Preparation method and application of mff binding mts-like peptide engineered targeting mitochondria-derived vesicles

Abstract

The application discloses a preparation method and application of MFF combined MTS-like peptide engineered mitochondria-derived vesicles, and relates to the technical field of biology.The application screens a polypeptide MTS-like targeting mitochondria TOM20, forms a recombinant protein MFF-MTS-like by combining the MTS-like with the MFF, constructs a recombinant plasmid expressing the recombinant protein, transfects cells for culture, extracts mitochondria-derived vesicles, extracts mtDNA from L-O2, introduces the mtDNA into MFF-MDV, and thus obtains engineered mitochondria-derived vesicles MFF-MDV rich in mtDNA.The results show that the MFF-MDV rich in mtDNA can effectively improve the level of mitochondrial oxidative phosphorylation, enhance the activity of antioxidant enzymes and reduce ROS, restore the mitochondrial membrane potential, and finally improve the mitochondrial network structure, and thus shows obvious potential of repairing mitochondria function and effect of improving mitochondrial dysfunction of aging cells.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a method for preparing MFF-binding MTS-like peptide-engineered targeted mitochondrial-derived vesicles and its application in anti-cellular aging. Background Technology

[0002] Mitochondrial dysfunction is a common phenomenon in the aging process. With age, the production of reactive oxygen species (ROS) in mitochondria increases, triggering oxidative stress and leading to oxidative damage to mitochondrial DNA, lipids, and proteins. Increased mutation rates in mitochondrial DNA can cause errors in the function of encoded enzyme subunits, impairing mitochondrial oxidative phosphorylation and resulting in insufficient cellular energy supply. Due to the secretion of age-related secretory phenotypes, the PINK1 / Parking ubiquitin pathway-mediated mitophagy function declines, leading to reduced clearance efficiency of damaged mitochondria. The decreased efficiency of mitophagy in senescent cells leads to the accumulation of damaged mitochondria, further exacerbating the cellular senescence process. There is a bidirectional relationship between mitochondrial dysfunction and cellular senescence; mitochondrial damage is not only a consequence of aging but also a driving factor. Therefore, improving mitochondrial dysfunction holds promise for delaying the progression of age-related diseases.

[0003] Mitochondrial DNA (mtDNA) mutations are a significant inducing factor of cellular senescence. mtDNA mutations disrupt the normal function of the mitochondrial electron transport chain, causing abnormal electron transport and leading to electron leakage. The leaked electrons react with oxygen molecules to generate large amounts of reactive oxygen species (ROS), such as superoxide anions and hydrogen peroxide. ROS are highly oxidizing and cause oxidative damage to intracellular macromolecules, such as DNA, proteins, and lipids. Furthermore, senescent cells may contain both normal and mutated mtDNA, exhibiting heterogeneity. The proportion of mutated mtDNA may change with cell division and proliferation; when this proportion exceeds a certain threshold, it can lead to cellular dysfunction and disease. Simultaneously, the high mtDNA mutation rate in senescent cells makes them prone to mitochondrial diseases. Therefore, interventions targeting mtDNA mutations and cellular senescence may provide new strategies for delaying aging and preventing disease.

[0004] Mitochondrial-derived vesicles (MDVs) are important intracellular membrane vesicles formed by the outer mitochondrial membrane, or both the inner and outer membranes and matrix contents, possessing a variety of biological functions. These functions include maintaining mitochondrial mass, enhancing antioxidant and anti-infection capabilities, promoting intercellular communication, and regulating cellular metabolism and immune responses, enabling them to play a crucial role in normal physiological processes and are also closely related to the occurrence and development of various diseases. Therefore, appropriate regulation of MDVs may be a potential therapeutic target for related diseases, which is of great significance for understanding mitochondrial biology and developing new disease treatment strategies. However, physiologically, MDV production is extremely low, insufficient to achieve therapeutic efficacy; therefore, increasing MDV production is a key issue for its potential as a drug.

[0005] Mitochondrial fission factor (MFF) is primarily responsible for regulating mitochondrial division. MFF binds to DRP1, promoting DRP1 aggregation and activity on the mitochondrial membrane. Subsequently, DRP1 drives mitochondrial membrane division through GTP hydrolysis. For example, when cells require more mitochondria to meet energy demands, MFF plays a role in promoting mitochondrial division. Mitochondrial vesicles (MDVs) are small vesicles formed by mitochondria under stress conditions to clear damaged components or perform specific signal transduction. MFF-promoted mitochondrial division can increase the production of outer membrane MDVs, thus increasing MDV yield. Mitochondrial targeting sequences (MTS-like sequences) are key signal peptides that guide the precise targeting of nuclear genome-encoded proteins to mitochondria. They are typically located at the N-terminus (20-80 amino acids), with a few located at the C-terminus or internally. They form an amphiphilic α-helix, rich in positively charged amino acids on one side and hydrophobic amino acids on the other. This structure allows the hydrophobic region to bind to the lipid bilayer of the mitochondrial membrane, while the hydrophilic region interacts with negatively charged components on the membrane surface, which is crucial for their targeting of mitochondria. MTS is recognized and bound to the transport complex (TOM complex) on the outer mitochondrial membrane. By binding to MTS-like peptides, it can effectively target mitochondria and precisely deliver drugs to the lesion site. In the application of mitochondrial-derived vesicle targeted engineering, the use of MTS for targeted modification can achieve precise targeted delivery of mitochondrial-derived vesicles, thereby improving therapeutic efficacy and reducing side effects, which has important scientific research and clinical significance. Summary of the Invention

[0006] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide the application of MFF engineered mitochondrial-derived vesicles rich in mtDNA in the preparation of drugs for treating mitochondrial dysfunction in senescent cells.

[0007] A second objective of this invention is to provide the use of MFF dual-gene engineered mitochondrial-derived vesicles rich in mtDNA in the preparation of medicaments for treating cellular senescence caused by mitochondrial dysfunction.

[0008] The above-mentioned objective of this invention is achieved through the following technical solution:

[0009] This invention screens out a peptide MTS-like protein that targets and binds to mitochondrial TOM20 by incubating a phage library with recombinant mitochondrial proteins. This peptide is then linked to the 3' end of the MFF protein using gene recombination technology to form a recombinant protein MFF-MTS-like protein, promoting MFF binding to mitochondria. Since mitochondria are molecular proteins naturally present in mitochondrial-derived vesicles, the peptide MTS-like protein can bind MDVs to mitochondria, thus helping MDVs target the mitochondria of the target cell. mtDNA is extracted from L-O2 and introduced into MFF-MDVs, resulting in engineered mitochondrial-derived vesicles (MFF-MDVs) rich in mtDNA. Specifically, a recombinant plasmid expressing the recombinant protein MFF-MTS-like protein is constructed (i.e., the coding gene for the peptide MTS-like protein is linked to the 3' end of the coding gene for the MFF protein to obtain a chimeric gene, and then a recombinant plasmid expressing this chimeric gene is constructed). The plasmid is transfected into cells for culture. The MDV with the peptide MTS-like protein linked to MFF can target mitochondria, promoting MDV binding to mitochondria.

[0010] L-O2 cells are derived from normal human liver tissue and have typical characteristics of hepatocytes, such as secreting albumin and synthesizing urea. They are often used to simulate the physiological or pathological processes of normal hepatocytes.

[0011] This invention constructs an MTS-like peptide coding gene linked to the 3' end of the MFF protein coding gene, i.e., the MTS-like peptide is linked to the C-terminus of the MFF protein. This invention constructs the pcDNA3.1-MFF-MTS-like-C recombinant plasmid, and links the MTS-like peptide to the C-terminus of the MFF protein using gene recombination technology, forming an MFF-MTS peptide fusion protein expression system. After transfecting cells (engineered cells) with the plasmid, engineered mitochondrial-derived vesicles rich in MFF (MFF / MTS-MDV) were successfully constructed.

[0012] This invention applies engineered mitochondrial-derived vesicles (MFF-MDVs) to an EtBr-induced senescent cell model of mitochondrial dysfunction, and detects the antioxidant activity indicators and mitochondrial signaling pathway protein expression in the cells. The results show that engineered mitochondrial-derived vesicles (MFF-MDVs) have a therapeutic effect on senescent model cells, indicating that MFF protein has successfully played a role in the treatment of mitochondrial dysfunction in senescent cells through the expression of mitochondrial-derived vesicles, providing a new target for the treatment of aging-related diseases.

[0013] Specifically, this invention incorporates the engineered mitochondrial-derived vesicles (MFF-MDVs) into an EtBr-induced mitochondrial dysfunction-induced senescent cell model, evaluating their effects on ATP content, mitochondrial membrane potential, ROS levels, and mtDNA content in the model cells. The expression levels of senescence-related proteins were also detected. The results show that the engineered mitochondrial-derived vesicles (MFF-MDVs) significantly promote mitochondrial oxidative phosphorylation, thus promoting mitochondrial function recovery, regulating the biological effects of various mitochondrial metabolic enzymes, and enhancing their antioxidant capacity. Therefore, they have application value in treating cellular senescence caused by mitochondrial dysfunction.

[0014] Therefore, the present invention first provides the application of the above-mentioned mtDNA-rich MFF engineered targeting mitochondrial-derived vesicles in the preparation of drugs to improve mitochondrial dysfunction in senescent cells.

[0015] The present invention also provides the use of the mtDNA-rich MFF engineered targeting mitochondrial-derived vesicles in the preparation of medicaments for treating cellular senescence associated with mitochondrial dysfunction.

[0016] Furthermore, the cellular senescence is caused by mitochondrial dysfunction.

[0017] Furthermore, the drug is used in the treatment of mitochondrial dysfunction in aging cells by promoting mitochondrial oxidative phosphorylation in subject cells, regulating the activity of mitochondrial metabolic enzymes and antioxidant enzymes, scavenging ROS, increasing ATP content, and enhancing mitochondrial network generation and restoring mitochondrial function.

[0018] Furthermore, the subjects were selected from mammals.

[0019] Furthermore, the mammals mentioned are selected from rats, cats, dogs, pigs, cattle, horses, sheep, and monkeys, etc.

[0020] Furthermore, the drug also includes other pharmaceutically acceptable excipients.

[0021] Furthermore, a recombinant plasmid overexpressing MFF was constructed. After transfecting L-O2 cells (engineered cells), mitochondrial-derived vesicles were extracted. MFF-engineered mitochondrial-derived vesicles rich in mtDNA were successfully constructed.

[0022] Furthermore, the amino acid sequence of the MTS-like polypeptide is shown in SEQ ID No. 1: MKLFKRQKRTLL. This invention screened two P12 polypeptide sequences from a phage peptide library that can exhibit affinity for recombinant mitochondrial proteins. Phage ELISA12 detection showed that the polypeptide with the sequence MKLFKRQKRTLL had stronger binding affinity.

[0023] Furthermore, the template plasmid of the recombinant plasmid is pcDNA3.1, that is, the pcDNA3.1-MFF-MTS-like recombinant plasmid is constructed.

[0024] Furthermore, the cells are hepatocytes.

[0025] Furthermore, the mitochondrial-derived vesicles are extracted using ultracentrifugation.

[0026] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the application of mtDNA-rich MFF-engineered targeted mitochondrial-derived vesicles in the preparation of drugs for treating anti-aging cell lines. The invention involves constructing an MFF / MTS-like fusion plasmid, infecting L-O2 cells, screening for MFF-L-O2, culturing the cells, collecting the culture medium, and centrifuging mitochondrial-derived vesicles (MDVs) with iodixanol at a gradient density to obtain mtDNA-rich MFF-engineered mitochondrial-derived vesicles. The results of this invention show that these engineered mitochondrial-derived vesicles (MFF-MDVs) significantly promote mitochondrial oxidative phosphorylation, stimulate the expression of cellular antioxidant enzymes, and improve mitochondrial network structure, thus promoting mitochondrial function recovery and improving and treating mitochondrial dysfunction in senescent cells. Therefore, this invention provides the application of mtDNA-rich MFF-engineered targeted mitochondrial-derived vesicles (MFF-MDVs) in the preparation of drugs for treating mitochondrial dysfunction in senescent cells. This invention successfully realizes the use of MFF protein in the form of mitochondrial-derived vesicles for the treatment of mitochondrial dysfunction in senescent cells. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0028] Figure 1 This refers to the screening of mitochondrial affinity peptides using phage ELISA in Example 1; wherein, p<0.05 p<0.01; Figure 2 This is a schematic diagram of the connection mode between the MTS sample and the MFF end in Example 1; Figure 3 For example, Co-IP detection of C in Example 1 + The binding of recombinant proteins to mitochondrial proteins; Figure 4 This refers to the expression levels of related proteins and mtDNA in MDVs from the Western Blot in Example 1; Figure 5 The particle size of MDVs was detected by nanoparticle tracking analysis in Example 1; Figure 6 Volcano plot of differentially expressed genes in MFF / MTS-like MDVs and WT-MDVs in Example 1; Figure 7 The image shows the KEGG and GO analysis results in the MFF / MTS samples-MDVs from Example 1. Figure 8 The internalization level of cells in each group in Example 2 (n=3, #: P<0.05); Figure 9 Western blot of senescence-related proteins P16 and P21 constructed from M293T senescent cells in Example 2; Figure 10 The relative mtDNA content (n=3) of M293T cells after treatment with MFF / MTS-MDVs in Example 2. P < 0.05 P < 0.01, ns: no significant difference compared with the normal group (no significant vs Normal)); Figure 11 The ATP levels of cells after internalizing MDVs in each group in Example 2 (n=3, P < 0.05 (P < 0.01, ns: no significant difference compared with the normal group). Figure 12 The relative ROS levels of cells in each group in Example 2 (n=3, P < 0.05 (P < 0.01, ns: no significant difference compared with the normal group). Figure 13 To detect the relative mitochondrial membrane potential of JC-1 cells after internalizing MDVs in each group in Example 2 (n=3, P < 0.05 P < 0.01 (P < 0.001, ns: no significant difference compared with the normal group). Figure 14The expression concentrations of senescence-related proteins P16 and P21 in cells after internalization of MDVs in each group in Example 2; (n=3, P < 0.05 P < 0.01, ns: no significant difference compared with the normal group. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0031] 1. Experimental materials Table 1. Cell and plasmid sources

[0032] 2. Experimental Methods (1) Experimental pretreatment Immerse the dried containers in an acidic potassium dichromate solution (concentrated sulfuric acid, potassium dichromate, and pure water in a 20:2:1 ratio) for 12 hours to remove impurities. After immersion, perform multi-stage cleaning: first rinse 20 times with running water, then rinse 10 times with ultrapure water. After cleaning, dry in an 80℃ hot air circulating oven. Then, autoclave the glass culture containers, pipettes, plastic caps, etc., to kill microorganisms, and finally dry them again for later use.

[0033] (2) Cell resuscitation Quickly remove the frozen samples from the cryopreservation device, thaw them in a 37°C water bath, and gently shake for 60 seconds. Sterilize the cryovials by wiping them with alcohol on a sterile work surface. Transfer the cell suspension to a centrifuge tube using a sterile pipette, and add 10 ml of nutrient culture medium. After observing the cell condition under an optical microscope, centrifuge at 8000 rpm for 5 minutes to precipitate the cells, discard the supernatant, resuspend the cells in culture medium, seed them into 25 ml culture flasks, and incubate at 37°C for amplification.

[0034] (3) Cell proliferation and passage When the cell coverage in the culture dish reaches 70%-80%, the cells enter the passage stage. Carefully remove the old culture medium and wash the cells twice with phosphate-buffered saline (PBS). Add 1 ml of 0.25% trypsin-EDTA mixture and digest at 37°C for 3 minutes. Observe under a microscope to confirm cell morphology changes; digestion is appropriate when cells detach from the surface. Add 3 ml of DMEM medium containing 10% fetal bovine serum to terminate digestion and prepare a single-cell suspension. Quantify the cells using a cell counter, seed equal volumes into multiple culture vessels, replenish culture medium, and label culture information. Incubate in a 5% CO2, 37°C incubator. Change the medium for the first time after 48 hours, and then every 2-3 days thereafter. Select well-growing cells for experiments and cryopreservation.

[0035] Example 1 Preparation of mtDNA-rich MFF engineered targeting mitochondrial-derived vesicles and isolation and characterization of MDVs I. Experimental Methods 1. Targeted peptide screening Screening was conducted according to the instructions of the phage display peptide library kit (Tec Biotech, China). This library contains billions of fd-tet phage clones, each displaying a random exogenous peptide sequence at the N-terminus of its fd-tet phage coat protein. The phage library was incubated at 37°C for 1 hour to remove phages specifically bound to the culture flask; pretreatment was then performed to remove non-specifically bound phages. The remaining phage library was incubated with TOM20 protein with attached magnetic beads at room temperature for 1 hour. TOM20 protein was washed 10 times with BSA / Tween buffer to remove unbound phages; bound phages were eluted for 10 minutes with low-pH elution buffer (0.1 N HCl, 1 mg / mL BSA, pH 2.2), and immediately neutralized with 1 M Tris-HCl (pH 8.8). The first eluent was concentrated using a Centricon 100 kDa ultrafiltration device, transferred to E. coli culture medium, and incubated at 37°C with shaking for 24 hours. Phage clones internalizing TOM20 protein were recovered using cell lysis buffer (such as 2% sodium dodecyl sulfate), centrifuged at 130 g for 10 minutes, the supernatant was discarded, and lysis buffer was added. The phages were amplified, and the screening process was repeated. After the third round of screening, the eluted phages were titrated, and 40 clones were randomly selected for sequencing to determine the TOM20 binding peptide sequence.

[0036] 2. Affinity verification This experiment used enzyme-linked immunosorbent assay (ELISA) to verify the binding ability and specificity of the selected phage to TOM20. 100 μL of a 100 μg / mL TOM20 protein solution was prepared by dissolving TOM20 in 0.1M NaHCO3. The 96-well plate was gently incubated at 4°C for 30 minutes with agitation to allow the TOM20 protein to initially attach to the well walls. Subsequently, the plate was incubated overnight at 4°C to allow the TOM20 protein to bind more firmly to the well walls. 2 × 10⁻⁶ phages were then added to the plate. 9 CFU-purified phage was added to blocking buffer. The blocking buffer containing the phage was added to a 96-well plate coated with TOM20 protein and incubated at room temperature for 1 hour. After incubation, unbound material needed to be washed away. The plate was washed three times with PBS containing 0.5% Tween20, followed by three more washes with PBS to further remove residual impurities and ensure the plate was clean. After washing, the plate was incubated at room temperature with alkaline phosphatase-conjugated anti-fd phage IgG (Abcam, MA, USA) for 1 hour. A phage-antibody-enzyme complex was formed. After washing the plate again, the substrate p-nitrophenol phosphate was added to the wells. The absorbance was measured at 405 nm using a plate reader.

[0037] 3. Plasmid synthesis and transfection This experiment aimed to construct a recombinant plasmid and transfect it into cells. The specific steps are as follows: Using pcDNA3.1 plasmid as a template, KpnI and XbaI restriction endonuclease sites were designed at both ends of the MFF-Flag-MTS-like sequence, successfully constructing a recombinant plasmid named pcDNA3.1-MFF-MTS-like-C (i.e., pcDNA3.1-MFF-Flag-MTS-like). L-O2 cells were evenly seeded in 6-well plates. When the cells reached a density of 30%-50%, subsequent transfection was performed. Two 1.5 mL centrifuge tubes were prepared. In one tube, 200 μL of serum-free medium was thoroughly mixed with 3 μg of plasmid; in the other tube, 200 μL of serum-free medium was mixed with 6 μL of transfect-mate. Lipofectamine 2000 was used as the transfection-mediating reagent. Both tubes were reacted at room temperature for 5 minutes each, then combined and reacted for another 20 minutes to form a transfection complex. The prepared transfection complex was added to a 6-well plate containing 2 mL of serum-free medium, gently mixed, and incubated. After 5 hours, the medium was replaced with complete medium, and the cells were cultured for another 48 hours, after which the cells were collected. Cells transfected with pcDNA3.1-MFF-MTS-like-C were named C1. + (Equivalent to L-O2-MFF).

[0038] 4. Co-immunoprecipitation (Co-IP) Cells were lysed after transfection, and proteins were extracted and incubated overnight at 4°C with primary antibody (anti-Flag) to form antigen-antibody complexes. Following Invitrogen guidelines, the mixture was incubated at 4°C for another 4 hours to complete immunoprecipitation. Magnetic beads were washed three times with 1×PBS to remove impurities, and 40 μL of elution buffer was used to elute proteins from the beads for subsequent Western blotting analysis. Sample buffer containing 5% β-mercaptoethanol was added to the eluted sample, and the sample was heated at 55°C for 15 minutes to promote protein denaturation. Western blotting was used to detect the binding of target proteins in the eluted sample. Detection proteins included MFF (1:1000, TBST buffer), TOM20 (1:1000, TBST buffer), and Flag (1:1000, TBST buffer).

[0039] 5. Extraction of extracellular vesicles by ultracentrifugation Collect the culture medium and seed cells in 75 cm² culture flasks. When the cell density reaches 80-90%, collect the culture medium. Centrifuge at 300 g for 20 min at 4°C, and collect the supernatant; then centrifuge at 2000 g for 20 min at 4°C, and collect the supernatant and discard the precipitate; next, centrifuge at 10000 g for 20 min at 4°C, seal and label the supernatant, and store at -80°C. Thaw the supernatant at 4°C using ultracentrifugation, transfer it to centrifuge tubes, and balance each tube pairwise (to a precision of 0.01 g). Place the tubes in an SW70Ti rotor and adapter, centrifuge at 100000 g for 70 min, discard the supernatant, and resuspend the precipitate in PBS. Transfer the precipitate to a 13.2 mL laminar flow cytometer tube, balance it, place it in an SW41Ti rotor, centrifuge at 10000 g for 70 min at 4°C, and discard the supernatant. Resuspend the precipitate in pre-cooled PBS, aliquot into sterile EP tubes, and store at -80°C, avoiding repeated freeze-thaw cycles.

[0040] 6. Iodixanol gradient density separation of MDVs Iodixanol gradient solutions were prepared, and the commercial OptiPrep solution containing 60% (wt / vol) iodixanol was pre-equilibrated using a Tris-HCl buffer system. Solution A: 0.17 g sucrose was dissolved in 2 mL of 60 mM Tris-HCl (pH 7.4), with a final sucrose concentration of 0.25 M. OWS: 1 mL of Solution A was added to 5 mL of OptiPrep to obtain 6 mL of 50% iodixanol working solution in 10 mM Tris-HCl (pH 7.4). Solution B: 1.28 g sucrose was dissolved in 15 mL of 10 mM Tris-HCl (pH 7.4), with a final sucrose concentration of 0.25 M. Iodixanol solutions of 40%, 20%, 15%, 13%, 11%, 9%, and 7% were added sequentially to centrifuge tubes, followed by extracellular vesicle solution. After balancing, SW41Ti rotors were used. Centrifugation was performed at 200,000 g for 16 h at 4 °C, and the liquids of different concentrations were aliquoted into EP tubes. The 20% layer solution was brought to a final volume of 10 mL with sterile PBS, centrifuged at 100,000 g for 70 min at 4 °C, and the supernatant was discarded. The MDVs precipitate was resuspended in pre-cooled PBS, aliquoted into sterile EP tubes, and stored at -80 °C, avoiding repeated freeze-thaw cycles.

[0041] 7. Western Blot analysis of MDV marker proteins isolated from cells stably overexpressing MDVs MDVs were separated from L-O2 and MFF-L-O2 cells. MDVs were grouped as follows: MDVs secreted by L-O2 cells were WT-MDVs; MDVs secreted by MFF-L-O2 cells were MFF / MTS-MDVs.

[0042] The concentration of collected samples was determined using the BCA method. Loading buffer was added to the protein sample at the concentration to be determined in a ratio of 5:1 (sample volume: 5 × loading buffer = 5:1) and incubated in a 100 °C metal bath for 10 min. Sample preparation was then completed. Western blotting was then used to detect the protein expression levels of the MDV markers TOM20 and TIM23.

[0043] 8. Nanoparticle size detection Nanoparticle tracking analysis was performed using a ZetaView PMX 110 (Particle Metrix) instrument and its accompanying software (ZetaView 8.02.28). Mitochondrial-derived vesicles were diluted in particle-free PBS and then injected into the sample chamber. The size and concentration of the mitochondrial-derived vesicles were then measured at a wavelength of 405 nm. Finally, the particle size of the mitochondrial-derived vesicles was quantitatively analyzed, and the relevant data were recorded.

[0044] 9. mtDNA introduction into MDV (1) mtDNA preparation: 293T cells were digested with trypsin and centrifuged at 12,000 rpm for 2 min at 4°C. The cell pellet was collected. 250 μL of solution A (on ice) was added, and the pellet was dispersed. Then, 250 μL of solution B (at room temperature) was added, and the mixture was stirred. The mixture was incubated on ice for 6 min. 350 μL of solution C (on ice) was added, and the mixture was stirred until a white precipitate formed. The mixture was incubated on ice for 25 min. The cells were centrifuged at 12,000 rpm for 10 min, and the supernatant was transferred to an adsorption column. After incubation for 5 min, the cells were centrifuged at 12,000 rpm for 1 min, and the waste liquid was discarded. 500 μL of wash buffer was added, and the cells were centrifuged at 12,000 rpm for 1 min. The centrifugation was repeated once, and the residual liquid was removed to obtain the mtDNA extract.

[0045] (2) mtDNA loading: Purified mtDNA was mixed with Lipofectamine 3000 at a ratio of 5:1 and incubated at room temperature for 30 min to form an mtDNA-liposome complex (RDL). The RDL was then mixed with isolated mitochondrial-derived vesicles at a particle ratio of 1:2 and incubated at room temperature for 30 min. The mixture was then frozen in liquid nitrogen for 15 min, thawed at 37°C for 20 min, and this freeze-thaw cycle was repeated three times to promote membrane fusion. Finally, the fusion product was separated by ultracentrifugation (100,000 × g, 4°C) and resuspended in sterile PBS to obtain mtDNA-loaded mitochondrial-derived vesicles.

[0046] II. Experimental Results 1. Two peptide sequences were screened from a 12-peptide phage peptide library, demonstrating affinity for the TOM20 recombinant protein. These peptide sequences and their binding frequencies are shown in Table 2. Phage ELISA analysis revealed that the peptide with the sequence MKLFKRQKRTLL exhibited stronger binding affinity, as shown in the results. Figure 1 As shown. This polypeptide sequence is named MTS-like.

[0047] Table 2. Peptide sequences and TOM20 binding frequencies selected by phage screening

[0048] 2. After confirming that the MTS-like sequence can specifically bind to TOM20, the MTS-like sequence, Flag tag sequence, and MFF protein-coding sequence were sequentially inserted into the recombinant plasmid. The MFF-MTS-like fusion protein was then overexpressed in cells, thereby achieving targeted localization of the fusion protein to TOM20. In this study, a Flag tag and the MTS-like sequence were tandemly linked to the end of the MFF protein sequence. Therefore, the fusion construction method was set as follows: fusion at the C-terminus was designated as C0. + Groups, see details on how to build them. Figure 2 .

[0049] The MTS-like coding sequence (36bp) is shown in SEQ ID No. 3: ATGAAACTGT TCAAACGTCA GAAACGTACC CTGCTG; The MFF protein coding sequence (1029 bp) is shown in SEQ ID No. 4: MFF The cDNA sequence of the gene (2174 bp) (NM_001277061.2) is shown in SEQ ID No. 5.

[0050] 3. The interaction between proteins in L-O2 cells was detected by co-immunoprecipitation (Co-IP) assay, and the results are as follows: Figure 3 As shown in the figure. The results showed that the blank cell group (L-O2) without plasmid transfection had no Flag protein band, and the expression level of endogenous MFF protein was significantly lower than that of C. + The high expression of the MFF-MTS-like fusion protein carrying the Flag tag in cells after plasmid transfection was due to this. After enrichment and purification with Flag-specific magnetic beads, the MFF-TOM20 complex protein with a molecular weight of 126 kDa was detected in the C+ group, indicating that the MTS-like fusion is located at the C-terminus of the MFF protein and can normally mediate the binding of the fusion protein to mitochondria, further verifying the mitochondrial targeting function of the MTS-like sequence.

[0051] 4. Western Blot of MDV-related proteins and MFF Western blotting analysis was performed on the MDV obtained above. The Western blotting analysis is as follows: Figure 4 As shown, the MFF / MTS-like-MDV contains the same mitochondrial marker protein as WT-MDV and highly expresses MFF. Furthermore, the generated MFF / MTS-MDV has a high expression of the mitochondrial outer membrane protein TOM20, indicating that this MDV is an outer membrane type.

[0052] 5. MDVs particle size analysis The extracted WT-MDV and MFF / MTS sample-MDV diameters were determined using a Nanosight particle size analyzer. The results are as follows: Figure 5 As shown, the particle size range is mainly 50-200 nm, and the average diameter of MDV is (98.45 ± 25.31 nm), which is consistent with the morphological characteristics of MDVs.

[0053] 6. MDV sequencing Compared to the WT-MDV group, the MFF / MTS sample-MDV group identified a total of 6449 DEGs (updated by 2849 and downdated by 3605). Figure 6The MFF gene was significantly upregulated, indicating that the vesicle was successfully enriched with MFF protein. Simultaneously, DRP1, a mitochondrial division-related gene closely related to MFF, was also upregulated, suggesting that MFF / MTS-like MDVs can promote the production of mitochondrial division proteins. KEGG and GO analyses showed significant activity in the MDV generation-related membrane fusion pathway, where MFF overexpression also activated mitochondrial division-related pathways. Figure 7 ).

[0054] Example 2 MDVs improve the effect of EtBr-induced mtDNA mutations on cellular senescence. I. Experimental Methods 1. Establishment of cell model 293T cells were seeded into 6-well plates containing DMEM medium supplemented with 10% FBS and cultured at 37°C with 5% CO2. When cell confluence reached 90%, the cells were transferred to 6-well plates containing fresh medium for further culture. Complete DMEM medium containing 25 ng / mL EtBr was prepared. EtBr (ethidium bromide) was used to induce the transformation of 293T cells into M293T cells. 293T cells were seeded into 6-well plates containing 25 ng / mL EtBr and cultured for 3 days. After induction, the cells were seeded into complete DMEM medium without EtBr for further growth and expansion. After 5 days, the cells had undergone a period of stable growth and expansion, and subsequent related studies could be conducted.

[0055] The cell experiments were divided into four groups: normal group, EtBr-induced 293T group (M293T), M293T group with internalized WT-MDV (WT-MDV), and M293T group with internalized MFF / MTS-MDV (MFF / MTS-MDV).

[0056] 2. Internalization detection of MDVs MDVs were extracted using the iodixanol gradient density method. Following the kit instructions: 100 μg of MDVs were labeled with 5 μL EvLINK505 and gently incubated in the dark at room temperature for 30 min. The purified samples were collected for subsequent experiments to assess cellular uptake of MDVs. Cells were seeded in confocal culture dishes and cultured for 24 h. Subsequently, M293T cells were incubated with EvLINK505-labeled MDVs for 24 h, 48 h, 72 h, and 96 h, and washed with PBS. To visualize the cell membrane, M293T cells were labeled with Cel-ILINK 555 and incubated in the dark at room temperature for 30 min. After incubation, cells were washed with PBS and fixed with 4% formaldehyde for 30 min. Then, they were stained with DAPI for 5 min. Internalization of labeled MDVs was observed using a laser confocal scanning microscope.

[0057] 3. Internalization of MDVs in each group reduced the expression levels of cellular senescence-related proteins P16 and P21. Western blot analysis was performed on senescence-related proteins P16 and P21 in cells after internalization of MDVs in each group.

[0058] 4. Detection of mtDNA content in cells Cells were digested with trypsin and centrifuged at 12,000 rpm for 2 min at 4°C. The cell pellet was collected. 250 μL of solution A (ice bath) was added, and the pellet was dispersed. Then, 250 μL of solution B (room temperature) was added, and the mixture was stirred. The mixture was incubated on ice for 6 min. 350 μL of solution C (ice bath) was added, and the mixture was stirred until a white precipitate formed. The mixture was incubated on ice for 25 min. The pellet was centrifuged at 12,000 rpm for 10 min, and the supernatant was transferred to an adsorption column. After incubation for 5 min, the pellet was centrifuged at 12,000 rpm for 1 min, and the waste liquid was discarded. 500 μL of wash buffer was added, and the pellet was centrifuged at 12,000 rpm for 1 min. The centrifugation was repeated once, and the residual liquid was removed to obtain the mtDNA extract.

[0059] Genomic DNA was extracted according to the DNA extraction kit. mtDNA was quantified using qPCR. Primers were designed based on NCBI and synthesized by Qingke Biotechnology. mtDNA was quantified using the ND1 subunit gene of NADH dehydrogenase, and nDNA was quantified using the β-actin gene. Each group was added to SYBR Green Master Mix and the corresponding primer preparation system for qPCR, with β-actin as an internal control gene. The corresponding primer sequences and PCR reaction conditions are shown in Tables 3 and 4. Three independent biological replicates were performed. Calculate the relative gene expression levels.

[0060] Table 3 PCR amplification primers

[0061] Table 4 PCR reaction conditions

[0062] 5. ATP level detection Add 200 μL of lysis buffer to each well of a 6-well plate, pipette, centrifuge at 12000 g for 5 min at 4°C, and collect the supernatant for analysis. Dissolve the standard solution reagent and dilute the ATP standard solution to prepare a series of solutions of 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 µM. Add 100 µL of detection working solution to a 96-well plate, let it stand at room temperature for 3-5 min, add 20 µL of the test solution or standard solution to each well, mix well, and measure the luminescence value using a microplate reader. Plot a standard curve to calculate the ATP content.

[0063] 6. ROS level measurement Dilute DCFH-DA to a 10 μM solution using serum-free medium. Wash cells with PBS, add 100 μL of diluent, and incubate at 37°C and 5% CO2 for 1 h. Wash three times with serum-free medium, add fresh medium, and incubate for another 1 h. Collect cells and observe fluorescence intensity using a laser confocal microscope (EX 488 nm, EM 525 nm).

[0064] 7. Mitochondrial membrane potential detection Dilute JC-1 with 1:160 ultrapure water, add 2 mL of 5×JC-1 staining buffer and mix well. Discard the culture medium, add staining working solution and incubate for 20 min, discard the supernatant, wash twice with pre-cooled 1× buffer, add fresh culture medium, observe and photograph under an inverted fluorescence microscope with excitation light at 490 nm and 525 nm, and analyze the ratio of red to green fluorescence intensity using ImageJ software.

[0065] 8. ELISA detection of aging-related protein expression levels Cells were treated with PMSF lysis buffer, centrifuged, and the supernatant was diluted. Standards, biotinylated antibody, HRP enzyme conjugate working solution, and washing buffer were prepared. Samples or standards were added to the ELISA plate and incubated at 37°C. The liquid was discarded. Biotinylated antibody working solution was added and incubated. The plate was washed. HRP enzyme conjugate working solution was added and incubated. The plate was washed again. TMB substrate was added for color development in the dark. Stop solution was added, and OD values ​​were measured at 450 nm. Protein concentration was calculated from the standard curve.

[0066] 9. Statistical Analysis Statistical analysis of continuous variables is expressed as mean ± standard deviation. GraphPad Prism 10.0 was used for statistical analysis. Independent samples t-tests or one-way ANOVA were used to analyze the statistical differences between groups. A p-value less than 0.05 was considered statistically significant.

[0067] II. Experimental Results 1. Internalization of MDVs M293T cells were co-cultured with MDV labeled with EvLINK505 and observed using laser confocal scanning microscopy at time points of 24 h, 48 h, 72 h, and 96 h. Figure 8 The results showed that MFF-MDV and MFF / MTS-MDV reached the peak of internalization after 72 hours of co-culture.

[0068] 2. Expression levels of aging markers P16 and P21 proteins mtDNA mutations lead to cellular senescence, and the expression of P16 and P21 proteins is upregulated during cellular senescence. To further confirm the successful construction of senescent cells, total protein was extracted from the cells and analyzed by Western blot. The results are as follows: Figure 9 As shown, the expression levels of P16 and P21 proteins in M293T cells were significantly increased, indicating that the senescent cell model was successfully constructed.

[0069] 3. Detection of mtDNA content in cells of each group To investigate the changes in mtDNA content after MDVs in each group were internalized to M293T, this study extracted mtDNA and nDNA from cells and detected the relative content of mtDNA in each group. The results are as follows: Figure 10 As shown, the mtDNA content in the MFF / MTS-MDV group cells increased significantly, indicating that MFF / MTS-MDV successfully carried mtDNA into M293T cells.

[0070] 4. Changes in ATP levels after MDVs internalized M293T cells in each group As cells age, mitochondrial function gradually declines, manifested as a decrease in mitochondrial number, an increase in the accumulation of mtDNA mutations, and damage to the electron transport chain. These changes lead to a weakened ATP production capacity. Isolated MDVs were internalized into M293T senescent cells, and the ATP levels in each group were measured. The results are as follows: Figure 11 As shown in the figure, the relative ATP level of the M293T group was significantly lower than that of the normal group, the relative ATP level of the WT-MDV group was also lower than that of the normal group but the difference was small, while the relative ATP level of the MFF / MTS-MDV group was not significantly different from that of the normal group.

[0071] 5. Changes in ROS levels after M293T cells were internalized by MDVs in each group Mitochondrial function declines in senescent cells, leading to increased ROS production. The results were obtained by internalizing MDVs from each group to M293T. Figure 12As shown, compared with the normal group, the relative ROS level of the WT-MDV group was significantly increased, and the differences were extremely significant; while the relative ROS level of the MFF / MTS-MDV group was closer to that of the normal group, and the ROS level of the MFF / MTS-MDVs group was decreased.

[0072] 6. Detection of mitochondrial membrane potential after MDVs internalize M293T cells in each group This study used the JC-1 fluorescent probe to label mitochondrial membrane potential and calculated the ratio of red fluorescent positive signal to green fluorescent positive signal. The results are as follows: Figure 13 As shown: Cells treated with MFF / MTS-MDV exhibited increased red fluorescence in mitochondria, indicating elevated membrane potential and mitochondrial damage repair. This suggests that MFF / MTS-MDV can significantly repair damaged mitochondria and promote the improvement of senescent cells.

[0073] 7. Expression levels of aging markers P16 and P21 proteins The results are as follows Figure 14 As shown, after internalizing MDVs in each group, the expression levels of aging-related proteins P16 and P21 in cells of the MFF / MTS-MDV group were significantly lower than those in other treatment groups.

[0074] In summary, the results indicate that MFF genetically engineered mitochondrial-derived vesicles can carry complete mtDNA into senescent cells for treatment, demonstrating a significant anti-cellular senescence effect.

[0075] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing MFF-bound MTS-like peptide-engineered mitochondrial-derived vesicles, characterized in that, Includes the following steps: 1) The coding gene of a polypeptide MTS, whose amino acid sequence is shown in any of SEQ ID No. 1 to SEQ ID No. 2, is linked to the 3' end of the MFF protein coding gene to obtain a chimeric gene; 2) Construct a recombinant plasmid expressing the chimeric gene; 3) The recombinant plasmid was transfected into cells for expression, and mitochondrial-derived vesicles were extracted; 4) The extracted and purified mtDNA was transferred into mitochondrial-derived vesicles using liposome transfection to obtain MFF-binding MTS-like peptide-engineered targeted mitochondrial-derived vesicles.

2. The preparation method according to claim 1, characterized in that, Step 1) The coding gene for the peptide MTS-like protein is linked to the C-terminus of the MFF protein coding gene via a Flag tag.

3. The preparation method according to claim 1, characterized in that, The amino acid sequence of the peptide MTS-like peptide in step 1) is shown in SEQ ID No.

1.

4. The preparation method according to claim 1, characterized in that, Step 2) The template plasmid used for the recombinant plasmid is pcDNA3.

1.

5. The preparation method according to claim 1, characterized in that, Step 4) The mtDNA mentioned is obtained by extraction and purification from L-O2 cells.

6. The use of MFF-bound MTS-like peptide-engineered mitochondrial-derived vesicles prepared by any one of the methods described in claims 1-5 in the preparation of drugs to improve senescent cells.

7. The application according to claim 6, characterized in that, The drug improves mitochondrial function in senescent cells by promoting ROS clearance in subject cells, protecting mitochondrial DNA, improving mitochondrial network structure, and promoting mitochondrial metabolic reprogramming.

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