or a membrane protein fused with a silk fibroin binding peptide and an MS2 domain; 2) Transfect cells with the lentiviral plasmid described in step 1) and collect the packaged lentiviral particles; 3) Infecting cells with the lentiviral particles packaged in step 2), culturing the positive cells obtained after screening, collecting the supernatant, and isolating the silk fibroin affinity engineered exosomes; The cells infected by the packaged lentiviral particles are human placenta-derived mesenchymal stem cells.

4. The construction method according to claim 3, characterized in that: The nucleotide sequence of the coding sequence of the membrane protein fused with the silk fibroin binding peptide in step 1) is shown in SEQ ID NO:

4.

5. The construction method according to claim 3, characterized in that: The transfection method in step 2) is to mix Preparation 1 and Preparation 2, add them to cells incubated in serum-free medium, replace them with serum-containing medium after 4-6 hours, and continue to culture for 48-72 hours; The preparation 1 is Opti-MEM culture medium containing Lipo2000; the volume ratio of Opti-MEM to Lipo2000 is 250:10; The preparation 2 is an Opti-MEM culture medium containing a miR146a plasmid and a lentiviral plasmid containing a coding sequence of a membrane protein fused with a silk fibroin binding peptide and an MS2 domain; the mass ratio of the Opti-MEM, miR146a plasmid and the lentiviral plasmid containing a coding sequence of a membrane protein fused with a silk fibroin binding peptide and an MS2 domain is 250:2:

2.

6. The construction method according to claim 5, characterized in that: The nucleotide sequence of the coding sequence of the membrane protein fused with the silk fibroin binding peptide and the MS2 domain is shown in SEQ ID NO:

5.

7. A functional exosome bound to silk fibroin, characterized in that: A complex formed by the silk fibroin affinity engineered exosomes according to claim 1 or 2 and silk fibroin; The mass ratio of the silk fibroin affinity engineered exosomes to the silk fibroin protein is 1:

10.

8. Use of the silk fibroin affinity engineered exosomes according to claim 1 or 2 or the functional exosomes bound to silk fibroin according to claim 7 in the preparation of a drug for tissue repair and regeneration.

9. The application according to claim 8, characterized in that: The tissue repair and regeneration is selected from one or more of the following: skin tissue damage repair and regeneration, cardiovascular tissue repair and regeneration, bone tissue repair and regeneration, and cartilage tissue repair and regeneration.

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