Engineered vesicle carrying mRNA targeting and application of engineered vesicle in improvement of fibroblast functionality

Abstract

The invention relates to the technical field of biology, in particular to an engineered vesicle carrying mRNA targeting and application of the engineered vesicle to improvement of fibroblast functionality. The iRGD polypeptide carried by the engineered vesicle is used as a targeting polypeptide of fibroblasts, specifically targets human dermal fibroblasts and can be combined with rich integrin alpha v and NRP-1 on cell membranes of the fibroblasts, meanwhile, the iRGD polypeptide and exosome surface mask protein emp2b construct a fusion protein, MS2 capsid protein is fused in exosome, and the human dermal fibroblasts can be specifically targeted to the human dermal fibroblasts. According to the present invention, with the combination of the mRNA containing the MS2 stem loop, the more target mRNA can be obtained, such that the more target mRNA can enter the senescent fibroblast in the targeting manner so as to improve the activity of the fibroblast and delay the senescence of the human skin;

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an engineered vesicle carrying mRNA targeting and its application in improving the functionality of fibroblasts. Background Art

[0002] Extracellular vesicles (EVs) are double-membrane spherical structures secreted by cells into the extracellular environment, secreted through endosomal and non-endosomal pathways. Researchers usually divide extracellular vesicles into three categories: exosomes, microvesicles, and apoptotic bodies. The diameter of exosomes is 30-150nm, the diameter of microvesicles is 150-1000nm, and the diameter of apoptotic bodies is 1-5μm. Extracellular vesicles can carry a variety of biological molecules, such as proteins, lipids, nucleic acids, and metabolites, and act as a means of intercellular communication to affect target cells. As an important medium for intercellular communication, EVs have made significant progress in the study of carrying mRNA in recent years. EVs can not only carry proteins, lipids, and metabolites, but also encapsulate and deliver mRNA molecules. This feature makes EVs show broad application prospects in the fields of gene expression regulation, disease diagnosis and treatment. Current studies have shown that the mRNA carried by extracellular vesicles can be translated into functional proteins in receiving cells, thereby affecting the physiological state of receiving cells. Therefore, exploring the potential of extracellular vesicles in carrying mRNA not only promotes the development of basic biological research, but also provides an important scientific basis for the development of new diagnostic and therapeutic methods. Dermal fibroblasts are divided into two subgroups, namely papillary fibroblasts and reticular fibroblasts. Their main function is to synthesize collagen, elastin and proteoglycans to maintain the integrity of the skin and its functions, and participate in wound repair. In the process of skin aging, taking photoaging as an example, ultraviolet rays induce enhanced oxidative stress in the skin, activate TNF-α and NF-κB signaling pathways, upregulate MMP, lead to collagen degradation and aging, and inhibit TGFbeta, which reduces the synthesis of new collagen, leading to skin aging.

[0003] Integrins are a class of transmembrane glycoprotein heterodimers composed of two subunits. Currently, 18 α subunits and 9 β subunits have been identified, which can be combined to form at least 20 different integrins. Integrins mediate the interaction of extracellular matrix proteins through fibronectin and are widely expressed on the surface of most cells in the body. Among them, αvβ3 integrin is one of the most important members of this family. It participates in intercellular regulation, signal transduction and cell movement, plays a key role in wound healing and platelet aggregation, and can recognize and bind to iRGD-containing polypeptides (endogenous RGD peptides). Integrins are mainly expressed in adult bone marrow stromal cells, including fibroblasts, smooth muscle cells and microvascular endothelial cells. iRGD polypeptide is a polypeptide containing an arginine-glycine-aspartic acid sequence, which is the recognition point of integrin αv. After recognizing integrin αv, iRGD polypeptide first hydrolyzes tumor vascular endothelial cells to expose the NRP-1 (Neuropilin-1) binding sequence, thereby mediating the entry of drugs into cells.

[0004] In recent studies, iRGD peptides have been found to have high expression of NRP-1 in fibroblasts and play a role in tumor metastasis. It is concluded that iRGD peptides can be combined with nanocarriers to effectively target cells with integrins, so it has important significance in future therapeutic research.

[0005] The MS2 capsid protein can interact with the structural dynamic region in the RNA of the MS2 phage and bind specifically to the MS stem-loop. Therefore, we can specifically bind and enrich the MS stem-loop on the target RNA with the MS2 capsid protein. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention provides an engineered vesicle carrying mRNA targeting to solve the problems in the prior art of being unable to specifically target fibroblasts and having a low mRNA loading amount.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] An engineered vesicle carrying mRNA targeting, simultaneously carrying iRGD polypeptide, lamp2b membrane protein, and MS2 capsid protein.

[0009] Preferably, the engineered vesicles are capable of safely and efficiently delivering mRNA to recipient cells.

[0010] Preferably, the engineered vesicles are secreted by HEK-293T cells.

[0011] Preferably, the iRGD polypeptide, lamp2b membrane protein and MS2 capsid protein are simultaneously expressed in HEK-293T cells.

[0012] An engineered vesicle carrying mRNA targeting is extracted by the following steps:

[0013] Step (1), constructing a target plasmid;

[0014] Select plasmid vectors: pcDNA3.1-HA-iRGD-10-Lamp2b plasmid and pcDNA3.1-10-Lamp2b-HA plasmid;

[0015] 1.1 Obtain the target gene;

[0016] According to the requirements of the constructed plasmid and the primer design principles, the corresponding primers were designed. The DNA sequences of the plasmid primers and the amino acid sequences of the lamp2b membrane protein, iRGD polypeptide, and MS2 capsid protein are as follows:

[0017] The upstream primer sequence of the pcDNA3.1-HA-iRGD-10-Lamp2b plasmid is shown in SEQ ID NO.1:

[0018] TGTCGTGGTGACAAAGGTCCCGACTGTGGCAGTGGATCTGGATCCGGT GG;

[0019] The downstream primer sequence of the pcDNA3.1-HA-iRGD-10-Lamp2b plasmid is shown in SEQ ID NO.2:

[0020] GGACCTTTGTCACCACGACATCCACTGCCCATAGTCGAGTTACCTGC; the upstream primer sequence of pcDNA3.1-10-Lamp2b-HA plasmid is shown in SEQ ID NO.3:

[0021] GGCAGTGGAGGCAGTGGATCTGGATCCGGTGG;

[0022] The downstream primer sequence of the pcDNA3.1-10-Lamp2b-HA plasmid is shown in SEQ ID NO.4:

[0023] TCCACTGCCTCCACTGCCCATAGTCGAGTTACCTGC;

[0024] The amino acid sequence of lamp2b membrane protein is shown in SEQ ID NO.5:

[0025] MNFTVRYETTNKTYKTVTISDHGTVTYNGSICGDDQNGPKIAVQFGPGFSWIANFTKAASTYSIDSVSFSYNTGDNTTFPDAEDKGILTVDELLAIRIPLNDLFRCSLSTLEKNDVVQHYWDVLVQAFVQNGTVSTNEFLCDKDKTSTVAPTIHTTVPSPTTTPTPKEKPEAGTYSVNNGN DTCLLATMGLQLNITQDKVASVININPNTTHSTGSCRSHTALLRLNSSTIKYLDFVFAVKNENRFYLKEVNISMYLVNGSVFSIANNNLSYWDAPLGSSYMCNKEQTVSVSGAFQINTFDLRVQPFNVTQGKYSTAQECSLDDDTILIPIIVGAGLSGLIIVIVIAYVIGRRKSYAGYQTL;

[0026] The amino acid sequence of the iRGD polypeptide is shown in SEQ ID NO.6:

[0027] CRGDKGPDC;

[0028] The amino acid sequence of MS2 capsid protein is shown in SEQ ID NO.7:

[0029] ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVR QSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCEL IVKAMQGLLKDGNPIPSAIAANSGIY;

[0030] 1.2 Recombination reaction;

[0031] 1.3 Transformation, plating and picking of single clones of the recombination reaction products;

[0032] 1.4 Plasmid extraction;

[0033] Step (2), cell transfection;

[0034] Step (3), vesicle extraction.

[0035] Preferably, an engineered vesicle carrying mRNA targeting as described above is used to improve the functionality of fibroblasts.

[0036] Preferably, the method for improving the functionality of fibroblasts using engineered vesicles carrying mRNA targeting comprises the following steps:

[0037] Step (1), staining the vesicles with a membrane dye PKH67;

[0038] 1.1 Preparation of solution A: Add 150 μL of Diluent C reagent to the vesicle suspension and pipette until mixed evenly;

[0039] 1.2 Preparation of B solution: Take a 5mL sterile EP tube, add 150μL Diluent C reagent, then add 0.6μL PKH26 dye to the sterile EP tube, pipette and beat until mixed evenly;

[0040] 1.3 Use a pipette to draw up solution A and add it to solution B. After pipetting evenly, incubate for 3 minutes to obtain a mixed solution.

[0041] 1.4 Add 300 μL FBS to the mixed solution, pipette evenly and allow to react until no obvious changes occur, then stop staining;

[0042] 1.5 Add 2 mL of 1×PBS solution to resuspend the vesicles, and transfer the resuspended vesicles to a sterile 15 mL ultracentrifuge tube. Rinse the EP tube 2-3 times with 1×PBS solution, collect the rinse solution into the ultracentrifuge tube, and perform ultracentrifugation. After ultracentrifugation, discard the supernatant and retain the green precipitate at the bottom of the ultracentrifuge tube. Resuspend the stained vesicles with 200 μL of 1×PBS solution to obtain PKH67 stained vesicles.

[0043] Step (2) confocal microscopy experiment;

[0044] 2.1 Soak the cell slide in 75% ethanol for 2 hours to sterilize it in advance, take out the slide with tweezers in the clean bench and dry it;

[0045] 2.2 Take a 24-well plate, place the cell slides in the small wells one by one, inoculate the fibroblasts on the cell slides, and set up 3 replicate wells for each group of experiments;

[0046] 2.3 When the cell confluence reaches 70%-80%, discard the culture medium, wash twice with 1× PBS solution, add 500 μL of free complete culture medium to each well, and add 30 μg of PKH67 dye vesicles for co-incubation;

[0047] 2.4 After the incubation, discard the culture medium, wash the cell slides 3 times with 1×PBS solution on a shaker, add 300 μL of 4% tissue cell fixative to each well and keep it in the dark for 30 minutes, and then wash the slides 3 times with 1×PBS solution on a shaker;

[0048] 2.5 Take a glass slide, add 50 μL of anti-fluorescence attenuation mounting medium on it, take out the cell slide with tweezers, and after drying, put the slide with cells facing down on the anti-fluorescence attenuation mounting medium and dry it in the dark;

[0049] 2.6 After the sealing is firm, the uptake of vesicles by fibroblasts is observed using a laser scanning confocal microscope.

[0050] Preferably, the method for improving the functionality of fibroblasts using engineered vesicles carrying mRNA targeting comprises the following steps:

[0051] Step (1), staining the vesicles with a membrane dye PKH67;

[0052] 1.1 Preparation of solution A: Add 150 μL of Diluent C reagent to the vesicle suspension and pipette until mixed evenly;

[0053] 1.2 Preparation of B solution: Take a 5mL sterile EP tube, add 150μL Diluent C reagent, then add 0.6μL PKH26 dye to the sterile EP tube, pipette and beat until mixed evenly;

[0054] 1.3 Use a pipette to draw up solution A and add it to solution B. After pipetting evenly, incubate for 3 minutes to obtain a mixed solution.

[0055] 1.4 Add 300 μL FBS to the mixed solution, pipette evenly and allow to react until no obvious changes occur, then stop staining;

[0056] 1.5 Add 2 mL of 1×PBS solution to resuspend the vesicles, and transfer the resuspended vesicles to a sterile 15 mL ultracentrifuge tube. Rinse the EP tube 2-3 times with 1×PBS solution, collect the rinse solution into the ultracentrifuge tube, and perform ultracentrifugation. After ultracentrifugation, discard the supernatant and retain the green precipitate at the bottom of the ultracentrifuge tube. Resuspend the stained vesicles with 200 μL of 1×PBS solution to obtain PKH67 stained vesicles.

[0057] Step (2), flow cytometry experiment;

[0058] 2.1 Fibroblasts were inoculated into 6-well plates, with 3 replicate wells set up for each group of experiments;

[0059] 2.2 When the cell confluence reaches 70%-80%, discard the medium in each well, rinse the cells twice with 1× PBS solution, add 2 mL of free complete medium to each well with a pipette, and then add 50 μg of PKH67 dye vesicles and incubate;

[0060] 2.3 Discard the cell culture medium, rinse the cells twice with 1× PBS solution, add 1 mL of trypsin to digest for 3 min, add 3 mL of culture medium to terminate digestion, centrifuge at 1000 rpm for 5 min, and then wash twice with 1× PBS solution to obtain cell pellets;

[0061] 2.4 Add 500 μL of 4% tissue cell fixative to each centrifuge tube, keep it away from light for 60 min to fully fix the cells, then add 5 mL of 1×PBS solution to each tube, discard the supernatant after centrifugation, repeat twice, then add 1 mL of 1×PBS solution to resuspend the cells, and use flow cytometry to observe the uptake of vesicles by fibroblasts.

[0062] Compared with the prior art, the present invention has the following beneficial effects:

[0063] Compared to natural vesicles that only carry mRNA, the engineered vesicles of the present invention can specifically and efficiently deliver mRNA to dermal fibroblast receptor cells;

[0064] Compared with natural vesicles that only carry mRNA, the engineered vesicles of the present invention can increase the mRNA loading capacity;

[0065] The engineered vesicles of the present invention are highly safe and will not affect the activity of receptor cells;

[0066] Compared with the vesicles carrying only LAMP2B protein, the engineered vesicles of the present invention can efficiently carry out membrane fusion with fibroblasts and have a higher membrane penetration efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 :The dyed vesicles were co-incubated with fibroblasts and observed using a laser scanning confocal microscope to obtain the uptake of vesicles by iRGD-Exo and blank-Exo fibroblasts after 3h and 6h of co-incubation;

[0068] Figure 2 :After the dyed vesicles were co-incubated with fibroblasts, flow cytometry was used to observe the uptake of vesicles by iRGD-Exo and blank-Exo fibroblasts after 3h and 6h of co-incubation;

[0069] Figure 3 : Figure 3 shows the results of protein immunoblotting of engineered vesicles carrying mRNA targeting extracted in Example 1. Implementation

[0071] The theoretical basis of the engineered extracellular vesicle principle proposed in the present invention is as follows:

[0072] First, the lamp2b-iRGD system in the engineered vesicles. The lamp2b membrane protein is a protein located on the lysosomal membrane. It plays an important role in the function of lysosomes and the degradation of intracellular substances in cells. It is a member of the lamp family. Its main functions include participating in the endocytosis and dissolution of intracellular substances, regulating the membrane structure and stability of lysosomes, and regulating the autophagy process. It is a common exosome membrane protein in exosome experiments and a commonly used exosome protein engineering design platform; iRGD peptide is a bifunctional peptide composed of CendR sequence and RGD sequence. It has multiple functions. It can enter the target cells through the CendR pathway and interact with integrins on the surface of target cells to achieve efficient targeted delivery. In its relationship with fibroblasts, iRGD polypeptide exhibits specific intracellular delivery ability and tissue permeability. iRGD can promote the entry of drugs and therapeutic molecules by binding to integrins on the surface of fibroblasts, thus having potential application prospects in drug delivery systems. Fibroblasts, as important connective tissue cells in the body, play an important role in this process, helping to transport and release drugs into target tissues or cells.

[0073] Secondly, MCP is a stable complex formed by the capsid protein of MS2 bacteriophage combined with a stem-loop with high affinity. MCP is usually used as an RNA tag, which can efficiently capture and enrich MS2 RNA in cells or cell exosomes; MS2 RNA is an RNA domain from Escherichia coli, which forms a stable complex with MCP; MS2 RNA is often used as an RNA tag, which is inserted into the RNA sequence of interest through gene editing technology for RNA tracking, enrichment and positioning; MCP is fused with exosome membrane proteins (such as CD9, CD63, etc.) to display MCP tags on the surface of exosomes; MS2 RNA (through MS2RNA domain tags) can bind to MCP to form a stable complex, thereby helping to study the release mechanism, biological function and application of exosomes as potential biomarkers. Therefore, the target mRNA can be brought to MS2 to specifically enter the vesicles we designed.

[0074] Example 1

[0075] This example discloses a method for extracting engineered vesicles carrying mRNA targeting. The experimental materials used are as follows: pcDNA3.1-GNSTM-3-RVG-10-Lamp2b-HA plasmid was purchased from Miaoling Company, HEK-293T cells were from the cell bank of the Chinese Academy of Sciences (SCSP-502), fetal bovine serum and DMEM culture medium were from Biological Industries (C04001-500 and C11995500), transfection reagent Exfect was from Thermo Fisher (L3000015), penicillin / streptomycin was from Beyotime (03-031-1B), poly-lysine was from Beyotime (P6407), ultrafiltration tube was from MilliporeSigma (UFC9100), DNA polymerase was from Vazyme (P505-d1);

[0076] The method for extracting the engineered vesicles carrying mRNA targeting comprises the following steps:

[0077] Step (1), construct the target plasmid

[0078] Select plasmid vectors: pcDNA3.1-HA-iRGD-10-Lamp2b plasmid and pcDNA3.1-10-Lamp2b-HA plasmid;

[0079] 1.1 Obtaining target genes

[0080] According to the requirements of the constructed plasmid and the primer design principles, the corresponding primers were designed. The DNA sequences of the plasmid primers and the amino acid sequences of the lamp2b membrane protein, iRGD polypeptide, and MS2 capsid protein are as follows:

[0081] The upstream primer sequence of the pcDNA3.1-HA-iRGD-10-Lamp2b plasmid is shown in SEQ ID NO.1:

[0082] TGTCGTGGTGACAAAGGTCCCGACTGTGGCAGTGGATCTGGATCCGGT GG

[0083] The downstream primer sequence of the pcDNA3.1-HA-iRGD-10-Lamp2b plasmid is shown in SEQ ID NO.2:

[0084] GGACCTTTGTCACCACGACATCCACTGCCCATAGTCGAGTTACCTGC

[0085] The upstream primer sequence of the pcDNA3.1-10-Lamp2b-HA plasmid is shown in SEQ ID NO.3:

[0086] GGCAGTGGAGGCAGTGGATCTGGATCCGGTGG

[0087] The downstream primer sequence of the pcDNA3.1-10-Lamp2b-HA plasmid is shown in SEQ ID NO.4:

[0088] TCCACTGCCTCCACTGCCCATAGTCGAGTTACCTGC

[0089] The amino acid sequence of lamp2b is shown in SEQ ID NO.5:

[0090] MNFTVRYETTNKTYKTVTISDHGTVTYNGSICGDDQNGPKIAVQFGPGFSWIANFTKAASTYSIDSVSFSYNTGDNTTFPDAEDKGILTVDELLAIRIPLNDLFRCSLSTLEKNDVVQHYWDVLVQAFVQNGTVSTNEFLCDKDKTSTVAPTIHTTVPSPTTTPTPKEKPEAGTYSVNNG NDTCLLATMGLQLNITQDKVASVININPNTTHSTGSCRSHTALLRLNSSTIKYLDFVFAVKNENRFYLKEVNISMYLVNGSVFSIANNNLSYWDAPLGSSYMCNKEQTVSVSGAFQINTFDLRVQPFNVTQGKYSTAQECSLDDDTILIPIIVGAGLSGLIIVIVIAYVIGRRKSYAGYQTL

[0091] The amino acid sequence of the iRGD polypeptide is shown in SEQ ID NO.6:

[0092] CRGDKGPDC.

[0093] The amino acid sequence of MS2 capsid protein is shown in SEQ ID NO.7:

[0094] ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY

[0095] The components of the PCR reaction system are shown in Table 1:

[0096] Table 1

[0097]

[0098] Among them, the template plasmid is pcDNA3.1-GNSTM-3-RVG-10-Lamp2b-HA plasmid;

[0099] The configuration and operation of the PCR reaction system was performed on ice;

[0100] 1.1.1 PCR reaction

[0101] The PCR instrument operation procedure is as follows: first, pre-denaturation is performed at 95°C for 1 min, then denaturation is performed at 95°C for 15 s to melt the DNA of the template plasmid; then an annealing step is performed, the temperature is controlled between 50-65°C, and maintained for 15 s to allow the primers to bind to the template plasmid DNA; followed by an extension step, a cycle reaction is performed at 72°C, and each kb (DNA kilobase pair) DNA fragment is extended for 60 s, for a total of 35 cycles; finally, a full extension is performed at 72°C for 10 min to ensure that all DNA segments are fully extended;

[0102] 1.1.2 Fragment recovery: Add 5 μL DNA Loading Buffer to the PCR product and linearized enzyme digestion vector, and then perform agarose gel electrophoresis; add Gelred dye to the electrophoresis solution, mix well, boil and cool to a suitable temperature, pour into a gel plate and wait for solidification; after solidification, put the gel plate into the electrophoresis tank containing electrophoresis solution, set the voltage to 85V, and the electrophoresis time to 30min to separate the target DNA fragment;

[0103] After electrophoresis, the gel was imaged in a gel imager to identify and cut out DNA fragments that matched the expected band size; these fragments were cut out and transferred to a pre-prepared 1.5 mL centrifuge tube, and 100 μL of Buffer GDP was added for every 100 mg of gel weight, and the gel was completely dissolved at 60°C. The dissolved gel solution was transferred to a DNA adsorption column to adsorb the DNA to the stationary phase of the column, and the column was centrifuged to remove the supernatant, and Buffer GDP and Buffer GW were added in sequence to remove residual salts and other contaminants;

[0104] Finally, use preheated Elution Buffer to elute the target DNA fragment, place the adsorption column in a new 1.5mL centrifuge tube, add 25μL preheated Elution Buffer, let it stand for 2min, then perform high-speed centrifugation at 12000g to collect the eluted solution, which is the PCR reaction product;

[0105] Furthermore, the concentration of the target gene in the elution solution can be measured using a micro-spectrophotometer Nano-500 to ensure that high-quality target gene fragments are obtained;

[0106] 1.2 Recombination reaction

[0107] In the recombination reaction, the following system was used: each reaction contained 4 μL of 5×CEⅡBuffer to provide an appropriate ionic environment and buffer conditions, 200 ng of PCR reaction product and the same amount of linearized enzyme digestion vector were added, 2 μL of ExnaseⅡ enzyme was added, which can effectively connect the PCR reaction product and the vector, and finally, the total volume was filled to 20 μL with deionized water to obtain the PCR recombination product;

[0108] The conditions for the recombination reaction were as follows: reaction in a 37°C water bath for 30 min;

[0109] 1.3 Transformation, plating and picking of single clones of recombination reaction products

[0110] 1.3.1 Add 20 μL of PCR recombinant product to 50 μL of DH5α competent cells for transformation and incubate on ice for 30 min to obtain a transformation mixture;

[0111] 1.3.2 Heat-activate the transformation mixture in a 42°C water bath for 60 seconds, then mix the transformation mixture with 1 mL of LB medium and shake on a shaker at 37°C for 45 minutes; take 100 μL of the shaken bacterial solution and spread it on a solid plate containing appropriate resistance using a sterile spreader stick, and invert and culture for 12 hours;

[0112] 1.3.3 After the culture is completed, pick a single clone from the plate, immerse it in 5 mL of LB liquid medium, and culture it on a shaker at 37°C for 12 hours;

[0113] 1.4 Plasmid extraction

[0114] 1.4.1 Centrifuge the bacterial solution obtained in step 1.3.3 at 3500rpm for 10min, discard the supernatant, add the precipitated bacteria to 250μL of Buffer P1, invert and mix, then add 250μL of Buffer P2, invert and mix again, add 350μL of Buffer P3, mix well, and centrifuge at 12000rpm for 10min;

[0115] Transfer the supernatant to the adsorption column, centrifuge at 12000 rpm for 60 s, and remove the supernatant; add 600 μL of Buffer PW2 to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and repeat this step once;

[0116] Place the adsorption column in a new sterile 1.5 mL EP tube, add preheated 50 μL Elution Buffer, let stand for 3 min, centrifuge at 12000 rpm for 2 min, measure the concentration of target plasmid DNA in the eluate using a micro-spectrophotometer Nano-500, and store the eluate in a -20 °C refrigerator;

[0117] Step (2), cell transfection

[0118] Prepare two EP tubes marked as A and B, add an equal amount of Opti-MEM medium to each EP tube according to the volume of cells to be transfected (700 μL for a 100 mm culture dish); add 10 μL of liposome transfection reagent to tube A; put 10 μg of target plasmid DNA in tube B (10 μg for a 100 mm culture dish); use a pipette to gently mix the solutions in tubes A and B, and incubate the mixture at room temperature for 15 min;

[0119] Add fresh complete medium to the culture dish, wait for the mixture to be added to the culture medium after incubation, shake gently, and then place in the cell culture incubator. After transfection for 8 hours, remove the medium containing liposome transfection reagent and replace with fresh DMEM serum-free medium;

[0120] Step (3), vesicle extraction

[0121] Since the extracted vesicle supernatant will be used for cell experiments later, it is necessary to ensure that the extracted vesicle supernatant is sterile. Therefore, the above liquid replacement operations are all carried out in a clean bench or a biological safety cabinet. In addition, the centrifuge tubes and EP tubes used are all sterile.

[0122] After culturing for 10 h in 6 100 mm culture dishes with transfected HEK-293T cells, the cells were replaced with an equal volume of serum-free medium for further culturing; after culturing for 48 h, the supernatant in each culture dish was collected and transferred to a 45 mL sterile centrifuge tube, and centrifuged at 3000 × g for 10 min; the cell culture supernatant was collected and transferred to a new centrifuge tube (do not destroy the precipitate), and placed on ice for later use;

[0123] If the collected cell culture supernatant cannot be used in time, it can be frozen at -20℃ or -80℃. When using, thaw it in a 25℃ water bath and place it on ice after it is completely dissolved.

[0124] 10 mL of cell culture supernatant was taken, and 2.5 mL of exosome extraction reagent (41201-A) was added. The mixture was vortexed and mixed for 1 min to obtain a mixed solution. The mixed solution was allowed to stand at 2°C to 8°C for 2 h. The centrifuge tube containing the mixed solution was taken out and centrifuged at 4°C and 10,000 × g for 60 min. The supernatant was discarded and the precipitate was collected.

[0125] Resuspend the exosomes: Take 100 μL 1× PBS and evenly blow the centrifuged precipitate to mix it thoroughly, and transfer it to a new 1.5 mL centrifuge tube;

[0126] Wash the exosomes: centrifuge the centrifuge tube at 4°C and 12,000×g for 2 minutes, discard the precipitate, and retain the supernatant to obtain purified exosomes, i.e., engineered vesicles carrying mRNA targeting;

[0127] The purified exosomes can be stored at 4°C for 3 days and at -80°C for long term to avoid repeated freezing and thawing.

[0128] Furthermore, the extracted engineered vesicles carrying mRNA targeting were subjected to protein identification:

[0129] The collected engineered vesicles (purified exosomes) carrying mRNA targeting were subjected to BCA protein assay and western blot (protein immunoblotting): add an appropriate amount of Loading Buffer and heat the sample in an 80°C water bath for 10 min. Fix the glass plate containing SDS-PAGE gel in the electrophoresis tank with a clamp, and make sure that 1× electrophoresis solution is filled in the middle of the clamp to cover the top of the gel to form a difference in the inner and outer liquid levels. Gently pull out the comb to avoid damaging the lane, then oscillate the prepared protein sample on an oscillator for 10 seconds and centrifuge; in order to avoid edge effects, discard the well samples at both ends, connect the electrophoresis device, note that the positive and negative electrodes should match the electrophoresis tank, set the constant voltage to 80V, and when the sample enters the separation gel and forms a straight line, increase the voltage to 130V, electrophoresis lasts for about 70 minutes, until the sample reaches the bottom edge of the gel. When the electrophoresis is about to end, prepare the filter paper, cut a 7×5cm PVDF membrane, cut off a small corner at the upper right corner, and then soak the membrane in anhydrous methanol for 30 seconds to activate the membrane function; then prepare a tray, pour in 1× transfer solution, carefully remove the SDS-PAGE gel from the glass plate, and build a "sandwich" structure in the order of sponge, filter paper, SDS-PAGE gel, PVDF membrane, filter paper, and sponge, paying attention to avoid the generation of bubbles; place the transfer clip in the transfer tank and add ice packs and enough transfer solution, connect the transfer device, ensure that the positive and negative electrodes match, put it in a foam box and cover it with sufficient ice cubes, set the current to 300mA, and the transfer time to 110min. After the transfer is completed, take out the PVDF membrane, put it into 5% skim milk with the front side facing up, and block it on a shaker for 90min; after blocking, cut the target band according to the molecular weight of the Marker, put it in a partition box, add the primary antibody diluent, and shake it in a refrigerator at 4℃ overnight; after incubation with the primary antibody, transfer the PVDF membrane to a new partition box, wash the membrane with 1×TBST solution 3 times, 15m each time in, wash on a shaker, recover the corresponding antibodies and store at -20°C; after washing, immerse the PVDF membrane in the secondary antibody dilution solution and incubate on a shaker at room temperature for 90 minutes; after the secondary antibody incubation is completed, recover the secondary antibody dilution solution and store at -20°C, wash the membrane again with 1×TBST solution for 3 times, 15 minutes each time, and keep the PVDF membrane wet after the final washing; then, prepare the chemiluminescent solution, mix solution A and solution B in a ratio of 1:1, place the PVDF membrane face up on the blackboard in a dark room, add the luminescent solution (about 100 μL per strip), and expose after 30 seconds; Figure 3 It can be seen that iRGD-Lamp2b and blank-Lamp2b proteins with HA tags are expressed on the exosome membrane, that is, the engineering design of the exosomes is successful.

[0130] Example 2

[0131] This embodiment discloses a method for improving the functionality of fibroblasts using engineered vesicles carrying mRNA targeting, comprising the following steps:

[0132] Step (1), staining the exosomes with the membrane dye PKH67;

[0133] The non-targeted exosomes blank-Exos and the targeted exosomes iRGD-Exos (engineered vesicles obtained with pcDNA3.1-HA-iRGD-10-Lamp2b plasmid as the target plasmid) were stained with the lipophilic green fluorescent dye PKH67 to track the uptake and localization of the two exosomes by fibroblasts; to prevent fluorescence quenching, the following steps were performed under light-proof conditions. The specific steps are as follows:

[0134] 1.1 Preparation of solution A: Add 150 μL Diluent C reagent to the corresponding exosome suspension and pipette repeatedly until mixed evenly;

[0135] The blowing process should be gentle to prevent damage to the membrane structure of the exosomes;

[0136] 1.2 Preparation of B solution: Take a 5mL sterile EP tube, add 150μL Diluent C reagent, then add 0.6μL PKH26 dye to the sterile EP tube, pipette and beat until mixed evenly;

[0137] 1.3 Use a pipette to draw solution A and add it to solution B. After pipetting evenly, incubate for 3 minutes to obtain a mixed solution.

[0138] 1.4 Add 300 μL FBS to the mixed solution, pipette evenly and react for 1 minute to stop staining;

[0139] 1.5 Add 2 mL of 1×PBS solution to resuspend the exosomes and transfer them to a sterile 15 mL ultracentrifuge tube. Rinse the EP tube three times with 1×PBS solution, collect the rinse solution into the ultracentrifuge tube, and perform ultracentrifugation (4°C, 100,000×g centrifugal force, 90 min). After ultracentrifugation, green precipitate can be observed at the bottom of the ultracentrifuge tube. Discard the supernatant and resuspend the stained exosomes with 200 μL of 1×PBS solution to obtain PKH67-blank-Exos and PKH67-iRGD-Exos. Store in a sterilized 1.5 mL EP tube away from light and place in a 4°C refrigerator for later use.

[0140] Step (2), confocal microscopy experiment;

[0141] 2.1 Soak the cell slide in 75% ethanol for 2 hours to sterilize it in advance, take out the slide with tweezers in the clean bench and dry it;

[0142] 2.2 Take a 24-well plate, place the cell slides in the small holes one by one, and inoculate the fibroblasts on the cell slides. Set up 3 replicate wells for each group of experiments. To prevent fluorescence quenching, the following operations are all carried out under light-proof conditions;

[0143] 2.3 When the cell confluence reached 75%, the culture medium was discarded, and the cells were washed twice with 1× PBS solution. 500 μL of free complete culture medium (serum-free, complete culture medium) was added to each well, and 30 μg of PKH67-blank-Exos and PKH67-iRGD-Exos were added respectively, and incubated for 6 h;

[0144] 2.4 After the incubation, discard the culture medium, wash the cell slides with 1×PBS solution for 3 times (5 min / time, shaker speed 60 r / min), add 300 μL 4% tissue cell fixative to each well and keep it in the dark for 30 min, and then wash the slides with 1×PBS solution for 3 times (5 min / time, shaker speed 60 r / min);

[0145] 2.5 Take a glass slide, remove the cell slide with tweezers, dry it slightly, add 50 μL of anti-fluorescence attenuation mounting medium (containing DAPI) on the glass slide, and put the cell side of the slide down on the mounting medium to avoid bubbles, and dry it away from light;

[0146] 2.6 When the seal is firm, turn on the laser scanning confocal microscope, adjust the parameters, observe the uptake of the two exosomes by fibroblasts and take pictures;

[0147] Depend on Figure 1 It can be seen that fibroblasts can take up targeted and non-targeted exosomes stained with PKH67. Obvious green fluorescence signals can be observed in the cells after 3 hours of co-incubation, and the fluorescence signal is further enhanced after 6 hours of co-incubation, indicating that both exosomes can enter the fibroblasts through endocytosis; under the condition of co-incubation for the same time, the enrichment degree of targeted exosomes (PKH67-iRGD-Exos) in fibroblasts is significantly higher than that of non-targeted exosomes (PKH67-blank-Exos), indicating that the exosomes modified with iRGD polypeptide can enhance the uptake ability of fibroblasts for exosomes.

[0148] Example 3

[0149] This embodiment also discloses a method for improving the functionality of fibroblasts using engineered vesicles carrying mRNA targeting, comprising the following steps:

[0150] Step (1), staining the exosomes with the membrane dye PKH67;

[0151] The non-targeted exosomes blank-Exos and the targeted exosomes iRGD-Exos were stained with the lipophilic green fluorescent dye PKH67 to track the uptake and localization of the two exosomes by fibroblasts; to prevent fluorescence quenching, the following steps were performed under light-proof conditions. The specific steps are as follows:

[0152] 1.1 Preparation of solution A: Add 150 μL Diluent C reagent to the corresponding exosome suspension and pipette repeatedly until mixed evenly;

[0153] The blowing process should be gentle to prevent damage to the membrane structure of the exosomes;

[0154] 1.2 Preparation of B solution: Take a 5mL sterile EP tube, add 150μL Diluent C reagent, then add 0.6μL PKH26 dye to the sterile EP tube, pipette and beat until mixed evenly;

[0155] 1.3 Use a pipette to draw solution A and add it to solution B. After pipetting evenly, incubate for 3 minutes to obtain a mixed solution.

[0156] 1.4 Add 300 μL FBS to the mixed solution, pipette evenly and react for 1 minute to stop staining;

[0157] 1.5 Add 2 mL of 1×PBS solution to resuspend the exosomes and transfer them to a sterile 15 mL ultracentrifuge tube. Rinse the EP tube three times with 1×PBS solution, collect the rinse solution into the ultracentrifuge tube, and perform ultracentrifugation (4°C, 100,000×g centrifugal force, 90 min). After ultracentrifugation, green precipitate can be observed at the bottom of the ultracentrifuge tube. Discard the supernatant and resuspend the stained exosomes with 200 μL of 1×PBS solution to obtain PKH67-blank-Exos and PKH67-iRGD-Exos. Store in a sterilized 1.5 mL EP tube away from light and place in a 4°C refrigerator for later use.

[0158] Step (2), flow cytometry experiment;

[0159] 2.1 The fibroblasts were inoculated in 6-well plates, with 3 replicate wells for each group of experiments. When the cell confluence reached 80%, the subsequent 2.2 experiment was started. To prevent fluorescence quenching, the following operations were performed under light-proof conditions;

[0160] 2.2 Discard the culture medium in each well, rinse the cells twice with 1× PBS solution, add 2 mL of free complete culture medium (remove the exosomes contained in FBS) to each well with a pipette, and then add 50 μg of PKH67-blank-Exos and PKH67-iRGD-Exos respectively, and incubate for 6 hours;

[0161] 2.3 Discard the cell culture medium, rinse the cells twice with 1× PBS solution, add 1 mL of trypsin to digest for 3 min, add 3 mL of culture medium to terminate digestion, centrifuge at 1000 rpm for 5 min, and then wash twice with 1× PBS solution to obtain cell pellets;

[0162] 2.4 Add 500 μL of 4% tissue cell fixative to each centrifuge tube, keep it in dark for 60 min to fully fix the cells, then add 5 mL of 1× PBS solution to each tube, discard the supernatant after centrifugation, repeat twice, then add 1 mL of 1× PBS solution to resuspend the cells and place them in flow cytometry sample tubes respectively, and mark the name, time, tube number and other information;

[0163] Depend on Figure 2 It can be seen that after 3 hours of co-incubation, the uptake efficiency of fibroblasts for PKH67-iRGD-Exos was 4.75%, and that of PKH67-blank-Exos was 2.33%; after 6 hours of co-incubation, the uptake efficiency of fibroblasts for PKH67-iRGD-Exos was 23.56%, and that of PKH26-blank-Exos was 10.75%; that is, under the condition of co-incubation for the same time, the uptake efficiency of fibroblasts for targeted exosomes was significantly higher than that for non-targeted exosomes.

[0164] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An engineered vesicle carrying mRNA targeting, characterized in that: It also carries iRGD polypeptide, lamp2b membrane protein, and MS2 capsid protein.

2. An engineered vesicle carrying mRNA targeting according to claim 1, characterized in that: The engineered vesicles were secreted by HEK-293T cells.

3. An engineered vesicle carrying mRNA targeting according to claim 2, characterized in that: The iRGD polypeptide, lamp2b membrane protein and MS2 capsid protein are simultaneously expressed in HEK-293T cells.

4. An engineered vesicle carrying mRNA targeting according to claim 1, characterized in that: The extraction process comprises the following steps: Step (1), constructing a target plasmid; Select plasmid vectors: pcDNA3.1-HA-iRGD-10-Lamp2b plasmid and pcDNA3.1-10-Lamp2b-HA plasmid; 1.1 Obtain the target gene; According to the requirements of the constructed plasmid and the primer design principles, the corresponding primers were designed. The DNA sequences of the plasmid primers and the amino acid sequences of the lamp2b membrane protein, iRGD polypeptide, and MS2 capsid protein are as follows: The upstream primer sequence of the pcDNA3.1-HA-iRGD-10-Lamp2b plasmid is shown in SEQ ID NO.1: TGTCGTGGTGACAAAGGTCCCGACTGTGGCAGTGGATCTGGATCCGGTGG; The downstream primer sequence of the pcDNA3.1-HA-iRGD-10-Lamp2b plasmid is shown in SEQ ID NO.2: GGACCTTTGTCACCACGACATCCACTGCCCATAGTCGAGTTACCTGC; The upstream primer sequence of the pcDNA3.1-10-Lamp2b-HA plasmid is shown in SEQ ID NO.3: GGCAGTGGAGGCAGTGGATCTGGATCCGGTGG; The downstream primer sequence of the pcDNA3.1-10-Lamp2b-HA plasmid is shown in SEQ ID NO.4: TCCACTGCCTCCACTGCCCATAGTCGAGTTACCTGC; The amino acid sequence of lamp2b membrane protein is shown in SEQ ID NO.5: MNFTVRYETTNKTYKTVTISDHGTVTYNGSICGDDQNGPKIAVQFGPGFSWIANFTKAASTYSIDSVSFSYNTGDNTTFPDAEDKGILTVDELLAIRIPLNDLFRCNSLSTLEKNDVVQHYWDVLVQAFVQNGTVSTNEFLCDKDKTSTVAPTIHTTVPSPTTTPTPKEKPEAGTYSVNNGN DTCLLATMGLQLNITQDKVASVININPNTTHSTGSCRSHTALLRLNSSTIKYLDFVFAVKNENRFYLKEVNISMYLVNGSVFSIANNNLSYWDAPLGSSYMCNKEQTVSVSGAFQINTFDLRVQPFNVTQGKYSTAQECSLDDDTILIPIIVGAGLSGLIIVIVIAYVIGRRKSYAGYQTL; The amino acid sequence of the iRGD polypeptide is shown in SEQ ID NO.6: CRGDKGPDC; The amino acid sequence of MS2 capsid protein is shown in SEQ ID NO.7: ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY; 1.2 Recombination reaction; 1.3 Transformation, plating and picking of single clones of the recombination reaction products; 1.4 Plasmid extraction; Step (2), cell transfection; Step (3), vesicle extraction.

5. An engineered vesicle carrying mRNA targeting according to claim 4, characterized in that: When obtaining the target gene in step (1), a PCR reaction is also included; The components of the PCR reaction system include 2×Phanta Buffer, upstream primer, downstream primer, dNTPMix, template plasmid, DNA polymerase, and deionized water; The template plasmid was pcDNA3.1-GNSTM-3-RVG-10-Lamp2b-HA plasmid; The PCR reaction process includes: pre-denaturation, denaturation, annealing, cycling reaction and extension.

6. Use of an engineered vesicle carrying mRNA targeting according to any one of claims 1 to 5 in improving the functionality of fibroblasts.

7. The use of the engineered vesicle carrying mRNA targeting according to claim 6 for improving the functionality of fibroblasts, characterized in that: The method for improving the functionality of fibroblasts using engineered vesicles carrying mRNA targeting comprises the following steps: Step (1), staining the vesicles with a membrane dye PKH67; 1.1 Preparation of Solution A: Add Diluent C reagent to the vesicle suspension and mix well; 1.2 Preparation of Solution B: Mix Diluent C reagent and PKH26 dye evenly; 1.3 Use a pipette to draw up solution A and add it to solution B, mix them evenly and incubate them together to obtain a mixed solution; 1.4 Add FBS to the mixed solution to stop staining; 1.5 Add 1× PBS solution to resuspend the vesicles, and perform ultracentrifugation on the resuspended vesicles, discard the supernatant, retain the green precipitate at the bottom of the ultracentrifuge tube, and resuspend the stained vesicles with 1× PBS solution to obtain PKH67 stained vesicles; Step (2) uptake of vesicles by fibroblasts; 2.1 Sterilize the cell slide and dry it; 2.2 Inoculate fibroblasts on the cell slide; 2.3 When the cell confluence reaches 70%-80%, discard the culture medium, wash with 1× PBS solution, add free complete culture medium again, and add PKH67 stained vesicles for co-incubation; 2.4 After the incubation, discard the culture medium, wash the cell slides with 1×PBS solution on a shaker, add tissue cell fixative and place in a dark place, and wash the slides with 1×PBS solution on a shaker again; 2.5 Add anti-fluorescence attenuation mounting medium on the slide, take out the cell slide, dry it, and then put the slide with cells facing down on the anti-fluorescence attenuation mounting medium and dry it in the dark; 2.6 When the sealing is firm, the fibroblasts have completed the uptake of vesicles.

8. The use of the engineered vesicle carrying mRNA targeting according to claim 6 for improving the functionality of fibroblasts, characterized in that: The method for improving the functionality of fibroblasts using engineered vesicles carrying mRNA targeting comprises the following steps: Step (1), staining the vesicles with a membrane dye PKH67; 1.1 Preparation of Solution A: Add Diluent C reagent to the vesicle suspension and mix well; 1.2 Preparation of Solution B: Mix Diluent C reagent and PKH26 dye evenly; 1.3 Use a pipette to draw up solution A and add it to solution B, mix them evenly and incubate them together to obtain a mixed solution; 1.4 Add FBS to the mixed solution to stop staining; 1.5 Add 1× PBS solution to resuspend the vesicles, and perform ultracentrifugation on the resuspended vesicles, discard the supernatant, retain the green precipitate at the bottom of the ultracentrifuge tube, and resuspend the stained vesicles with 1× PBS solution to obtain PKH67 stained vesicles; Step (2) uptake of vesicles by fibroblasts; 2.1 Inoculation of fibroblasts; 2.2 When the cell confluence reaches 70%-80%, discard the culture medium, wash with 1× PBS solution, add free complete culture medium again, and add PKH67 stained vesicles for co-incubation; 2.3 After the incubation, the culture medium was discarded, the cells were washed with 1× PBS solution, digested with trypsin, centrifuged, and then washed with 1× PBS solution to obtain cell pellets; 2.4 Add tissue cell fixative to the cell pellet, keep it in a dark place, then add 1×PBS solution, centrifuge and discard the supernatant, repeat the process of adding 1×PBS solution-centrifugation, add 1×PBS solution to resuspend the cells, and complete the uptake of vesicles by fibroblasts.

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