Application of OPA1-enriched exosomes in the preparation of drugs to improve mitochondrial dysfunction

Abstract

The present invention discloses the use of OPA1-enriched exosomes in the preparation of drugs for improving mitochondrial dysfunction. The present invention obtains the CD9-targeting polypeptide P13 by screening using phage display technology, forms a fusion protein OPA1-P13 with OPA1, then constructs a recombinant plasmid expressing the fusion protein and transfects cells for co-expression, extracts exosomes, and obtains OPA1-enriched exosomes OPA1-Exo. OPA1-Exo is used to treat mitochondrial dysfunction. The results show that OPA1-Exo can activate antioxidant enzymes to reduce ROS levels in cells, promote the oxidative phosphorylation pathway to increase mitochondrial ATP content, significantly improve the mitochondrial network, and restore mitochondrial dysfunction, thereby showing obvious mitochondrial function repair potential. The present invention successfully realizes the use of OPA1 protein in the form of exosomes for the treatment of mitochondrial dysfunction.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and more specifically, relates to the use of OPA1-enriched exosomes in the preparation of a drug for improving mitochondrial dysfunction. Background Art

[0002] When the body undergoes oxidative metabolism, the ETC in the mitochondria is prone to reducing oxygen molecules to form reactive oxygen species (ROS) due to a large amount of electron leakage. Excessive ROS leads to mtDNA mutations, causing mitochondrial dysfunction, affecting mitochondrial homeostasis, and triggering various diseases.

[0003] Exosomes are tiny vesicles secreted by cells that contain nucleic acids, proteins, and lipids. They have high targeting and ability to penetrate biological barriers, low immunogenicity and high biocompatibility, multifunctional payloads and potential for combination therapy, reduced side effects and enhanced stability, natural origin, and scalability. Therefore, they can be used as drug delivery vehicles for the treatment of various diseases.

[0004] Optic Atrophy 1 (OPA1) is a mitochondrial inner membrane fusion protein involved in regulating mitochondrial fusion. Studies have shown that OPA1 plays a key role in regulating mitochondrial fusion and fission, which is crucial for maintaining mitochondrial bioenergy production and cellular metabolic homeostasis. Mitochondrial morphological changes directly affect their function. OPA1 deficiency leads to mitochondrial fragmentation, which impairs cellular energy metabolism and viability. OPA1 can mitigate cellular oxidative stress and improve diseases associated with mitochondrial dysfunction. However, OPA1 protein itself is not present in exosomes, limiting its use in exosome-based treatments for mitochondrial dysfunction. Summary of the Invention

[0005] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art and provide the use of OPA1-enriched exosomes in the preparation of a drug for improving mitochondrial dysfunction.

[0006] A second object of the present invention is to provide a use of OPA1-enriched exosomes in the preparation of a drug for treating diseases related to mitochondrial dysfunction.

[0007] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0008] The present invention first prepares OPA1-enriched exosomes (OPA1-Exo), which are first formed into a fusion protein OPA1-P13 by polypeptide P13 and OPA1, then constructing a recombinant plasmid expressing the fusion protein OPA1-P13 and transfecting cells for culture, and extracting exosomes to obtain OPA1-enriched exosomes OPA1-Exo; that is, the coding sequence of polypeptide P13 is connected to the 3' end or 5' end of the OPA1 protein coding sequence to obtain an OPA1-P13 fusion gene, then constructing a recombinant plasmid expressing the OPA1-P13 fusion gene, and then transfecting the recombinant plasmid into cells for co-expression, and extracting exosomes to obtain OPA1-enriched exosomes OPA1-Exo; the amino acid sequence of the polypeptide P13 is shown in any one of SEQ ID Nos. 1 to 4.

[0009] Peptide is a molecule formed by amino acids connected by peptide bonds. It can promote the interaction between proteins, thereby forming a stable ternary complex or enhancing existing protein-protein interactions.

[0010] CD9, also known as tetraspanin 9, belongs to the tetraspanin superfamily and is an important membrane protein. CD9 is a hallmark protein of exosomes, naturally present on the exosome membrane. It is commonly used for the detection, isolation, and identification of exosomes, and is particularly important in the targeted application of engineered exosomes.

[0011] The present invention screens out a peptide P13 that targets and binds to CD9 by incubating a phage library with CD9 protein. This peptide is then linked to the end of the OPA1 protein through genetic recombination technology to form a fusion protein, OPA1-P13, to promote the binding of OPA1 to CD9. Since CD9 is a transmembrane protein already present in exosomes, the peptide P13 can bind to the OPA1 protein and CD9, thereby helping the OPA1 protein to achieve trans-organelle transport from the cell nucleus to the exosomes, enabling it to be enriched in the exosomes. Specifically, a recombinant plasmid expressing the fusion protein OPA1-P13 is constructed (i.e., the coding sequence of the peptide P13 is linked to the 3' or 5' end of the OPA1 protein coding sequence to obtain the OPA1-P13 fusion gene, and then a recombinant plasmid expressing the OPA1-P13 fusion gene is constructed), and cells are transfected and cultured; the peptide P13 can link to CD9 in the OPA1-targeted exosomes, promoting the binding of OPA1 to CD9, and obtaining OPA1-enriched exosomes (OPA1-Exo). When the amino acid sequences of the OPA1 protein and polypeptide P13 are known, those skilled in the art can obtain the coding gene sequences of the OPA1 protein and polypeptide P13 using conventional technical means and optimize them according to the host codon preference.

[0012] Furthermore, the method involves constructing a recombinant plasmid expressing the vesicle secretion-promoting gene RAB31. The recombinant plasmid expressing the OPA1-P13 fusion gene and the exosome secretion-promoting RAB31 recombinant plasmid are then co-transfected into cells for co-expression. Exosomes are then extracted to produce OPA1-enriched exosomes (OPA1-RAB31-Exo). RAB31 can further increase exosome yield, thereby increasing the amount of OPA1 protein per milliliter of exosomes. RAB31 is a small GTPase belonging to the RAS oncogene family (RAB subfamily) that plays a key role in intracellular membrane trafficking and signaling regulation. As a member of the Rab family, RAB31 is activated by GTP binding, recruiting downstream effector proteins to regulate vesicle formation, trafficking, and fusion with target membranes. It is crucial for the functional integrity of the Golgi apparatus and the trans-Golgi network (TGN). When the amino acid sequence of the RAB31 protein is known, those skilled in the art can obtain the coding gene sequence of the RAB31 protein using conventional techniques and optimize it according to the codon preference of the host.

[0013] The present invention applied the OPA1-enriched exosomes OPA1-Exo to a 293T cell model of H2O2-induced oxidative damage, and detected the antioxidant activity indicators of cell mitochondria. The results showed that OPA1-enriched exosomes OPA1-Exo had a significant improvement effect on mitochondrial dysfunction, indicating that OPA1 protein successfully plays a role in the treatment of mitochondrial dysfunction through exosome expression.

[0014] Specifically, the present invention added the OPA1-enriched exosomes OPA1-Exo to a 293T cell model of H2O2-induced oxidative damage to evaluate its effect on cellular antioxidant stress. Indicators such as the activity of intracellular antioxidant enzymes SOD1, SOD2, CAT, and GSH-Px, as well as ROS and ATP levels were measured. The results showed that OPA1-Exo reduced ROS levels in 293T cells by activating antioxidant enzymes, increased ATP content in mitochondria by promoting the oxidative phosphorylation pathway, and improved mitochondrial network structure, restoring mitochondrial function. This indicates that OPA1-Exo has application value in improving mitochondrial dysfunction or treating related diseases caused by mitochondrial dysfunction.

[0015] Therefore, the present invention first provides the use of the above-mentioned OPA1-enriched exosomes OPA1-Exo in the preparation of a drug for improving mitochondrial dysfunction.

[0016] The present invention also provides the use of the OPA1-enriched exosomes OPA1-Exo in the preparation of drugs for treating related diseases caused by mitochondrial dysfunction.

[0017] Preferably, the present invention also provides the use of the aforementioned OPA1-enriched exosomes, OPA1-RAB31-Exo, in the preparation of a drug for ameliorating mitochondrial dysfunction or for the preparation of a drug for treating diseases caused by mitochondrial dysfunction. Because RAB31 can further increase the amount of OPA1 protein in exosomes, OPA1-RAB31-Exo can further enhance the therapeutic effect on mitochondrial dysfunction compared to OPA1-Exo.

[0018] Furthermore, the mitochondrial dysfunction is mitochondrial dysfunction caused by oxidative damage.

[0019] Furthermore, the drug eliminates ROS by upregulating the expression of antioxidant enzymes in the subject's body, promotes the oxidative phosphorylation pathway in the subject's body to increase ATP content, improves the subject's mitochondrial network structure and restores mitochondrial function, thereby improving mitochondrial dysfunction.

[0020] Furthermore, the subject is selected from mammals.

[0021] Furthermore, the mammal is selected from mice, cats, dogs, pigs, cows, horses, sheep, monkeys and humans.

[0022] Furthermore, the medicine also includes other pharmaceutically acceptable excipients.

[0023] Furthermore, the coding sequence of the polypeptide P13 is connected to the coding sequence of the OPA1 protein via a Flag tag, that is, the polypeptide P13 is connected to the OPA1 protein via a Flag tag.

[0024] Furthermore, the coding sequence of the polypeptide P13 is connected to the 3' end of the coding sequence of the OPA1 protein, that is, the polypeptide P13 is connected to the C-terminus of the OPA1 protein. The present invention constructs pcDNA3.1-OPA1-P13-C and pcDNA3.1-OPA1-P13-N recombinant plasmids, and connects the polypeptide P13 to the C-terminus or N-terminus of the OPA1 protein through genetic recombination technology to form an OPA1-polypeptide P13 fusion protein expression system. After transfecting cells (engineered gene cells), OPA1-enriched exosomes were successfully constructed. By analyzing the content of OPA1 in the N+ and C+ groups in transfected cells (engineered gene cells), exosomes (engineered exosomes), and target cells (target cells), C + There was more co-localization of OPA1 with mitochondria in the WT group, indicating that the C-terminal linked peptide P13 can more effectively promote the translocation of OPA1 to mitochondria in target cells.

[0025] Furthermore, the amino acid sequence of the polypeptide P13 is shown in SEQ ID No. 1: GKYCMATCCSMMA. The present invention screened four polypeptide sequences from a 12-peptide phage peptide library, which were able to produce affinity with the CD9 recombinant protein. Phage ELISA testing showed that the polypeptide with the sequence GKYCMATCCSMMA had stronger binding ability.

[0026] Furthermore, the template plasmid of the recombinant plasmid is pcDNA3.1, that is, pcDNA3.1-OPA1-P13 and pcDNA3.1-RAB31 recombinant plasmids are constructed.

[0027] Furthermore, the cells are 293T cells.

[0028] Furthermore, the exosomes are extracted by ultracentrifugation.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention provides the use of OPA1-enriched exosomes in the preparation of a drug for ameliorating mitochondrial dysfunction. This method involves incubating a phage library with CD9 recombinant protein to screen for the targeting peptide P13. The peptide P13 is then combined with the OPA1 protein to form a fusion protein, OPA1-P13. A recombinant plasmid expressing the fusion protein, OPA1-P13, is then transfected into cells for culture. The peptide P13 can bind to CD9 in OPA1-targeted exosomes, promoting OPA1-CD9 binding and enabling transcellular transport and enrichment of the OPA1 protein from the cell interior to the exosomes. The exosomes are then extracted to obtain OPA1-enriched exosomes, OPA1-Exo. OPA1-Exo has been used to treat mitochondrial dysfunction. Results show that OPA1-Exo can activate antioxidant enzymes, reduce ROS levels in cells, promote the oxidative phosphorylation pathway, and increase mitochondrial ATP content. This significantly improves the mitochondrial network and restores mitochondrial dysfunction, demonstrating significant potential for mitochondrial functional restoration. This invention successfully utilizes OPA1 protein in the form of exosomes for the treatment of mitochondrial dysfunction. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The results are shown for screening CD9 affinity peptides using phage ELISA in Example 1. *: p<0.05, **: p<0.01.

[0032] Figure 2 Schematic diagram of two modes of connection between P13 and the end of OPA1 in Example 1.

[0033] Figure 3 For the Co-IP detection N in Example 1 + and C+ Protein binding of recombinant protein to CD9.

[0034] Figure 4 The ELISA method for determining the content of Flag and OPA1 in Example 1 is as follows. Figure 4 A shows the Flag level in 293T-OPA1-transfected cells; B shows the Flag and OPA1 levels in exosomes secreted by 293T-OPA1 cells; C shows the Flag level in 293T cells after 3 days of co-culture with exosomes. *: p < 0.05, ns: p > 0.05.

[0035] Figure 5 This is the detection of engineered exosomes in Example 1. Figure 5 Figure (A) shows the microstructure of Exo, OPA1-Exo, and OPA1-RAB31-Exo cells under transmission electron microscopy (scale bar = 100 nm). Figure (B) shows the secretion levels of Exo, OPA1-Exo, and OPA1-RAB31-Exo cells as assessed by BCA assay (ns: p > 0.05). Figure (C) shows the particle size distribution of Exo, OPA1-Exo, and OPA1-RAB31-Exo cells as assessed by nanoparticle tracking. Figures (D) and (E) show Western blot analysis of exosome markers and OPA1 protein in Exo, OPA1-Exo, and OPA1-RAB31-Exo cells, as well as quantitative analysis of OPA1 expression based on grayscale values. #: p < 0.05 compared with the 293T group; *: p < 0.05 compared with the Exo group; **: p < 0.01 compared with the Exo group.

[0036] Figure 6 The expression level of antioxidant enzymes and ROS content in Example 2 were detected. Figure 6 Figure (A) shows the detection of SOD1, SOD2, CAT, and GSH-Px activities in normal 293T cells (Control group), oxidatively damaged 293T cells (H2O2 group), and oxidatively damaged 293T cells treated with OPA1-RAB31-Exo (H2O2-OPA1-RAB31-Exo group); Figure (B) shows the determination of cellular ROS by staining with a CM-H2DCFDA probe and measuring it by immunofluorescence and FACS (Scale bar = 50 μm). *: P < 0.05, **: P < 0.01.

[0037] Figure 7 This is the ATP level detection in Example 2. Wherein, **: P < 0.01.

[0038] Figure 8This is a diagram of the fluorescence detection (Mito-Tracker Green fluorescent probe) of the mitochondrial network structure in Example 2. Figure 8 A is the mitochondrial structure of 293T cells induced by H2O2; B is the mitochondrial structure of 293T cells after OPA1-Exo treatment, scale bar = 50 μm.

[0039] Figure 9 Schematic diagram of the present invention. DETAILED DESCRIPTION

[0040] 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.

[0041] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0042] 1. Experimental Materials

[0043] Table 1 Cell sources

[0044] cell Feature Description source 293T human embryonic kidney cells Merck KGaA <![CDATA[293T-OPA1(N + )]]> Human embryonic kidney cells, transfected with pcDNA3.1-OPA1-P13-N plasmid The present invention is constructed <![CDATA[293T-OPA1(C + )]]> Human embryonic kidney cells, transfected with pcDNA3.1-OPA1-P13-C plasmid The present invention is constructed 293T-OPA1-RAB31 Human embryonic kidney cells, transfected with pcDNA3.1-OPA1-P13-C and pcDNA3.1-RAB31 plasmids The present invention is constructed E. coli DH5α Plasmid construction host bacteria Laboratory collection

[0045] Table 2 Plasmid sources

[0046] plasmids Genetic markers and construction source pcDNA3.1-OPA1-P13-N plasmid KpnI and XbaI restriction endonuclease sites were designed at both ends of the P13-Flag-OPA1 sequence. Wuhan Jinkairui pcDNA3.1-OPA1-P13-C plasmid KpnI and XbaI restriction endonuclease sites were designed at both ends of the OPA1-Flag-P13 sequence. Wuhan Jinkairui pcDNA3.1-RAB31 plasmid KpnI and XbaI restriction endonuclease sites were designed at both ends of the RAB31 sequence Wuhan Jinkairui

[0047] DMEM powder was purchased from Gibco; antioxidant enzyme activity assay kit was purchased from Dojindo Molecular Technology Inc; fetal bovine serum (FBS) was purchased from ThermoFisher; and HEK293T cells were stored in the laboratory.

[0048] 2. Experimental methods

[0049] 2.1 Cell Culture

[0050] (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.

[0051] (2) Cell culture conditions: 293T cells were cultured in DMEM. Complete culture medium was prepared by adding 50 mL of filtered fetal bovine serum (FBS) and 5 mL of penicillin-streptomycin (DST) to every 445 mL of DMEM. The cells were cultured in a sterile cell culture incubator at 37°C and 5% CO2. Cell growth was regularly observed to determine whether further treatment was necessary. After the cells reached an appropriate density, they were digested with sterile trypsin and then passaged, plated, and frozen for subsequent experimental needs.

[0052] (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.

[0053] (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.

[0054] (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.

[0055] Example 1 Preparation of OPA1-enriched exosomes

[0056] 1. Experimental Methods

[0057] 1. Targeting peptide screening

[0058] 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 CD9 recombinant protein for 1 hour at room temperature. Unbound phage were removed by washing the CD9 protein 10 times with bovine serum albumin (BSA) / Tween wash buffer. Phage bound to the CD9 protein 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 CD9 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 described above. After the third round of screening, the eluted phage were titered, and 40 clones were randomly selected for sequencing to determine the sequence of the CD9-binding peptide.

[0059] 2. Affinity Verification

[0060] The binding capacity and specificity of the selected phages for CD9 were determined by enzyme-linked immunosorbent assay (ELISA). 150 μL of 100 μg / mL CD9 (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.

[0061] 3. Plasmid synthesis and transfection

[0062] Using the pcDNA3.1 plasmid as a template, KpnI and XbaI restriction endonuclease sites were designed at both ends of the P13-Flag-OPA1 and OPA1-Flag-P13 sequences. Recombinant plasmids, designated pcDNA3.1-OPA1-P13-C (i.e., pcDNA3.1-OPA1-Flag-P13) and pcDNA3.1-OPA1-P13-N (i.e., pcDNA3.1-P13-Flag-OPA1), were constructed and named pcDNA3.1-OPA1-P13-N (i.e., pcDNA3.1-P13-Flag-OPA1). 293T cells were evenly seeded in a 6-well plate and cultured to a cell density of 30% to 50%. In a 1.5 mL centrifuge tube, 200 μL of serum-free medium and 3 μg of each of the above recombinant plasmids were added. In another centrifuge tube, 200 μL of serum-free medium and 6 μL of transfect-mate were mixed. The transfection method mediated by Lipofectamine 2000 was used. The reaction was carried out at room temperature for 5 minutes. After that, the contents of the two tubes were combined and the reaction was continued for 20 minutes. The transfection complex was added to a 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 the cells were collected after 48 hours of continuous culture for subsequent experiments. The cells after pcDNA3.1-OPA1-P13-N transfection were named N + The cells transfected with pcDNA3.1-OPA1-P13-C were named C +(Same as the subsequent 293T-OPA1.) Simultaneously, BstXⅠ and NotⅠ restriction sites were introduced at both ends of the RAB31 gene. After double digestion of the plasmid pcDNA3.1 with the corresponding endonucleases, it was ligated with the target gene RAB31 fragment to construct the pcDNA3.1-RAB31 recombinant plasmid. Following the above method, the pcDNA3.1-OPA1-P13-C and pcDNA3.1-RAB31 recombinant plasmids were co-transfected into cells, and the resulting cells were named 293T-OPA1-RAB31.

[0063] 4. Co-immunoprecipitation (Co-IP)

[0064] Fusion expression of OPA1 and CD9 was detected using co-immunoprecipitation. Transfected cells were lysed, proteins were extracted, and incubated with the primary antibody (anti-Flag) overnight at 4°C. Incubation was performed 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 tested were OPA1 (diluted 1:1000 in TBST buffer), CD9 (diluted 1:1000 in TBST buffer), and Flag (diluted 1:1000 in TBST buffer).

[0065] 5. Exosome Extraction

[0066] After incubating 293T-OPA1 cells, 293T-OPA1-RAB31 cells, and 293T 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 0.22 μm filter from Merck Millipore. 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. The exosomes extracted from 293T were named Exo, the exosomes extracted from 293T-OPA1 were named OPA1-Exo, and the exosomes extracted from 293T-OPA1-RAB31 were named OPA1-RAB31-Exo.

[0067] 6. ELISA detection of mitochondrial internalization

[0068] 293T cells (serving as target cells) were seeded into culture dishes and cultured to an appropriate confluence (70%-90%) to ensure optimal cell growth and exosome uptake during co-culture. Exosomes extracted from the N+ and C+ engineered cells were added to the 293T cell culture system, and co-cultured in a cell culture incubator. After co-culture, the transfected cells (N+ and C+ engineered cells) and target cells (293T cells) were collected. Mitochondrial proteins were extracted from both transfected and target cells using a mitochondrial extraction kit. Proteins were then extracted from the transfected cells, exosomes, and target cells.

[0069] The Flag and OPA1 protein levels in each sample were determined using the Flag and OPA1 ELISA kit instructions. The absorbance of the ELISA plate was read using a microplate reader, and the concentrations of Flag and OPA1 in each sample were calculated based on the standard curve.

[0070] 7. Western Blot

[0071] (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.

[0072] (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.

[0073] (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.

[0074] (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.

[0075] (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.

[0076] (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.

[0077] (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.

[0078] According to the recommendations of the "Information Standards for Exosome Research (MISEV2018)", the present invention uses Western Blot to detect exosome positive markers OPA1, CD9 and ALIX, and the corresponding antibodies used are diluted at ratios of 1:1000, 1:500 and 1:1000, respectively.

[0079] 8. Nanoparticle Tracking Analysis

[0080] 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.

[0081] 9. Transmission Electron Microscopy

[0082] 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.

[0083] 10. Statistical analysis

[0084] 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.

[0085] 2. Experimental Results

[0086] 1. Four peptide sequences were screened from a 12-peptide phage peptide library, which were able to produce affinity with CD9 recombinant protein. The peptide sequences and binding frequencies are shown in Table 3. After phage ELISA detection, the peptide with the sequence GKYCMATCCSMMA had stronger binding ability. Figure 1 This polypeptide sequence was named P13.

[0087] Table 3 Phage-screened peptide sequences and peptide-CD9 binding frequencies

[0088] Peptide sequence Peptide binding frequency GKYCMATCCSMMA (SEQ ID No.1) 25 AGMTCMASCCKYM (SEQ ID No.2) 19 GKMTCYCMASCMG (SEQ ID No.3) 16 GCASMKYCCMMTS (SEQ ID No.4) 10

[0089] 2. After confirming that P13 can bind to CD9, the P13 sequence, Flag tag, and OPA1 protein coding sequence were connected to the recombinant plasmid, and the OPA1-P13 recombinant protein was expressed in 293T cells to allow the protein to bind to CD9 in a targeted manner. Since previous studies have not reported whether OPA1-linked polypeptides affect its protein structure, it is planned to connect Flag and P13 from the ends of the protein sequence, but it is not certain whether to connect to the N-terminus or C-terminus of OPA1. Therefore, these two connection methods were studied. P13 connected to the N-terminus of OPA1 is named N + Group, connected to the C end is named C + Group, such as Figure 2 shown.

[0090] 3. After constructing the above recombinant plasmid and transfecting 293T cells, the protein-protein interaction in the cells was detected by Co-IP. The results are as follows Figure 3 As shown in the figure, there is no Flag band in the untransfected cells (293T group), and the OPA1 content is significantly less than that in the N + and C + The reason is that the OPA1-P13 with Flag was overexpressed after plasmid transfection. After separation using Flag magnetic beads, the 122 kDa OPA1-CD9 complex was found in N + and C + In the group, it was shown that P13 could bind to CD9 no matter which end of OPA1 it was connected to, which clarified the role of P13.

[0091] 4. In order to clarify N + and C + The content of OPA1 in transfected cells (engineered gene cells), exosomes (engineered exosomes), and target cells (target cells) was analyzed from N + and C +Exosomes were extracted from the cells of the target group, and the exosomes were co-cultured with 293T cells. Mitochondria of the transfected cells and target cells were extracted. The ELISA results of Flag and OPA1 are shown in Figure 2. Figure 4 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 4 In exosomes, there was no statistical difference in the levels of Flag and OPA1 ( Figure 4 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 OPA1-P13 molecules in exosomes enter mitochondria in large quantities ( Figure 4 The above results suggest that C-terminal connection with P13 may promote the transport of OPA1 to mitochondria in target cells, so theoretically C + group is more conducive to mitochondrial targeted therapy.

[0092] 5. In subsequent experiments, the recombinant plasmid pcDNA3.1-OPA1-P13-C with P13 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 5 As shown in A. The level of cell secretion of exosomes was detected by BCA method, and the results were as shown in Figure 5 As shown in Figure B, in a T25 culture flask with a cell density of 80%, 39.55±7.25 μg of Exo was isolated per 1 mL of culture medium, while the yield of OPA1-Exo was 42.87±6.97 μg / mL and the yield of OPA1-RAB31-Exo was 40.77±6.5 μg / mL, with no significant statistical difference between the three. The results of nanoparticle size tracking detection are shown in Figure 2. Figure 5 As shown in Figure C, the particle size range of Exo, OPA1-Exo and OPA1-RAB31-Exo is 40~150 nm, and there is no significant difference in the particle size concentration of the three. The markers contained in the exosomes were detected by Western blot. The results are as follows Figure 5As shown in Figure D, in 293T, 293T-OPA1, and 293T-OPA1-RAB31 cells, compared with Exo, OPA1-Exo, and OPA1-RAB31-Exo, the expression levels of CD9 and ALIX of the cells were lower, while the expression level of GAPDH was higher. This is because at a unit concentration, exosomes contain more related markers, while exosomes basically do not contain housekeeping proteins. OPA1 protein was affected by plasmid overexpression in 293T-OPA1 and 293T-OPA1-RAB31 cells, and was 40.34% and 39.22% higher than that in 293T cells, respectively. In exosomes, the OPA1 content in OPA1-Exo exosomes was 11 times that of Exo, and the OPA1 content in OPA1-RAB31-Exo exosomes was further increased compared with OPA1-Exo exosomes, which was 16.1 times that of Exo ( Figure 5 This result indicates that the peptide P13-based strategy has successfully constructed OPA1-enriched engineered exosomes.

[0093] Example 2 In vitro study of OPA1-enriched exosomes to improve H2O2-induced mitochondrial dysfunction in 293T cells

[0094] 1. Experimental Methods

[0095] 1. Establishment of cell model

[0096] 293T cells were cultured in DMEM (GIBCO BRL) containing 10% heat-inactivated fetal bovine serum (FBS) and 1% antibiotic / antimycotic solution (GIBCO BRL). 293T cells were exposed to 500 μM H2O2 in FBS-free DMEM for 30 min to induce oxidative stress, and then the protection group was treated with 1×10 7 cfu / ml were suspended in DMEM without FBS. 6 cells / 100 mm 2 The cells were seeded in culture dishes at a density of 100 μg / mL and then cultured with OPA1-Exo.

[0097] The cell experiment was divided into three groups: blank group (Control), H2O2 group, and H2O2 plus OPA1-Exo group (H2O2-OPA1-Exo).

[0098] 2. Detection of antioxidant enzymes and ROS

[0099] SOD1, SOD2, CAT, and GSH-Px activity were measured according to the kit instructions (Dojindo Molecular Technology Inc.). Intracellular ROS production was quantified using the cell-permeable, oxidation-sensitive fluorescent probe CM-H2DCFDA (Molecular Probes). 293T cells co-cultured with OPA1-Exo were treated with up to 500 μM H2O2 for 16 hours. 293T cells were plated at 2.5 × 10 cells per well. 5 Cells were seeded into six-well plates in complete culture medium at a density of 100 cells / well and pretreated with DMEM containing 1 μM CM-H2DCFDA for 30 minutes at 37°C. Cells were harvested, washed twice with DMEM, resuspended in DMEM, and analyzed on a FACSCalibur flow cytometer (BD Biosciences). Cells were cultured in 11 cm 2 Cells were plated in normal culture medium (BD Biosciences) and treated with 1 μM CM-H2DCFDA for 30 minutes. The cells were washed three times in normal culture medium and analyzed using a Leica laser scanning confocal microscope at 37°C using an excitation wavelength of 488 nm and an emission wavelength of 500–540 nm.

[0100] 3. ATP level detection

[0101] Follow the ATP assay kit instructions. First, aspirate the culture medium and add 200 μL of lysis buffer to each well of a 6-well plate. After repeated pipetting, lyse the cells and centrifuge at 12,000 g for 5 minutes at 4°C. Remove the supernatant for subsequent measurement. Thaw the reagents on ice and dilute the ATP standard solution with ATP assay lysis buffer to concentrations of 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 μM. Add 100 μL of ATP assay working solution to a dark-protected 96-well plate and let it stand at room temperature for 3-5 minutes to deplete background ATP and reduce background. Add 20 μL of the test solution or standard solution to each well and quickly mix. Detect chemiluminescence using a full-wavelength multi-function microplate reader using the luminometer function. Plot a standard curve and calculate the ATP content of each cell group based on the luminescence values.

[0102] 4. Fluorescence detection of mitochondrial network structure (Mito-Tracker Green fluorescent probe)

[0103] Use anhydrous DMSO to prepare Mito-Tracker Green solution into a 1 mM stock working solution and store it at -20 °C in the dark for future use. 5The cells were transferred to culture dishes at a density of 100 μg / mL and cultured in a conventional manner. After culturing for 24 h, the culture medium was removed and Mito-Tracker Green staining solution preheated to 37°C was added for incubation for 2 h. After the incubation, the staining solution was replaced with fresh culture medium and the mitochondrial network structure was observed under a laser confocal microscope.

[0104] 5. Statistical processing

[0105] 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.

[0106] 2. Experimental Results

[0107] 1. OPA1-Exo activates antioxidant enzymes to reduce ROS levels in 293T cells

[0108] In order to study the effect of OPA1-Exo on the antioxidant activity of mammalian cell mitochondria, 293T cells were first treated with hydrogen peroxide to establish an oxidative damage cell model. Then, OPA1-Exo and H2O2 were added to the 293T cell culture as the experimental group and the control group, respectively. Figure 6 As shown in the figure, it was found that the antioxidant enzyme SOD1 in the experimental group was restored compared with the control group, and the expression levels of SOD2 and CAT were significantly increased ( Figure 6 A in the figure), CM-H2DCFDA probe staining was used to detect cell ROS, and the quantitative analysis graph showed that ROS in the experimental group was significantly reduced ( Figure 6 Therefore, our data suggest that OPA1-Exo can clear intracellular ROS by upregulating the expression of antioxidant enzymes, and its action pathway is as follows Figure 9 shown.

[0109] 2. OPA1-Exo promotes the oxidative phosphorylation pathway to increase ATP content

[0110] Due to mitochondrial dysfunction, the oxidative phosphorylation pathway of cells is impaired, the anaerobic glycolysis pathway of cells is enhanced, and the ATP content decreases accordingly. Therefore, we studied whether OPA1-Exo affects the oxidative phosphorylation pathway to increase ATP content. To this end, OPA1-Exo was transferred into 293T cells. The ATP level in the cells was measured using an ATP kit. The results are shown in Figure 2. Figure 7 As shown, the ATP level in cells was significantly increased after co-culture with OPA1-Exo, indicating that OPA1-Exo strengthened the oxidative phosphorylation pathway of cells and thus increased the ATP content.

[0111] 3. OPA1-Exo improves mitochondrial network structure

[0112] Mitochondrial dysfunction can lead to the destruction of mitochondrial network structure, and the mitochondrial morphology usually changes from an elongated tubular structure to a fragmented spherical structure. After we performed Mito-Tracker Green fluorescence staining on each group of cells, we observed the following under fluorescence microscopy: Figure 8 As shown in A, the mitochondria of M293T cells induced by H2O2 were fragmented and incomplete in structure. However, when OPA1-Exo was transferred into 293T cells, it was found that the mitochondria were interconnected and showed a network shape, as shown in Figure 1. Figure 8 As shown in Figure B, OPA1-Exo can significantly improve the mitochondrial network and restore mitochondrial dysfunction. Further in vitro studies of mitochondrial dysfunction using OPA1-RAB31-Exo, following the above method, showed that OPA1-RAB31-Exo can better ameliorate H2O2-induced mitochondrial dysfunction compared to OPA1-Exo. This present invention successfully utilizes OPA1 protein in the form of exosomes for the treatment of mitochondrial dysfunction.

Claims

1. An OPA1-enriched exosome, characterized in that The preparation method of the OPA1-enriched exosomes is to connect the coding sequence of the polypeptide P13 to the 3' end or 5' end of the OPA1 protein coding sequence to obtain an OPA1-P13 fusion gene, then construct a recombinant plasmid expressing the OPA1-P13 fusion gene, and then transfect the recombinant plasmid into cells for co-expression, and extract exosomes to obtain the exosomes; the amino acid sequence of the polypeptide P13 is shown in SEQ ID No.

1.

2. The OPA1-enriched exosomes according to claim 1, characterized in that The preparation method of the OPA1-enriched exosomes further includes constructing a RAB31 recombinant plasmid, then co-transfecting the recombinant plasmid expressing the OPA1-P13 fusion gene and the RAB31 recombinant plasmid into cells for co-expression, and extracting exosomes to obtain OPA1-enriched exosomes.

3. The OPA1-enriched exosomes according to claim 1, characterized in that The amino acid sequence of the polypeptide P13 is GKYCMATCCSMMA.

4. The OPA1-enriched exosomes according to claim 1, characterized in that The coding sequence of the polypeptide P13 is connected to the 3' end of the coding sequence of the OPA1 protein.

5. The OPA1-enriched exosomes according to claim 1, characterized in that The coding sequence of the polypeptide P13 is connected to the coding sequence of the OPA1 protein via a Flag tag.

6. The OPA1-enriched exosomes according to claim 1, characterized in that The template plasmid of the recombinant plasmid is pcDNA3.1.

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