Target protein-enriched exosome based on polypeptide molecular glue and preparation method of target protein-enriched exosome

Abstract

The invention discloses a target protein-enriched exosome based on polypeptide molecular glue and a preparation method of the target protein-enriched exosome. The preparation method comprises the following steps: incubating and culturing a phage library and HSP60 recombinant protein to screen HSP60-targeted polypeptide molecular glue G12, constructing recombinant plasmids for expressing target protein-polypeptide molecular glue G12 fusion protein based on the polypeptide molecular glue G12, and transfecting cells for culture; the polypeptide molecule glue G12 is linked with HSP60 in the target protein targeted exosome to realize trans-organelle transport, so that the target protein is enriched into the exosome, and then the exosome rich in the target protein is obtained through extraction. The invention provides a theoretical basis for clinically delivering the target protein which does not exist in the exosome in a natural state so that the target protein can play a therapeutic role through the exosome.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and more specifically, relates to a target protein-enriched exosome based on polypeptide molecular glue and a preparation method thereof. Background Art

[0002] Exosomes are tiny vesicles secreted by cells that contain nucleic acids, proteins, and lipids. They can be taken up by target cells and transport their contents. Exosomes have high targeting and ability to penetrate biological barriers, low immunogenicity and high biocompatibility, multifunctional payloads and combined therapeutic potential, reduced side effects and enhanced stability, natural origin and scalability, and therefore can be used as drug delivery vehicles for the treatment of various diseases. SIRT3 protein has a strong NAD + SIRT3, ROMO1, and TOM40 are proteins that regulate mitochondrial redox homeostasis, regulate mitochondrial metabolism, control stress responses, and maintain mitochondrial genome stability. Reactive oxygen species modulator 1 (Romo1) increases cellular reactive oxygen species levels and participates in regulating processes such as cell proliferation, apoptosis, and oxidative stress. Its abnormal expression is associated with various diseases. Mitochondrial outer membrane translocase 40 (TOM40) is a major member of the mitochondrial outer membrane complex and a key protein channel in the mitochondrial outer membrane, crucial for regulating mitochondrial function. However, proteins such as SIRT3, ROMO1, and TOM40 are not naturally present in exosomes, making it difficult to deliver these proteins therapeutically via exosomes. Summary of the Invention

[0003] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a method for preparing exosomes enriched with target protein based on polypeptide molecular glue.

[0004] The second object of the present invention is to provide exosomes rich in target protein prepared by the above preparation method.

[0005] The above-mentioned object of the present invention is achieved through the following technical solutions: A method for preparing exosomes enriched with a target protein based on a polypeptide molecular glue comprises linking the coding gene of polypeptide molecular glue G12 having an amino acid sequence as shown in any one of SEQ ID Nos. 1 to 4 to the 3' end or 5' end of the target protein coding gene to obtain a chimeric gene, then constructing a recombinant plasmid expressing the chimeric gene, and then transfecting the recombinant plasmid into cells for expression, extracting exosomes, and obtaining exosomes enriched with the target protein (target protein-Exo); the target protein is a protein that is not naturally present in exosomes.

[0006] Molecular glue is a class of artificially synthesized substances. Molecular glue can be enzymes, transcription factors, structural proteins, etc. Molecular glue can promote interactions between proteins, thereby forming stable ternary complexes or enhancing existing protein-protein interactions.

[0007] Heat shock protein 60 (HSP60) is a mitochondrial chaperone protein encoded by the nuclear genome. It plays an important role in the transport and folding of mitochondrial proteins and is a key mitochondrial marker. Studies have found that HSP60 inactivation affects key mitochondrial functions, reducing cellular respiration and ATP levels.

[0008] The present invention is a protein directional transport technology mediated by molecular glue. By incubating and culturing a phage library with HSP60 recombinant protein, a polypeptide molecular glue G12 that targets and binds to HSP60 is screened out, and then it is connected to the ends of SIRT3, ROMO1 and TOM40 to be enriched target proteins through genetic engineering technology to form a recombinant protein. The polypeptide molecular glue G12 is used to promote the binding of the target protein to be enriched with HSP60. Since HSP60 is a heat shock protein that exists in exosomes, SIRT3, ROMO1 and TOM40 to be enriched target proteins are bound to HSP60 through polypeptide molecular glue G12, which can help the target protein to be enriched to achieve cross-organelle transport from the cell nucleus to the exosomes, so that it can be enriched in the exosomes. Specifically, by constructing a recombinant plasmid expressing a target protein-polypeptide molecular glue G12 fusion protein (i.e., linking the gene encoding polypeptide molecular glue G12 to the 3' or 5' end of the target protein encoding gene to obtain a chimeric gene, and then constructing a recombinant plasmid expressing this chimeric gene), transfecting cells, culturing, and expressing the chimeric gene, polypeptide molecular glue G12 can bind to HSP60 in exosomes, promoting the binding of the target protein to be enriched with HSP60, and obtaining engineered exosomes enriched with the target protein. If the amino acid sequences of the target protein and polypeptide molecular glue G12 are known, those skilled in the art can obtain the coding gene sequences of the target protein and polypeptide molecular glue G12 using conventional techniques and optimize them based on the host codon preference.

[0009] Furthermore, the coding gene of the polypeptide molecular glue G12 is connected to the target protein coding gene via a Flag tag, i.e., the polypeptide molecular glue G12 is connected to the target protein via the Flag tag, which can be expressed as target protein-Flag-G12 or G12-Flag-target protein. The Flag tag serves as an epitope tag to facilitate subsequent protein detection.

[0010] Furthermore, the coding gene of the polypeptide molecular glue G12 is connected to the 3' end of the target protein coding gene, that is, the polypeptide molecular glue G12 is connected to the C-terminus of the target protein, which is expressed as target protein-G12. The present invention constructs recombinant plasmids such as pcDNA3.1-SIRT3-G12-C and pcDNA3.1-SIRT3-G12-N, and connects the polypeptide molecular glue G12 to the C-terminus or N-terminus of SIRT3 and other proteins through gene recombination technology to form a SIRT3-polypeptide molecular glue G12 fusion protein expression system, and successfully constructs engineered exosomes rich in target proteins such as SIRT3. By + and C + The SIRT3 content in transfected cells (engineered gene cells), exosomes (engineered exosomes), and target cells (target cells) was analyzed. C + The results showed that there was more co-localization of SIRT3 with mitochondria in the 3-D group, indicating that connecting the peptide molecular glue G12 to the C-terminus of the target protein can more effectively promote the transport of target proteins such as SIRT3 to mitochondria in target cells.

[0011] Furthermore, the amino acid sequence of the polypeptide molecular glue G12 is shown in SEQ ID No. 1: GCMYKASMTCCM. The present invention screened four polypeptide sequences from a 12-peptide phage peptide library, which were able to produce affinity with the HSP60 recombinant protein. Phage ELISA testing showed that the polypeptide with the sequence GCMYKASMTCCM had stronger binding ability.

[0012] Furthermore, the target protein is a therapeutic protein that is not naturally expressed in exosomes.

[0013] Preferably, the target protein includes but is not limited to SIRT3, ROMO1 or TOM40.

[0014] Furthermore, the recombinant plasmid uses a template plasmid including but not limited to pcDNA3.1; for example, using pcDNA3.1, a pcDNA3.1-target protein-G12 recombinant plasmid is constructed.

[0015] Furthermore, the cells include but are not limited to cardiomyocytes.

[0016] Preferably, the cardiomyocytes include but are not limited to rat cardiomyocytes, and the rat cardiomyocytes include but are not limited to H9C2 cells.

[0017] Furthermore, the method for extracting the exosomes includes but is not limited to ultracentrifugation.

[0018] The present invention also provides engineered exosomes rich in target protein (target protein-Exo) obtained by any of the above preparation methods.

[0019] The present invention also provides a product containing the above-mentioned engineered exosomes rich in target protein (target protein-Exo).

[0020] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a molecular glue-based target protein enriched exosome and a preparation method thereof. The present invention screens out the polypeptide molecular glue G12 targeting HSP60 by incubating and culturing a phage library with HSP60 recombinant protein, and then constructs a recombinant plasmid expressing the target protein-polypeptide molecular glue G12 fusion protein based on the polypeptide molecular glue, transfects cells and cultures, extracts exosomes, and obtains engineered exosomes rich in the target protein; the polypeptide molecular glue G12 can link the target protein to HSP60 in the exosomes, promote the binding of the target protein to HSP60, thereby helping the target protein, which is not naturally present in the exosomes, to be transported and enriched in the exosomes, thereby obtaining engineered exosomes rich in the target protein. The engineered exosomes of the present invention can significantly increase the content of the target protein in the exosomes, providing a theoretical basis for the clinical delivery of target proteins that are not naturally present in exosomes so that they can exert therapeutic effects through exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The results are shown for the screening of HSP60 affinity peptides by phage ELISA in Example 1. *: p<0.05, **: p<0.01.

[0022] Figure 2 Schematic diagram of two modes of connection between G12 and SIRT3 terminal in Example 1.

[0023] Figure 3 This is the protein binding effect of the molecule in Example 1 on the SIRT3 recombinant protein modified with G12 at the N-terminus or C-terminus and HSP60.

[0024] Figure 4 For the Co-IP detection N in Example 1 + and C + Protein binding of recombinant proteins to HSP60.

[0025] Figure 5 The ELISA method for determining the levels of Flag and SIRT3 in Example 1 was used. Figure 5A shows the Flag level in H9C2-SIRT3-transfected cells; B shows the Flag and SIRT3 levels in exosomes secreted by H9C2-SIRT3 cells; C shows the Flag level in H9C2 cells after 3 days of hypoxia-reperfusion coculture with exosomes; D shows the immunofluorescence staining and colocalization quantification of SIRT3 and MitoTracker in H9C2 cells after 3 days of hypoxia-reperfusion coculture with exosomes (Scale bar = 100 μm). *: p < 0.05, ns: p > 0.05.

[0026] Figure 6 This is the construction and identification of engineered exosomes in Example 1. Figure 6 Figure (A) shows the microstructure of exosomes under transmission electron microscopy (scale bar = 100 nm). Figure (B) shows the BCA assay for exosome secretion levels in each group. (ns: p > 0.05). Figure (C) shows the size distribution of various exosomes detected by nanoparticle tracking. Figure (EF) shows Western blot analysis of exosome markers and SIRT3 protein in Exo and SIRT3-Exo cells, as well as quantification of SIRT3 expression based on band grayscale values. (#) shows p < 0.05 compared with the H9C2 group; (**) shows p < 0.01 compared with the Exo group. Figure (GH) shows ELISA analysis of ROMO1 and TOM40 protein levels in Exo, ROMO1-Exo, and TOM40-Exo cells. (**) shows p < 0.01 compared with the H9C2 group; (***) shows p < 0.001 compared with the Exo group.

[0027] Figure 7 The exosome biogenesis structure in H9C2 co-localizes with SIRT3 in Example 1. Figure 7 A shows the immunofluorescence imaging of Rab5a, Rab7a, and CD63 with SIRT3 (scale bar = 10 μm); B shows the quantitative analysis of the 488 and 594 nm fluorescence intensities on the selected paths within the field of view based on the immunofluorescence images.

[0028] Figure 8 The exosome biogenesis structure and Flag co-localization in H9C2-SIRT3 in Example 1. Figure 8 Figure A shows immunofluorescence imaging of Rab5a, Rab7a, and CD63 with Flag, respectively (scale bar = 10 μm); Figure B shows quantitative analysis of the 488 and 594 nm fluorescence intensities along the selected path within the field of view based on the immunofluorescence images.

[0029] Figure 9The results were obtained by using nanoflow cytometry to label Flag-FITC / Rab5a-PC5 and Flag-FITC / Rab7a-PC5, respectively, and quantitative analysis of the SIRT3-G12 content in double-positive endosomes (H9C2-SIRT3 endosomes) in Example 1. ***: p < 0.001.

[0030] Figure 10 This is a schematic diagram of the present invention, taking SIRT3 as an example. DETAILED DESCRIPTION

[0031] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0032] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0033] 1. Experimental Materials Table 1 Cell sources cell Feature Description source H9C2 Rat cardiomyocytes Merck KGaA <![CDATA[H9C2-SIRT3(N + )]]> Rat cardiomyocytes, transfected with pcDNA3.1-SIRT3-G12-N plasmid <![CDATA[H9C2-SIRT3(C + )]]> Rat cardiomyocytes, transfected with pcDNA3.1-SIRT3-G12-C plasmid <![CDATA[H9C2-ROMO1(C + )]]> Rat cardiomyocytes, transfected with pcDNA3.1-ROMO1-G12-C plasmid The present invention is constructed <![CDATA[H9C2-TOM40(C + )]]> Rat cardiomyocytes, transfected with pcDNA3.1-TOM40-G12-C plasmid E. coli DH5α Plasmid construction host bacteria Laboratory collection Table 2 Plasmid sources plasmids Genetic markers and construction source pcDNA3.1-SIRT3-G12-N plasmid KpnI and XbaI restriction endonuclease sites were designed at both ends of the G12-Flag-SIRT3 sequence Wuhan Jinkairui pcDNA3.1-SIRT3-G12-C plasmid KpnI and XbaI restriction endonuclease sites were designed at both ends of the SIRT3-Flag-G12 sequence Wuhan Jinkairui pcDNA3.1-ROMO1-G12-C plasmid KpnI and XbaI restriction endonuclease sites were designed at both ends of the ROMO1-Flag-G12 sequence Wuhan Jinkairui pcDNA3.1-TOM40-G12-C plasmid KpnI and XbaI restriction endonuclease sites were designed at both ends of the TOM403-Flag-G12 sequence Wuhan Jinkairui DMEM powder was purchased from Gibco; antioxidant enzyme activity assay kits were purchased from Dojindo Molecular Technology Inc; fetal bovine serum (FBS) was purchased from ThermoFisher; and H9C2 cells were preserved in the laboratory.

[0034] 2. Experimental methods 2.1 Cell Culture (1) Preparation of the cell room: If the cell room is being disinfected with UV light, turn it off and turn on the fluorescent light. Turn on the 37°C water bath and place the DMEM culture medium and cell treatment PBS taken out of the 4°C chromatography cabinet into the water bath to heat; put on a white coat and slippers for the cell room, wear a mask and a hat, spray the sleeves of the white coat with 75% ethanol and disinfect your hands. Use an alcohol cotton ball to disinfect the microscope stage. After UV irradiation, start ventilation and fluorescent lighting, and pull the workbench glass to the appropriate position. Take out the DMEM culture medium and PBS that have returned to room temperature and disinfect them, then place them on the clean bench for use; take out the sterilized pipette tips from the 55°C oven, disinfect them with 75% ethanol, and place them on the left side of the clean bench for use. Turn on the cell room centrifuge. Carefully take out the cells to be processed and place them on the sterilized microscope stage to observe the cell growth status and whether there is contamination. When everything is ready, start processing the cells.

[0035] (2) Cell culture conditions: H9C2 cardiomyocytes are adherent cells and are cultured in DMEM. Complete culture medium is prepared by adding 50 mL of filtered fetal bovine serum (FBS) and 5 mL of penicillin-streptomycin (DPS) to every 445 mL of DMEM. The cells are cultured in a sterile cell culture incubator at 37°C and 5% CO2. Cell growth is regularly observed to determine whether further processing is necessary. After the cells have grown to an appropriate density, they are digested with sterile trypsin and then passaged, plated, and frozen for subsequent experimental needs.

[0036] (3) Cell recovery: Take a 15 mL centrifuge tube and add 3 mL of complete culture medium for later use. Remove the frozen cells from liquid nitrogen and heat them in a 37°C water bath. After thawing, disinfect the surface of the cryotube. Quickly transfer the thawed cell suspension to the prepared complete culture medium in a clean bench; centrifuge at 800 rpm for five minutes. After centrifugation, discard the supernatant in the clean bench and resuspend the cells in complete culture medium. Transfer the cells to a T25 cell culture flask at a 1:1 ratio, shake gently, and then culture in an incubator.

[0037] (4) Cell passaging: Remove the cell culture flask and discard the old culture medium, and wash it once with PBS; add an appropriate amount of trypsin, and after an appropriate time, observe under a microscope. If the cells slowly become independent spheres, immediately add 4 times the volume of trypsin to terminate the digestion; slowly and gently blow off the digested cells, transfer them to a 15 mL centrifuge tube, and centrifuge at 800 rpm for 5 min; retrieve the cells after centrifugation, discard the supernatant in the centrifuge tube in an ultra-clean workbench, add an appropriate amount of complete culture medium to resuspend, and gently blow evenly, and transfer them to a T25 cell culture flask at a ratio of 1:3 to continue culture.

[0038] (5) Cell freezing: Remove the cell culture flask and discard the old culture medium, wash once with PBS; add an appropriate amount of trypsin for digestion. After the digestion is terminated, gently blow the cells and transfer them to a 15 mL centrifuge tube, centrifuge at 800 rpm for 5 minutes; retrieve the cells after centrifugation, discard the supernatant in the centrifuge tube in the clean bench, add 1 mL of cell freezing solution (FBS: DMSO = 9:1) to resuspend, and gently blow evenly, then transfer to a pre-prepared cryopreservation tube; finally, perform a gradient cooling of the cells (4℃, 30 min; -20℃, 1.5h; -80℃, 12h) and transfer to liquid nitrogen for storage for later use.

[0039] Example 1 Construction of SIRT3, ROMO1 and TOM40-enriched exosomes based on molecular glue 1. Experimental Methods 1. Targeting peptide screening Targeted peptide screening was performed according to the instructions of a phage display peptide library kit (Tec Biotech, China). The library contains billions of fd-tet phage clones, each displaying a random exogenous peptide sequence at the N-terminus of the fd-tet phage coat protein. The phage library was first incubated in a flask at 37°C for 1 hour to remove phage that specifically bound to the flask. Then, after pretreatment to remove nonspecifically bound phage, the remaining phage library was incubated with HSP60 recombinant protein for 1 hour at room temperature. Unbound phage were washed 10 times with bovine serum albumin (BSA) / Tween wash buffer to remove HSP60 protein. Phage bound to HSP60 were eluted for 10 minutes using a low-pH elution buffer (0.1 N HCl, 1 mg / mL BSA, adjusted to pH 2.2 with glycine). The elution buffer was immediately neutralized with 1 M Tris-HCl (pH 8.8). The eluate from the first round of elution was concentrated using a Centricon 100 kDa ultrafiltration device (MerckMillipore, Germany). The concentrated eluate was then transferred to E. coli culture medium and incubated at 37°C with constant shaking for 24 hours. Phage clones that internalized the HSP60 protein were recovered using cell lysis buffer (2% sodium dodecyl sulfate, 10 mM Tris-HCl, 2 mM EDTA, pH 8.0). The cells were centrifuged at 130 g for 10 minutes, the supernatant removed, and cell lysis buffer added. The phage were then amplified and further screened, similar to the first round of screening. After the third round of screening, the eluted phage were titrated, and 40 clones were randomly selected for sequencing to determine the sequence of the HSP60-binding peptide.

[0040] 2. Affinity Verification The binding capacity and specificity of the selected phages to HSP60 were determined by enzyme-linked immunosorbent assay (ELISA). 150 μL of 100 μg / mL HSP60 (dissolved in 0.1 M NaHCO3, pH 8.6) was prepared and coated on a 96-well plate, then incubated at 4°C with gentle shaking for 30 minutes, followed by overnight incubation at 4°C. Subsequently, 2×10 9 CFU-purified phage were incubated in blocking buffer at room temperature for 1 hour. The plates were then washed three times with PBS containing 0.5% Tween 20 and then three more times with PBS. The plates were then incubated with alkaline phosphatase-conjugated anti-FD phage IgG (Abcam, MA, USA) at room temperature for 1 hour. After washing, the substrate p-nitrophenol phosphate was added to the wells, and the absorbance was measured at 405 nm using a plate reader.

[0041] 3. Molecular docking The AlphaFold2 algorithm was used to predict the structure of the SIRT3-G12 protein sequence, revealing its complete three-dimensional configuration. The structural model was verified using the PROCHECK program, demonstrating that the dihedral angles of each amino acid residue in the simulated structure were within reasonable ranges and adhered to stereochemical energy criteria. The SIRT3-G12 protein model constructed by homology modeling served as a starting point for subsequent molecular docking studies. Using AutoDock Tools v1.5.6 software, the intrinsic charge properties of the protein were preserved, and the pdbqt format file required for docking was generated. The HSP60 protein structure was obtained from the PDB database as a docking receptor and processed using AutoDock Tools to generate a docking file. Finally, molecular docking was performed using the ZDOCK software package.

[0042] 4. Plasmid Synthesis and Transfection Using the pcDNA3.1 plasmid as a template, KpnI and XbaI restriction endonuclease sites were designed at both ends of the G12-Flag-SIRT3 and SIRT3-Flag-G12 sequences. Recombinant plasmids, named pcDNA3.1-SIRT3-G12-C (i.e., pcDNA3.1-SIRT3-Flag-G12) and pcDNA3.1-SIRT3-G12-N (i.e., pcDNA3.1-G12-Flag-SIRT3), were constructed and named. H9C2 cardiomyocytes were evenly seeded in 6-well plates and cultured to a cell density of 30%–50%. In a 1.5 mL centrifuge tube, 200 μL of serum-free medium and 3 μg of plasmid were added. In another centrifuge tube, 200 μL of serum-free medium and 6 μL of transfect-mate were mixed. Transfection was performed using Lipofectamine 2000 at room temperature for 5 minutes. The contents of the two tubes were then combined and the reaction continued for 20 minutes. The transfection complex was added to a pre-laid 6-well plate containing 2 mL of serum-free medium and shaken. The cells were cultured in a cell culture incubator for 5 hours, replaced with complete medium, and cultured for 48 hours before harvesting the cells for subsequent experiments. The cells transfected with pcDNA3.1-SIRT3-G12-N were named N + The cells transfected with pcDNA3.1-SIRT3-G12-C were named C + (Same as H9C2-SIRT3 in the following.) Similarly, pcDNA3.1-ROMO1-G12-C (i.e., pcDNA3.1-ROMO1-Flag-G12) and pcDNA3.1-TOM40-G12-C (i.e., pcDNA3.1-TOM40-Flag-G12) plasmids were constructed and transfection experiments were performed.

[0043] 5. Co-immunoprecipitation (Co-IP) Transfected cells were lysed, proteins were extracted, and incubated with the primary antibody (anti-Flag) overnight at 4°C. Incubation was continued for 4 hours at 4°C according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). After immunoprecipitation, the beads were washed three times with 1× phosphate-buffered saline (PBS). Proteins were then eluted from the beads with 40 μL of elution buffer and analyzed by immunoblotting. Samples were supplemented with sample buffer containing 5% β-mercaptoethanol and heated at 55°C for 15 minutes. Binding to the target protein was then detected by western blotting. The target proteins were SIRT3 (diluted 1:1000 in TBST buffer), HSP60 (diluted 1:1000 in TBST buffer), and Flag (diluted 1:1000 in TBST buffer).

[0044] 6. Exosome Extraction After incubating H9C2-SIRT3 and H9C2 cells in exosome-free medium for 48 hours, the culture supernatants were collected. First, the samples were centrifuged at 300 g for 10 minutes, followed by ultracentrifugation at 2000 g for 10 minutes. Next, ultracentrifugation was performed at 10,000 g for 30 minutes. The resulting cell pellet, membranes, and debris were discarded, and the supernatant was filtered through a Merck Millipore 0.22 μm filter. Finally, exosomes were isolated by ultracentrifugation at 120,000 g for 90 minutes. These exosomes were then resuspended in PBS and washed at 120,000 g for 90 minutes. Exosomes extracted from H9C2 were designated Exo, and exosomes extracted from H9C2-SIRT3 were designated SIRT3-Exo. Similarly, H9C2-ROMO1 and H9C2-TOM40 cells were incubated, and ROMO1-Exo and TOM40-Exo were extracted.

[0045] 7. Mitochondrial internalization Exosomes and SIRT3-Exosomes were labeled using the PKH26 kit (Sigma-Aldrich) for fluorescent detection. 6 μL of PKH26 staining solution was added to 1 mL of exosome sample diluted in diluent. After gentle mixing, the sample was incubated at room temperature for 5 minutes. The staining reaction was stopped by adding 2 mL of 10% BSA, and the volume was adjusted to 8.5 mL with serum-free medium. The resulting mixture was transferred to an ultracentrifuge tube and centrifuged at 120,000 g for 2 hours at 4°C. The PKH26-labeled exosomes were carefully resuspended in DPBS in the dark and stored at -80°C in the dark. To examine the in vitro uptake of PKH26-labeled exosomes by H9C2 cells, 200 μg / mL of labeled exosomes were introduced into H9C2 culture medium (DMEM supplemented with 10% FBS, 1% penicillin and streptomycin, seeded at 60% density) and incubated for 12, 24, 48, 72, and 96 hours. The cells were then washed with DPBS and fixed with 4% formaldehyde for immunostaining. 4',6-diamidino-2-phenylindole (DAPI) was used to visualize cell nuclei. Three independent samples were collected from each group and imaged using an LSM880 confocal laser scanning microscope (Zeiss). The captured images were analyzed using ImageJ software (Media Cybernetics Inc., USA) to determine relative fluorescence intensity.

[0046] 8. Western Blot (1) After one cell passage, Western Blot analysis was performed, and some protein lysates were extracted from the cells for Western Blot analysis. First, the cells were washed with pre-cooled PBS at 4°C for 1 minute each time, and a total of 3 times to ensure the complete removal of residual liquid. Then, lysis buffer was prepared according to the ratio of 1 mL RIPA plus 10 μL PMSF (100 mM), and the mixture was shaken and placed on ice. Subsequently, 400 μL of lysis buffer was added to each bottle of cells and lysed on ice for 30 minutes, with the culture bottle shaken intermittently during the period to promote sufficient cell reaction. After the lysis was completed, the cells were quickly scraped to the side of the culture bottle on ice using a cell scraper, and the cell fragments and lysate were transferred to a 1.5 mL EP tube using a pipette. Then, these EP tubes were centrifuged at 8000 g for 10 minutes at 4°C. After centrifugation, the supernatant was taken and stored at -20°C for subsequent detection.

[0047] (2) Subsequently, the BCA protein concentration was determined. First, BSA was dissolved in PBS to prepare a series of standard concentrations, including 5, 2.5, 1, 0.5, 0.25, 0.125, 0.05, and 0.025 mg / mL. Then, 20 μL of each concentration of standard and total protein sample were taken and added to a 96-well plate. Two replicate wells were set for each standard and sample. Next, the A solution and B solution of the BCA kit were mixed in a volume ratio of 50:1 to form a working solution. After adding 200 μL of the working solution to each well, the 96-well plate was placed in a 37°C incubator and incubated for 30 minutes. Finally, the OD value at a wavelength of 562 nm was read using a microplate reader, and a standard curve was drawn based on the OD value and concentration of the standard to obtain the protein concentration of the total protein sample.

[0048] (3) Next, an SDS-PAGE gel was prepared, consisting of a 15% separation gel and a 5% stacking gel. First, the separation gel was injected into the gap between the glass plates to a depth of 1.5 cm from the upper edge, and an appropriate amount of 75% ethanol was added to the upper layer. After the separation gel solidified, the upper layer of ethanol was poured out and the stacking gel was injected. Then, a comb was inserted and allowed to dry naturally.

[0049] (4) Before electrophoresis, heat the total protein sample in a 95°C water bath for 5 minutes and mix with the protein loading buffer. Then, pour the electrophoresis buffer into the electrophoresis tank and add 10 μL of protein maker to the lanes on both sides, and add 15 μL of sample to each lane. During the electrophoresis process, first use 90 V voltage for 30 minutes in the stacking gel stage, and then use 160 V voltage for electrophoresis in the separation gel stage until the bromophenol blue runs to the bottom of the gel.

[0050] (5) After electrophoresis, transfer was performed. First, filter paper of appropriate size and 0.22 μm PVDF membrane were cut and the PVDF membrane was activated with methanol for 1 minute. Then, the transfer clamp was assembled in the order of "sponge-filter paper-gel-PVDF membrane-filter paper-sponge", and it was ensured that there were no bubbles. After assembly, the transfer clamp was inserted into the transfer tank and the transfer solution was poured in. Under ice bath conditions, the transfer operation was performed at a constant current of 200 mA for 60 minutes.

[0051] (6) After the transfer, the antibody incubation was performed. First, the membrane was washed with TBST solution for 5 minutes and blocked with 5% skim milk powder (prepared with PBS solution) at room temperature for 1 hour. Subsequently, the membrane was washed with TBST solution 3 times, each time for 5 minutes. Next, the primary antibody diluted with primary antibody diluent was added and incubated overnight at 4°C. The next day, the membrane was washed with TBST solution 3 times, each time for 5 minutes, and then the secondary antibody diluted with secondary antibody diluent was added and incubated on a shaker at room temperature for 1 hour. Finally, the membrane was washed with TBST solution again 3 times, each time for 5 minutes.

[0052] (7) Finally, luminescence detection was performed. After adding ECL luminescence solution and incubating for 3 minutes, exposure imaging was performed. Finally, the grayscale values of the internal reference gene and the target gene were determined using ImageJ software, thus completing the entire Western Blot detection process.

[0053] SIRT3-transfected cells and exosomes were detected by Western Blot for the detection of exosome positive markers SIRT3, CD9, and ALIX. The corresponding antibodies were diluted at ratios of 1:1000, 1:500, and 1:1000, respectively.

[0054] 9.ELISA ROMO1 and TOM40 protein levels in transfected cells and exosomes were measured using an experimental ELISA. First, protein was extracted from cells and exosomes. ROMO1 and TOM40 standards were diluted to various concentrations and added to the wells of an ELISA plate to prepare a standard curve. Protein extract was added to the experimental wells, while the diluent blank was added to the control wells. The plates were incubated at 37°C for 30 minutes, then discarded. The plates were washed three times and patted dry. After adding 100 μL of enzyme-linked reagent and incubating at 37°C for 30 minutes, the plates were discarded, washed, and patted dry. After adding 100 μL of colorimetric solution, the plates were incubated at room temperature in the dark for 15 minutes. After adding 50 μL of stop solution, the absorbance was measured at 450 nm using a microplate reader. Standard curves were then constructed to calculate concentrations.

[0055] 10. Immunofluorescence colocalization H9C2 and H9C2-SIRT3 cells were seeded in confocal microplates. When cells reached the appropriate density, they were washed three times with PBS, fixed with 4% paraformaldehyde for 30 minutes, and washed three times with PBS. The cells were permeabilized with Triton X100 for 15 minutes and washed three times with PBS. The cells were blocked with 1% BSA for 1 hour and washed three times with PBS. The cells were incubated overnight at 4°C with primary antibodies against Flag (1:1000) or SIRT3 (1:1000), as well as primary antibodies against Rab5a (1:1000) or Rab7a (1:1000) or CD63 (1:800) of another species. The next day, after washing with PBS, the cells were incubated with AlexaFluor 488- and AlexaFluor 594-conjugated secondary antibodies corresponding to the species of the primary antibody, sequentially for 1 hour at room temperature and washed with PBS. Three independent samples were collected for each group and imaged using an LSM880 confocal laser scanning microscope. Finally, ImageJ software was used to mark specific areas of the image and perform fluorescence colocalization analysis at 488 nm and 594 nm wavelengths.

[0056] 11. Nanoparticle Tracking Analysis Nanoparticle tracking analysis (NTA) was performed using a ZetaView PMX 110 (Particle Metrix) and its software (ZetaView 8.02.28). Exosomes were diluted in particle-free PBS and placed in the sample chamber. Size and concentration were then measured at a wavelength of 405 nm, and the exosome size was quantified and recorded.

[0057] 12. Transmission Electron Microscope 10 μL of exosome sample was dropped onto a copper grid and incubated at room temperature for 10 minutes. The sample was then negatively stained with 2% uranyl acetate and allowed to dry on the edge of the filter paper. The prepared sample was imaged using an H-7650 transmission electron microscope (Hitachi) at 80 kV. Mitochondria were detected using the same method as described above.

[0058] 13. Endosome Extraction H9C2-SIRT3 cells were seeded in a 6-well plate. When the cells reached an appropriate density, they were digested and resuspended in DMEM medium. The cells were centrifuged at 300 g for 10 minutes, and the supernatant was discarded. Pre-chilled HepengBio washing buffer was added, and the mixture was gently pipetted and centrifuged again at 300 g for 10 minutes. The supernatant was discarded. Pre-chilled HepengBio lysis buffer was added, vortexed for 1 minute, and then subjected to differential centrifugation in a benchtop centrifuge: 3800 g at 4°C for 10 minutes. The supernatant was retained. The supernatant was then transferred to an ultracentrifuge tube and centrifuged at 285,000 g at 4°C for 60 minutes. The supernatant was discarded. The pellet was resuspended in HepengBio high-density buffer. Next, medium-density buffer, and low-density buffer were slowly added on top of the high-density buffer, followed by lysis buffer until the tube was completely filled. Avoid shaking the tube during this process. The cells were then centrifuged at 285,000 g for 60 minutes at 4°C. A pipette was then slowly inserted into the interface between the lysis buffer and the low-density buffer, and then the low-density buffer and the medium-density buffer to obtain endosomal vesicle samples. The cells were again brought to volume with HepengBio lysis buffer and centrifuged at 285,000 g for 60 minutes at 4°C. The endosomal vesicles were then resuspended in 1 mL of HepengBio lysis buffer and stored at -80°C until further use.

[0059] 14. Endosome Nanoflow Cytometry H9C2-SIRT3 endosomes were detected by nanoflow cytometry. Endosomes were labeled with Flag-FITC and primary antibodies against Rab5a or Rab7a, respectively, and then centrifuged at 285,000 g for 60 minutes at 4°C before being recollected. Endosomes were labeled with a secondary antibody and then centrifuged at 285,000 g for 60 minutes at 4°C before being recollected. After resuspending in PBS, the endosomes were detected using 488 nm and 647 nm lasers. This assay was provided by Guangzhou Ruibei Medical Technology Co., Ltd.

[0060] 15. Statistical analysis Continuous variables are expressed as mean ± standard deviation. Statistical analyses were performed using GraphPad Prism 10.0. Comparisons between groups were analyzed using independent sample t-tests or one-way ANOVA. Differences were considered statistically significant when p < 0.05.

[0061] 2. Experimental Results 1. Four peptide sequences were screened from a 12-peptide phage peptide library, which were able to produce affinity with HSP60 recombinant protein. The peptide sequences and binding frequencies are shown in Table 3. After phage ELISA detection, the peptide sequence GCMYKASMTCCM had stronger binding ability. Figure 1 This polypeptide sequence was named G12, i.e. molecular glue G12.

[0062] Table 3 Phage-screened peptide sequences and peptide-HSP60 binding frequencies Peptide sequence Peptide binding frequency GCMYKASMTCCM (SEQ ID No. 1) 22 MCAKLGSASCCM (SEQ ID No. 2) 18 VCKPLASGHCTV (SEQ ID No. 3) 12 VCTAMGSASCCM (SEQ ID No. 4) 7 2. After confirming that G12 can bind to HSP60, the G12 sequence, Flag tag, and SIRT3 protein coding sequence were connected to the recombinant plasmid, and the SIRT3-G12 recombinant protein was expressed in cardiomyocytes to allow the protein to bind to HSP60 in a targeted manner. Since previous studies have not reported whether SIRT3-linked polypeptides have an effect on its protein structure, it is planned to connect Flag and G12 from the end of the protein sequence, but it is not certain whether to connect to the N-terminus or C-terminus of SIRT3. Therefore, these two connection methods were studied. G12 connected to the N-terminus of SIRT3 is named N + Group, connected to the C end is named C + Group, such as Figure 2 Molecular docking prediction shows that G12 can bind to HSP60 regardless of which end of SIRT3 it is connected to. Figure 3 shown.

[0063] 3. After constructing the above recombinant plasmid and transfecting H9C2 cells, the protein-protein interaction in the cells was detected by Co-IP. The results are as follows Figure 4 As shown in the figure, there is no Flag band in the untransfected cells (H9C2 group), and the SIRT3 content is significantly less than that in the N + and C + The reason is that SIRT3-G12 with Flag was overexpressed after plasmid transfection. After separation using Flag magnetic beads, it was found that the 100kDa SIRT3-HSP60 complex appeared in N + and C + In the group, it was shown that, as predicted by molecular docking, G12 could bind to HSP60 regardless of which end of SIRT3 it was connected to, which clarified the molecular glue function of G12.

[0064] 4. In order to clarify N + and C + The SIRT3 content in transfected cells (engineered gene cells), exosomes (engineered exosomes), and target cells (target cells) was analyzed. + and C + Exosomes were extracted from the cells of the target group and co-cultured with H9C2 cells treated with hypoxia and reperfusion. Mitochondria of the transfected cells and target cells were extracted. The ELISA results of Flag and SIRT3 are shown in Figure 2. Figure 5 As shown: In the transfected cells, there was no significant difference in the total Flag content between the two groups. +The Flag in the mitochondria of group A was slightly higher than that of group B + groups, but there was no statistically significant difference ( Figure 5 In exosomes, the levels of Flag and SIRT3 were not statistically different ( Figure 5 B in the target cell). However, in the target cell, C + The number of Flag in group mitochondria was significantly higher than that in group N + group, indicating that SIRT3-G12 molecules in exosomes enter mitochondria in large quantities ( Figure 5 C in the figure). Immunofluorescence also confirmed the results. SIRT3 staining was performed on the target cells, and C + There was more co-localization of SIRT3 with mitochondria in the group ( Figure 5 The above results suggest that C-terminal connection of G12 molecular glue may promote the transport of SIRT3 to mitochondria in target cells, so theoretically C + group is more conducive to mitochondrial targeted therapy.

[0065] 5. In subsequent experiments, the recombinant plasmids pcDNA3.1-SIRT3-G12-C, pcDNA3.1-ROMO1-G12-C and pcDNA3.1-TOM40-G12-C with G12 linked to the C-terminus will be used. Under transmission electron microscopy, exosomes exhibit vesicle-like structures, and double-layer membrane structures can be seen in some fields of view, such as Figure 6 As shown in A; the level of cell secretion of exosomes was detected by BCA method, and the results were as shown in Figure 6 As shown in Figure B, in a T25 culture flask with a cell density of 80%, 43.82±8.55 μg of Exo could be isolated per 1 mL of culture medium, while the yields of SIRT3-Exo, ROMO1-Exo and TOM40-Exo were 40.17±7.92, 39.20±8.92 and 41.55±6.54 μg / mL, respectively, with no statistical difference; the results of nanoparticle size tracking detection are shown in Figure 4. Figure 6 C and Figure 6 As shown in D in Figure 3, the particle size of exosomes ranges from 40 to 180 nm, and there is no significant difference in the particle size concentration between the two. The markers contained in the exosomes were detected by Western blot, and the results are shown in Figure 3. Figure 6As shown in Figure E, in H9C2 and H9C2-SIRT3 cells, compared with Exo and SIRT3-Exo, the expression levels of CD9 and ALIX in cells are lower, while the expression level of GAPDH is higher. This is because at a unit concentration, exosomes contain more related markers and basically no housekeeping proteins. SIRT3 protein in H9C2-SIRT3 cells is affected by plasmid overexpression and is 42.35% higher than that in H9C2 cells. In exosomes, the SIRT3 content in SIRT3-Exo exosomes is 13.58 times that of Exo (H9C2 cells not transfected with SIRT3) ( Figure 6 F in Figure 3); Similarly, the results of ROMO1 and TOM40 measured by ELISA showed the same trend ( Figure 6 G and Figure 6 H) These results indicate that molecular glue G12 was successfully used to construct engineered exosomes enriched with SIRT3, ROMO1, and TOM40, allowing proteins that are not naturally present in exosomes to be enriched in exosomes.

[0066] 6. After clarifying the effects of G12 attachment ends and G12 modification on the target level in exosomes, we further observed how SIRT3-G12 recombinant protein enters exosomes in H9C2-SIRT3 cells. By observing the co-localization relationship between early endosome (Rab5a), late endosome (Rab7a), and multivesicular body markers (CD63) and SIRT3 protein in H9C2 cells, and comparing the co-localization relationship between these markers and SIRT3-G12 (Flag) protein in H9C2-SIRT3 cells, the results are as follows: Figure 7 As shown, SIRT3 rarely colocalizes with Rab5a, Rab7a, or CD63 in H9C2 cells. Furthermore, SIRT3 protein, localized in mitochondria, exhibits a ribbon-like and filamentous mitochondrial morphology. This result is consistent with previous studies that have not found SIRT3 in exosomes.

[0067] In H9C2-SIRT3 cells, SIRT3-G12 with a Flag tag did not co-localize with the early endosome marker Rab5a, but its co-localization relationship with the late endosome marker Rab7a and the multivesicular body marker CD63 became increasingly stronger. Figure 8 This indicates that SIRT3-G12 mainly enters the multivesicular bodies during the stage of sorting and loading cytoplasmic proteins, and is highly enriched in the exosomes released subsequently.

[0068] Endosomes (early endosomes and late endosomes) were isolated and extracted from H9C2-SIRT3 cells using a kit and labeled with Flag-FITC and Rab5a-PC5, Flag-FITC and Rab7a-PC5, respectively. Nanoflow cytometry analysis revealed that the number of double-positive stained vesicles increased significantly in the late endosomes. Figure 9 Due to the lack of multivesicular body extraction reagents and methods, it is impossible to determine the level of SIRT3-C12 in multivesicular bodies. Figure 8 The results suggest that SIRT3-G12 is gradually loaded into multivesicular bodies during the stage of late endosome budding to form multivesicular bodies, causing CD63 + The strongest fluorescence co-localization phenomenon was observed in the multivesicular bodies of SIRT3-G12. Thus, at the organelle level, it was found that the SIRT3-G12 recombinant protein was sorted and loaded into the multivesicular bodies, resulting in a significant increase in its concentration in SIRT3-Exo exosomes.

[0069] In summary, the principle of enriching exosomes with the target protein of the present invention is as follows: Figure 10 As shown, taking SIRT3 as an example, molecular glue G12 promotes SIRT3 to bind to HSP60, so that SIRT3 is transported from the cell nucleus to the exosomes, realizing trans-organelle transport, thereby obtaining SIRT3-enriched exosomes. That is, based on the characteristics of polypeptide molecular glue targeting and binding to HSP60, the present invention can enrich the target protein that does not exist in the exosomes in the natural state into the exosomes through trans-organelle transport, thereby obtaining the target protein-enriched exosomes.

Claims

1. A method for preparing exosomes enriched with target protein based on polypeptide molecular glue, characterized in that: The gene encoding the polypeptide molecular glue G12 having an amino acid sequence as shown in any one of SEQ ID Nos. 1 to 4 is linked to the 3' end or 5' end of the target protein encoding gene to obtain a chimeric gene, and then a recombinant plasmid expressing the chimeric gene is constructed. The recombinant plasmid is then transfected into cells for expression, and exosomes are extracted to obtain the target protein; the target protein is a protein that does not exist in exosomes in its natural state.

2. The preparation method according to claim 1, characterized in that The coding gene of the polypeptide molecular glue G12 is connected to the coding gene of the target protein via a Flag tag.

3. The preparation method according to claim 1, characterized in that: The coding gene of the polypeptide molecular glue G12 is connected to the 3' end of the coding gene of the target protein.

4. The preparation method according to claim 1, characterized in that The amino acid sequence of the polypeptide molecular glue G12 is GCMYKASMTCCM.

5. The preparation method according to claim 1, characterized in that: The target protein includes SIRT3, ROMO1 or TOM40.

6. The preparation method according to claim 1, characterized in that: The template plasmid used in the recombinant plasmid is pcDNA3.

1.

7. Engineered exosomes rich in target protein prepared by the preparation method according to any one of claims 1 to 6.

8. A product, characterized in that Containing the engineered exosomes rich in the target protein according to claim 7.

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