A stem cell exosome capable of delaying cell aging and a preparation method and application thereof

Abstract

The application discloses an engineered exosome targeting human urokinase-type plasminogen activator receptor uPAR and a preparation method and application thereof. Firstly, a high-affinity anti-human uPAR single-domain antibody VHH is prepared; then, a human umbilical cord mesenchymal stem cell engineering cell line expressing a VHH-PTGFRN fusion protein is constructed, and the engineered exosome is prepared and purified by combining three-dimensional hypoxic culture, chemical induction and size-exclusion chromatography technology. The engineered exosome comprises four comparative samples, wherein the core sample Exo^(VHH / miR) can simultaneously display the anti-uPAR VHH on the membrane surface to realize targeted delivery and enrich miR-193b-3p in the chamber to realize gene silencing, and the synergistic effect of the two can efficiently remove the uPAR high-expression senescent cells and inhibit the senescence-associated secretory phenotype SASP. The engineered exosome has the advantages of standardized preparation process, high yield and high purity, strong targeting, excellent curative effect and good safety, and can be used for preparing a treatment preparation for anti-aging and aging-related diseases, and has important clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a stem cell exosome that can delay cell aging, its preparation method, and its application. Background Technology

[0002] Cellular senescence is a key biological basis and driving factor for the decline of bodily functions and most age-related diseases, such as osteoarthritis, idiopathic pulmonary fibrosis, and atherosclerosis. Senescent cells enter an irreversible state of cell cycle arrest, while continuously secreting large amounts of inflammatory cytokines, chemokines, and extracellular matrix degrading enzymes. This characteristic is defined as the senescence-associated secretory phenotype (SASP). SASP not only disrupts the local tissue microenvironment homeostasis and triggers chronic low-grade inflammation, but also induces senescence in neighboring normal cells through paracrine mechanisms, thereby creating a cascade amplification effect and accelerating tissue function loss. Based on this, "Senolytic therapy," which specifically eliminates senescent cells, has become an important research direction for anti-aging and the treatment of age-related diseases.

[0003] First-generation senolytic drugs are primarily small-molecule inhibitors, such as the combination of dasatinib and quercetin. They induce apoptosis in senescent cells by interfering with overactive pro-survival signaling pathways like BCL-2 or PI3K / AKT. However, these drugs generally suffer from significant off-target effects, a narrow therapeutic window, and low oral bioavailability for some. Furthermore, they typically require intermittent dosing to mitigate systemic toxicity from continuous administration, limiting their long-term safety and patient compliance.

[0004] Mesenchymal stem cell-derived exosomes (MSC-Exo), as natural nanoscale vesicles, carry abundant bioactive substances such as proteins, lipids, and functional nucleic acids (e.g., microRNA and mRNA). Studies have shown that MSC-Exo can regulate signaling pathways related to aging, inflammation, and repair in recipient cells by mimicking the paracrine function of their parent cells, exhibiting the potential to inhibit cellular senescence, reduce inflammation, and promote tissue regeneration, while also possessing advantages such as low immunogenicity, good stability, and scalability for production. However, unengineered natural exosomes lack active targeting ability after systemic administration, and are mainly cleared rapidly by the mononuclear phagocytic system (e.g., liver, spleen), resulting in extremely low accumulation efficiency in specific pathological sites (e.g., articular cartilage, lung interstitium). To improve efficacy, high doses are often required, which not only increases production costs but may also lead to unpredictable side effects due to nonspecific distribution.

[0005] To improve delivery efficiency, genetic engineering strategies have been applied to modify exosomes. A common method is to fuse targeted ligands (such as specific peptides or antibody fragments) with endogenous exosomal membrane proteins (such as Lamp2b, CD63, or PTGFRN). Among these, single-domain antibodies (VHHs, also known as nanobodies) are considered superior to traditional single-chain antibody fragments (scFvs) due to their small molecular weight (approximately 15 kDa), strong tissue penetration, high structural stability, and ease of gene fusion. Existing literature has reported cases of using VHH-modified exosomes for tumor-targeted delivery. However, applying this platform technology to the field of clearing senescent cells still faces several key scientific issues and technical challenges: First, it is necessary to screen and verify a target molecule that is stably and highly expressed on the surface of senescent cells from multiple tissue sources and plays a core role in their pathological function; second, it is necessary to go beyond the traditional role of exosomes as "passive carriers" and construct an intelligent system that can actively intervene in target function and achieve integrated "recognition and treatment"; finally, the ideal system should be able to avoid or adapt to possible changes in target expression under treatment stress in order to achieve a lasting and stable therapeutic effect.

[0006] Urokinase-type plasminogen activator receptor (uPAR, also known as CD87) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein. In recent years, numerous studies have confirmed that uPAR is stably and significantly upregulated on the surface of various types of senescent cells (including fibroblasts, endothelial cells, and epithelial cells) in vitro and in vivo, making it a highly conserved cell surface marker of aging. More importantly, uPAR is not a passive 'tag'. Studies have shown that its activated downstream signaling pathways (such as the ERK and AKT pathways) directly participate in regulating the generation and maintenance of aging-associated secretory phenotypes (SASPs), serving as a 'hub' molecule connecting aging recognition and phenotype maintenance. This dual characteristic of highly specific membrane localization and core functional regulation makes uPAR an ideal therapeutic target for achieving integrated 'targeted delivery' and 'functional intervention'. Although the targeting value of uPAR has been recognized, there are currently no reports on the genetic engineering fusion of single-domain antibodies targeting uPAR with MSC exosomes for use in anti-aging therapy. Summary of the Invention

[0007] To address the above technical problems, this invention provides a stem cell exosome that can delay cell aging, its preparation method, and its application.

[0008] (I) Technical Solution of the Invention

[0009] The core of this invention lies in providing a bifunctional engineered exosome targeting uPAR and its preparation method, specifically including the following steps:

[0010] 1. Preparation and screening of anti-human uPAR single-domain antibody VHH: Healthy adult alpacas were selected and immunized with recombinant human uPAR protein as antigen to construct a library with a capacity of 3.2 × 10⁻⁶. 8 The phage display antibody library of CFU was used; through three rounds of solid-phase antigen panning, high-affinity and high-specificity anti-uPAR VHH was obtained and named VHH-uPAR-A3, whose amino acid sequence is SEQ ID NO 1 in the sequence listing.

[0011] 2. Construction of engineered human umbilical cord mesenchymal stem cell line: VHH-uPAR-A3, flexible linker peptide (GGGGS)3, and PTGFRN protein fragments were synthesized to construct the VHH-PTGFRN fusion protein coding sequence, the nucleotide sequence of which is shown in SEQ ID NO 2 of the sequence listing. This sequence was cloned into a lentiviral vector, packaged into lentivirus, and transduced into human umbilical cord mesenchymal stem cells. After selection with puromycin and flow cytometry sorting, an engineered cell line MSC^(PGFRN-uPAR-VHH) stably expressing the VHH-PTGFRN fusion protein was obtained. At the same time, a control cell line MSC^(PGFRN-Control-VHH) expressing unrelated VHH was constructed.

[0012] 3. Preparation and purification of engineered exosomes: Parental mesenchymal stem cells, MSC^(PGFRN-Control-VHH), and MSC^(PGFRN-uPAR-VHH) were subjected to three-dimensional hypoxia culture. In the later stage of culture, sodium valproate (VPA) was added to MSC^(PGFRN-uPAR-VHH) cells to induce miR-193b-3p enrichment. The cell culture supernatant was collected and purified by centrifugation, filtration, tangential flow concentration and size exclusion chromatography to obtain four groups of exosome samples, namely, natural unmodified Exo-Native, Exo-Control expressing irrelevant target proteins, Exo-VHH showing only anti-uPAR VHH, and ExoVHH / miR showing anti-uPAR VHH and enriched with miR-193b-3p (also referred to as Exo^(VHH / miR) in the examples).

[0013] 4. Functional validation of engineered exosomes: Flow cytometry was used to verify the targeted binding ability of exosomes to senescent cells with high uPAR expression; Western blotting was used to verify the inhibitory effect of exosomes on uPAR protein expression in target cells; senescence β-galactosidase staining and enzyme-linked immunosorbent assay were used to verify the effect of exosomes in clearing senescent cells and inhibiting SASP secretion; and natural aging mouse models were used to verify the in vivo anti-aging efficacy and safety of exosomes.

[0014] (II) Key Technical Features of the Invention

[0015] 1. Targeted modification strategy: The anti-uPAR VHH is fused with the exosome membrane protein PTGFRN through the flexible linker peptide (GGGGS)3, so that VHH is stably displayed on the surface of exosomes, thereby achieving active targeted recognition of senescent cells with high uPAR expression and avoiding non-specific binding to normal cells.

[0016] 2. Dual-function synergistic design: Exo^(VHH / miR) has both "membrane targeting" and "cavity silencing" functions. VHH on the membrane surface improves the delivery efficiency of exosomes to senescent cells, while miR-193b-3p enriched in the cavity can specifically inhibit uPAR gene expression. The two work synergistically to enhance the clearance effect of senescent cells and solve the problem of insufficient efficacy of single targeting or single gene silencing.

[0017] 3. Standardized preparation process: A purification process of "three-dimensional hypoxia culture + chemical induction + size exclusion chromatography" was established, which can stably obtain high yield (about 300 μg / 100 mL culture medium) and high purity (purity >95%, no obvious cell debris and nucleic acid contamination) engineered exosomes. The physicochemical properties of the four groups of samples are highly consistent, which facilitates subsequent functional comparison and large-scale production.

[0018] 4. High safety profile: The exosomes are derived from human umbilical cord mesenchymal stem cells, exhibiting good biocompatibility and low immunogenicity. In vivo experiments have demonstrated that the engineered exosomes do not cause abnormal weight gain, liver and kidney function damage, or pathological changes in major organs in mice, and are well tolerated.

[0019] (III) Beneficial Effects: Compared with the prior art, the present invention has the following significant beneficial effects:

[0020] 1. High targeting and specificity: Engineered exosomes can specifically recognize and bind to senescent cells with high uPAR expression through surface anti-uPAR VHH. The binding signal is 4.9-5.26 times higher than that of natural exosomes, and can be competitively inhibited by free VHH, effectively avoiding damage to normal young cells and reducing side effects.

[0021] 2. High efficiency in clearing senescent cells and significant synergistic effect: Exo^(VHH / miR) achieves a clearance rate of 70.7% for senescent cells, which is significantly higher than Exo-VHH (50.9%) with only targeting function and the commonly used D+Q drug combination in preclinical trials (41.1%). At the same time, it can downregulate uPAR protein expression by 62% and reduce the secretion of SASP factors such as IL-6 and MMP-3 by more than 77%, with efficacy superior to existing technologies.

[0022] 3. Standardized and scalable preparation process: The optimized three-dimensional hypoxic culture and purification process can stably obtain high yields and high purity exosomes. Approximately 300 μg of exosome protein can be obtained from every 100 mL of culture medium, and the physicochemical properties are uniform, meeting the needs of subsequent industrial production and clinical application.

[0023] 4. Good safety and excellent biocompatibility: The exosomes are derived from human umbilical cord mesenchymal stem cells and have no obvious immunogenicity. In vivo experiments have shown that after continuous intravenous injection for 8 weeks, the liver and kidney functions and major organ structures of mice were not abnormal, and the weight gained steadily. The tolerance was significantly better than that of chemical senescent cell scavengers.

[0024] 5. Broad application prospects: This engineered exosome can efficiently clear senescent cells and inhibit SASP secretion, significantly improve the physiological function of naturally aging mice, reduce the aging burden of multiple tissues, and can be used to prepare anti-aging agents and treat a variety of age-related diseases. It has important clinical application value and industrialization potential.

[0025] 6. Strong technical stability: The constructed engineered cell lines can stably express fusion proteins (FLAG positivity rate >98%), the prepared exosomes have complete morphology, clear marker proteins, stable targeting function and miRNA enrichment effect, and can be prepared repeatedly.

[0026] (iv) Scope of application: The engineered exosomes of this invention (especially Exo^(VHH / miR)) can be widely used in the biomedical field, specifically including:

[0027] 1. Anti-aging treatment: Used to clear senescent cells in the body, reduce the aging burden on the body, and improve age-related physiological function decline (such as decreased muscle strength, loss of skin collagen, etc.).

[0028] 2. Treatment of aging-related diseases: Used to treat chronic inflammation, cardiovascular diseases, neurodegenerative diseases, osteoporosis, skin aging and other diseases caused by the accumulation of senescent cells.

[0029] 3. Targeted delivery vector: It can be used as a universal targeted delivery vector to deliver other therapeutic molecules (such as drugs, siRNA, miRNA, etc.) to cells that highly express uPAR (such as senescent cells and tumor cells), thereby improving treatment efficiency. Attached Figure Description

[0030] Figure 1 The SDS-PAGE results of purified VHH-uPAR-A3 are shown in Figure 1, where 1 represents VHH-uPAR-A3.

[0031] Figure 2Western blot results of VHH-uPAR-A3, where 1 is the lysate of HEK293T cells transfected with the human uPAR gene, and 2 is the lysate of cells transfected with the empty vector.

[0032] Figure 3 Flow cytometry results of VHH-uPAR-A3 cells incubated with HT1080 human fibrosarcoma cells that highly express uPAR.

[0033] Figure 4 Western blot results of four groups of exosome samples, where 1 to 4 are Exo-Native, Exo-Control, Exo-VHH, and Exo^(VHH / miR), respectively. Detailed Implementation

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0035] 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.

[0036] Example 1: Preparation, screening and functional identification of anti-human uPAR single-domain antibody (VHH)

[0037] 1.1 Construction of Immune and Antibody Libraries

[0038] A healthy adult alpaca was selected and immunized with recombinant human uPAR protein (abcam, ab290079). The immunization procedure was as follows: For the first immunization, 200 μg of antigen was emulsified with Freund's complete adjuvant and injected subcutaneously at multiple sites in the neck; subsequent booster immunizations were performed every two weeks, using 100 μg of antigen emulsified with Freund's incomplete adjuvant, for a total of four booster immunizations. One week after the last immunization, peripheral blood lymphocytes were collected, and total RNA was extracted using the TRIzol method, followed by reverse transcription to synthesize cDNA.

[0039] The variable region (VHH) gene fragment of alpaca heavy chain antibody was amplified by nested PCR. The purified VHH gene fragment was cloned into the phage display vector pMECS, which allows VHH to be displayed on the phage surface as a pIII fusion protein carrying ampicillin resistance and an HA tag. The recombinant plasmid was electroporated into *E. coli* TG1 competent cells to construct an initial immune antibody library, the size of which was determined by titration to be 3.2 × 10⁻⁶.8 CFU meets diversity requirements.

[0040] 1.2 Phage Display Screening

[0041] A three-round biopanning process of adsorption-elution-amplification was employed using a solid-phase antigen method. Recombinant human uPAR protein was coated onto immunotubes (10 μg / mL in the first round, decreasing to 5 μg / mL and 2 μg / mL in subsequent rounds to increase the screening pressure). In each round, the phage antibody library was incubated with the coated antigen, followed by rigorous washing (PBS containing 0.1% Tween-20) to remove non-specifically bound phages. Specifically bound phages were then eluted with glycine-hydrochloric acid buffer (pH 2.2) and immediately neutralized with Tris-HCl (pH 9.1). The eluted phages were then used to infect logarithmically growing TG1 Escherichia coli for amplification, which was used in the next round of screening. With each round of screening, phages specifically bound to the antigen were significantly enriched; the titer of the eluent after the third round of screening was 1.2 × 10⁻⁶ higher than that of the first round. 4 times.

[0042] 1.3 Identification and Sequence Analysis of Positive Clones

[0043] Ninety-six single clones were randomly selected from the plates after the third round of screening for soluble expression. Clones that specifically bind to uPAR protein and do not bind to irrelevant proteins (BSA) were initially screened using enzyme-linked immunosorbent assay (ELISA). Strongly positive clones (32 in total) with OD450 values ​​more than 5 times higher than the negative control were selected for plasmid extraction and DNA sequencing.

[0044] Sequencing results were compared and analyzed using the IMGT database. Clustering was performed based on differences in complementarity-determining region (CDR) sequences, ultimately yielding 12 unique VHH sequences. Comparison revealed that one clone (named VHH-uPAR-A3) exhibited the strongest signal in ELISA, and its encoded amino acid sequence is shown in SEQ ID NO: 1.

[0045] 1.4 Antibody expression, purification, and affinity assay

[0046] The VHH-uPAR-A3 gene was cloned into the expression vector pET-22b(+) and transformed into *E. coli* BL21(DE3) expression strain. Expression was induced for 20 hours at 16°C using 0.5 mM IPTG. After bacterial lysis, the protein was purified using a nickel affinity chromatography column using its C-terminal 6×His tag, and further purified using a Superdex 75 Increase gel filtration chromatography column to obtain high-purity monomeric VHH protein (SDS-PAGE purity >95%). Figure 1 ).

[0047] The affinity of VHH-uPAR-A3 for uPAR protein was determined using surface plasmon resonance (SPR) technology. Human uPAR protein was immobilized on a CM5 chip, and purified VHH was passed through it at different concentrations. Kinetic curves were fitted using BIAevaluation software, and the equilibrium dissociation constant of VHH, KD = 1.8 × 10⁻⁶, was calculated. -9 M (i.e., 1.8 nM) indicates that it has nanomolar-level high affinity. This affinity is significantly superior to that of traditional monoclonal antibodies (such as ab317150) or single-chain antibody fragments targeting other targets (whose KD is typically around 10). -8 M level).

[0048] 1.5 Specificity and Cross-Reactivity Validation

[0049] The specificity of VHH-uPAR-A3 was verified by Western blotting. The results showed that VHH specifically recognized a band of approximately 55 kDa (consistent with the molecular weight of glycosylated uPAR) in the lysate of HEK293T cells transfected with the human uPAR gene, but did not recognize the lysate of cells transfected with the empty vector. Figure 2 ).

[0050] Furthermore, flow cytometry was used to verify its binding ability to uPAR on the cell surface in its native state. Biotin-labeled VHH-uPAR-A3 was detected using streptavidin-APC. The results showed that VHH strongly and positively bound to human fibrosarcoma cells HT1080 (MFI: 25,430) that highly expressed uPAR, while the binding signal to human breast cancer cells MCF-7 (MFI: 1,250) with low uPAR expression was very weak, consistent with the binding trend of commercially available anti-human uPAR monoclonal antibodies (such as ab317150), confirming its excellent cell-level specificity. Figure 3 ).

[0051] Example 2: Preparation, purification and systematic characterization of engineered exosomes

[0052] 2.1 Construction of engineered cell lines

[0053] (1) Construction of integrated expression carriers

[0054] Gene synthesis: GenScript Biotech Ltd. was commissioned to synthesize a DNA fragment encoding VHH-uPAR-A3 (with optimized codons for mammalian cell expression and an N-terminal FLAG (DYKDDDDK) tag added), as well as a DNA fragment encoding flexible linker peptide (GGGGS)3 and amino acids 448 to 730 of human PTGFRN protein (UniProt ID: Q9P2B2). The protein expressed by this fragment was named VHH-PTGFRN fusion protein. Therefore, the nucleotide sequence encoded by the VHH-PTGFRN fusion protein is shown in SEQ ID NO:2.

[0055] Gibson Assembly ligation: Using NEBuilder® HiFi DNA Assembly Master Mix, the three fragments described above were homologously recombinated with the linearized pLVX-EF1α-IRES-Puro lentiviral vector, which had been double-digested with XhoI / HindIII, at 50°C for 60 minutes to construct the final plasmid `pLVX-PGFRN-uPAR-VHH`. Simultaneously, a control plasmid `pLVX-PGFRN-Control-VHH` was constructed, in which the VHH fragment was replaced with an irrelevant VHH sequence encoding anti-green fluorescent protein (GFP). All plasmids were verified to be correct by Sanger sequencing.

[0056] (2) Lentiviral packaging and cell transduction

[0057] Virus packaging: 10 μg `pLVX-PGFRN-uPAR-VHH` (or control plasmid), 7.5 μg psPAX2 packaging plasmid, and 2.5 μg pMD2.G envelope plasmid were mixed with 50 μL Lipofectamine 3000 and co-transfected into HEK293T cells (cultured in 10 cm dishes) that had reached 70-80% confluence. The medium was replaced with fresh medium 6 hours after transfection, and the virus-containing supernatant was collected at 48 and 72 hours.

[0058] Virus concentration and titer determination: The collected supernatant was filtered through a 0.45 μm filter membrane and then analyzed using Lenti-X. TM The virus was concentrated overnight at 4°C using a concentrator. After centrifugation, the viral pellet was resuspended in serum-free medium. Lenti-X was used. TM Viral genome titers were determined using a qRT-PCR titer kit. Results showed that the titer of the pLVX-PGFRN-uPAR-VHH virus stock solution was 2.1 × 10⁻⁶. 8 TU / mL, control virus was 1.8 × 10⁻⁶ 8 TU / mL.

[0059] Cell transduction and screening: Fifth-generation human umbilical cord mesenchymal stem cells (hUC-MSCs, commercial products or laboratory-derived and cultured, such as those from Shanghai Hongshun Biotechnology Co., Ltd.) were used at a rate of 2 × 10⁻⁶ cells / year. 5 Cells were seeded per well in 6-well plates. When the cell density reached 30%, an appropriate amount of virus solution (MOI=30) and 6 μg / mL Polybrene were added. After incubation for 12 hours, the medium was replaced with complete medium. 72 hours after transduction, medium containing 1.5 μg / mL puromycin was added for continuous selection for 14 days.

[0060] Cell sorting: Since the extracellular region of the VHH-PTGFRN fusion protein contains a FLAG-tagged epitope, cells successfully transduced and expressing this fusion protein were specifically identified and sorted by flow cytometry using an anti-FLAG-tagged monoclonal antibody. Accordingly, surviving cells were digested with Accutase, stained with anti-FLAG (DYKDDDDK) Tag Monoclonal Antibody (1:200 dilution) at 4°C for 30 minutes, washed with PBS, and then the top 10% of cells with the highest FLAG signal intensity were sorted by flow cytometry. Results showed that after sorting, the proportion of FLAG-positive cells in the `MSC^(PGFRN-uPAR-VHH)` cell line was >99%, with a mean fluorescence intensity (MFI) of 15,230; the FLAG positivity rate in the `MSC^(PGFRN-Control-VHH)` cell line was >98%, with an MFI of 14,850.

[0061] 2.2 Induction, production, and purification of engineered exosomes

[0062] (1) The following four groups of cells were cultured to produce exosomes:

[0063] Group 1 (Exo-Native): Parental hUC-MSCs.

[0064] Group 2 (Exo-Control): MSC^(PGFRN-Control-VHH) cells.

[0065] Group 3 (Exo-VHH): MSC^(PGFRN-uPAR-VHH) cells.

[0066] Group 4 (Exo^(VHH / miR)): MSC^(PGFRN-uPAR-VHH) cells.

[0067] (2) Cell culture and induction

[0068] After resuscitation of the stable cell lines constructed above and the parental hUC-MSCs, they were cultured and expanded using standard methods. For exosome production, all cells were transferred to a three-dimensional culture system: using Cytodex 3 microcarriers, cells were cultured at 5 × 10⁻⁶ cells / mL in a 125 mL stirred bioreactor. 5 Inoculate at a density of cells / mL.

[0069] Using a chemically defined exosome-free mesenchymal stem cell culture medium (ScienCell), the cells were cultured at 37°C, 5% CO2, and 3% O2 ​​(hypoxia) with a stirring speed of 40 rpm.

[0070] On day 3 (when cell confluence was approximately 80%), the `Exo^(VHH / miR)` production group was induced by replacing the medium with fresh exosome-free medium containing 1.0 mM sodium valproate (VPA, Sigma). The `Exo-Native`, `Exo-Control`, and `Exo-VHH` groups were replaced with an equal volume of medium without inducers. All groups were cultured for another 48 hours.

[0071] (3) Exosome isolation and purification

[0072] Collection and Pretreatment: Collect conditioned medium from all groups (approximately 100 mL / group) and immediately place on ice. Perform the following steps in sequence: centrifuge at 300 × g for 10 minutes to remove live cells; centrifuge at 2,000 × g for 20 minutes to remove dead cells; centrifuge at 10,000 × g for 30 minutes to remove cell debris.

[0073] Concentration: After vacuum filtration through a 0.22 μm PES membrane, the supernatant was concentrated to 5 mL using a tangential flow filtration system equipped with a hollow fiber column with a molecular weight cutoff of 100 kDa.

[0074] Fine purification: Load the concentrate onto a qEV original 35 nm size exclusion column and elute with sterile PBS (pH 7.4) as the mobile phase. Collect the 7th-9th mL fraction using an automated fraction collector according to the instructions.

[0075] Concentration determination and dispensing: using Pierce TM The total protein concentration of purified exosome samples was determined using the BCA Protein Assay Kit. Nucleic acid contamination was assessed by measuring the A260 / A280 ratio using NanoDrop OneC. All samples were adjusted to a uniform protein concentration (1.0 mg / mL) with PBS, aliquoted into 100 μL tubes, and stored at -80°C for later use.

[0076] The results showed that the average exosome protein yield per 100 mL of culture medium was: `Exo-Native` 285 ± 32 μg; `Exo-Control` 298 ± 28 μg; `Exo-VHH` 312 ± 35 μg; and `Exo^(VHH / miR)` 305 ± 30 μg. The A260 / A280 ratios of each group were between 1.7 and 1.9, consistent with the characteristics of pure exosomes.

[0077] 2.3 Exosome Characterization and Quality Analysis

[0078] (1) Nanoparticle tracking analysis

[0079] Method: All exosome samples were diluted 1000-fold with sterile PBS to the appropriate detection concentration (approximately 1×10⁻⁶). 8 Particles / mL). A NanoSight NS300 system equipped with a 488 nm laser and an sCMOS camera was used. Five 60-second video segments were automatically captured for each sample at 25°C, and particle concentration and size distribution were analyzed using NTA 3.4 software.

[0080] The results are shown in Table 1: There was no statistically significant difference in particle concentration among the four groups of exosomes (p > 0.05), indicating that genetic engineering (fusion protein expression) and chemical induction (VPA treatment) did not significantly affect the basal exosome secretion capacity of cells, ensuring dose equivalence in subsequent functional comparison experiments. The average particle size, key indicators characterizing distribution width (D10, D50, D90), and polydispersity index (PDI) of all groups of exosomes were highly consistent, and all PDI values ​​were less than 0.2, indicating that all preparations were uniformly sized and well-dispersed nanoparticles. This demonstrates that the engineering modifications of this invention (including membrane protein fusion and intraluminal miRNA enrichment) did not alter the core physical properties of exosomes; their size remains within the typical extracellular vesicle range, which is beneficial for maintaining their natural biodistribution and cellular uptake characteristics.

[0081] Table 1 Summary of key parameters for NTA analysis of four groups of exosome samples (mean ± standard deviation, n=5 independent measurements)

[0082]

[0083] (2) Protein immunoblotting analysis

[0084] Methods: 20 μg of each exosome protein sample was added to 1× Laemmli loading buffer (containing 5% β-mercaptoethanol) and boiled at 95°C for 5 minutes. Electrophoresis was performed using a 4-20% gradient Tris-Glycine precast gel, followed by wet transfer to a PVDF membrane. The membrane was incubated with mouse anti-human CD9 antibody (1:1000), anti-CD63 antibody (1:1000), anti-TSG101 antibody (1:1000), and rabbit anti-FLAG-tagged antibody (1:2000), respectively, and then incubated with the corresponding HRP-labeled secondary antibody. Chemiluminescent substrate was used for development, and images were acquired using an imaging system.

[0085] The results show that: Figure 4 As shown, all four exosome samples exhibited clear strong positive bands at the expected locations (CD9: ~24 kDa, CD63: ~50 kDa, TSG101: ~46 kDa), confirming that the purified vesicles are typical exosomes. Crucially, only in the `Exo-Control`, `Exo-VHH`, and `Exo^(VHH / miR)` groups were a single, clear FLAG-tagged positive band detected at approximately 70–90 kDa (a molecular weight consistent with the expected molecular weight of the glycosylated fusion protein), perfectly matching the expected molecular weight of the VHH-PTGFRN fusion protein. This band was absent in the `Exo-Native` group. These results demonstrate that the engineered fusion protein has been successfully expressed and specifically integrated into the membrane of the engineered exosomes.

[0086] (3) Quantitative analysis of miR-193b-3p in exosomes

[0087] Methods: Total RNA was isolated from equal volumes (100 μL, 1 mg / mL) of exosome samples using the miRNeasy Serum / Plasma Kit. Before reverse transcription, 1 × 10⁻⁶ ppm of the serum was added to each sample. 8 The synthesized cel-miR-39 was used as an exogenous reference to correct for extraction efficiency. Reverse transcription was performed using the miScript II RT Kit. qPCR reactions were performed on a real-time quantitative PCR system using miR-193b-3p specific miScript Primer Assay and cel-miR-39 primers. Relative quantification was performed using 2... −ΔΔCq Using the `Exo-Native` group as the calibration sample, the relative enrichment fold of miR-193b-3p among the groups was calculated.

[0088] The results showed that, after correction with cel-miR-39, the absolute copy number of miR-193b-3p (mean ± SD, n=4 independent batches) / μg exosomal protein was: Exo-Native: (4.2±0.7)×10 7 ;Exo-Control: (4.5±0.6)×10 7 Exo-VHH: (5.1±0.9)×10 7 ;Exo^(VHH / miR): (2.8±0.4)×10 8 .

[0089] Univariate ANOVA combined with Tukey's post-hoc test showed that the miR-193b-3p content in the `Exo^(VHH / miR)` group was significantly higher than that in the other three groups (p < 0.001). The content in the `Exo^(VHH / miR)` group was approximately 5.5 times that in the `Exo-VHH` group, demonstrating that sodium valproate induction treatment can effectively and specifically enrich the target miRNA into the exosome cavity.

[0090] 2.4 Summary

[0091] This study obtained an hUC-MSCs cell line that stably expresses uPAR-targeting (or control) VHH-PTGFRN fusion protein through genetic engineering, laying the foundation for the large-scale and standardized production of exosomes in the future.

[0092] This study established and optimized a standardized production and purification process combining three-dimensional hypoxic culture, chemical induction, and size exclusion chromatography. This process can reliably obtain high yields (~300 μg / 100 mL culture medium) and high purity (typical morphology, well-defined marker proteins, and low nucleic acid contamination) of exosomes.

[0093] In this study, we obtained four sets of comparative samples that were highly consistent in basic physicochemical properties but showed clear differences in core functional elements: `Exo-Native` (natural and unmodified), `Exo-Control` (expressing irrelevant target proteins), `Exo-VHH` (exhibiting only anti-uPAR VHH), and `Exo^(VHH / miR)` (exhibiting anti-uPAR VHH and enriching uPAR repressive miRNAs). This lays the foundation for subsequent research.

[0094] Example 3: In vitro targeted binding and synergistic inhibition of uPAR expression by engineered exosomes

[0095] 3.1 Materials and Methods

[0096] 3.1.1 Analysis of exosome targeting and binding capacity (flow cytometry)

[0097] Exosome fluorescent labeling: Take 100 μg of each exosomal protein (Exo-Native, Exo-Control, Exo-VHH, Exo^(VHH / miR)) and label them using pHrodo. TM The cells were labeled using the Red SE extracellular vesicle labeling kit.

[0098] Combined with experiments: IMR-90 cells (highly expressing uPAR) induced by 10 Gy X-rays on day 7 of senescence were used at 2×10-1 5 Cells were seeded at 5 x 10⁶ cells / well in a 24-well plate and incubated overnight. Cells were washed twice with pre-chilled PBS. An equal number of particles (5 x 10⁶) were added to each well. 9 Fluorescently labeled exosomes (dissolved in 200 μL of PBS containing 2% BSA in an ice bath) were incubated on ice at 4°C for 1 hour (to inhibit cell memory activity and detect only surface binding). After incubation, the cells were gently washed three times with pre-cooled PBS at 4°C.

[0099] Flow cytometry: Cells were digested with 0.25% trypsin-EDTA, resuspended in 400 μL of ice-cold PBS, and immediately analyzed. At least 10,000 live-cell events were collected for each sample, and pHrodo Red fluorescence (representing bound exosomes) was detected using flow cytometry. Background fluorescence was set using the PBS-treated group without exosomes.

[0100] Competition inhibition experiment: To verify the binding specificity, before adding Exo-VHH, cells were pre-incubated with 20 times molar excess of free anti-uPAR VHH (prepared in Example 1) at 4°C for 30 minutes before the binding experiment was performed.

[0101] 3.1.2 Effect of exosome treatment on uPAR protein expression in target cells (Western Blot)

[0102] Cell treatment: Senescent IMR-90 cells were seeded in 6-well plates (5 × 10⁶ cells / well). 5 Cells / well). After cell adhesion, replace with exosomes containing different concentrations (final concentration 1×10⁻⁶). 10 Complete culture medium containing particles / mL was used. The following groups were established: untreated senescent group, Exo-Native group, Exo-Control group, Exo-VHH group, and Exo^(VHH / miR) group. Each group had 4 replicates. Treatment lasted 48 hours.

[0103] Protein sample preparation: After treatment, wash cells twice with pre-chilled PBS, add 150 μL of RIPA lysis buffer to each well, and lyse on ice for 30 minutes. Collect the lysis buffer, centrifuge at 14,000g for 15 minutes at 4°C, and collect the supernatant. Determine protein concentration using the BCA method.

[0104] Western Blot analysis: 30 μg of total protein was loaded, electrophoresed, and transferred to a membrane. Blocking was performed with 5% skim milk at room temperature for 1 hour. Primary antibody incubation conditions: rabbit anti-human uPAR polyclonal antibody (prepared in our laboratory, 1:1000) overnight at 4°C; mouse anti-β-actin monoclonal antibody (1:5000) for 1 hour at room temperature. Secondary antibody was incubated at room temperature for 1 hour using the corresponding HRP-labeled antibody. After ECL development, the grayscale values ​​of the uPAR band (approximately 55 kDa) and the β-actin band (42 kDa) were quantified using software. The relative expression levels of each treatment group were calculated by normalizing the uPAR / β-actin ratio of the untreated senescent group to 1.0.

[0105] 3.2 Results

[0106] 3.2.1 Specificity analysis of exosome targeting and binding

[0107] Flow cytometry was used to quantitatively detect the surface binding ability of exosomes to senescent cells at 4°C. Specific data are shown in Table 2. The binding signal intensity (MFI) of Exo-VHH to Exo^(VHH / miR) group was significantly higher than that of Exo-Native and Exo-Control groups (p < 0.001), indicating that anti-uPAR VHH mediated efficient specific targeting. There was no statistically significant difference in MFI between the Exo-VHH and Exo^(VHH / miR) groups (p > 0.05), demonstrating that VPA induction and miRNA enrichment do not affect the targeting function of VHH on the exosome surface. The binding signal of the Exo-Control (non-related VHH) group was not different from that of the Exo-Native group, ruling out non-specific adsorption; pre-addition of free anti-uPAR VHH almost completely blocked the binding of Exo-VHH (signal intensity decreased to baseline levels), further confirming that the binding is achieved through VHH-uPAR specific interaction.

[0108] Table 2 Results of exosome-targeted binding specificity analysis

[0109]

[0110] 3.2.2 Cooperative inhibition of uPAR protein expression in target cells

[0111] The results of Western blot quantitative analysis are shown in Table 3: The Exo-Native and Exo-Control groups had no effect on uPAR expression in senescent cells. Exo-VHH treatment downregulated uPAR expression by approximately 29% (p < 0.01), which may be due to VHH binding interfering with uPAR membrane cycling or promoting its endocytic degradation. Synergistic effect: The Exo^(VHH / miR) group showed the strongest inhibitory effect, downregulating uPAR expression by 62%. More importantly, its downregulation effect was significantly better than that of the Exo-VHH group with only targeting function (p < 0.01).

[0112] Table 3 Summary of Western Blot quantitative analysis results

[0113]

[0114] 3.3 In summary, this embodiment demonstrates through quantitative experiments that:

[0115] This study demonstrates that engineered exosomes targeting uPAR VHH can specifically and with high affinity bind to senescent cells with high uPAR expression, and this binding can be competitively inhibited by free VHH, confirming the specificity of the targeting mechanism. Crucially, Exo^(VHH / miR) can more efficiently downregulate uPAR protein expression in target cells, significantly outperforming Exo-VHH which only has a targeting function. This data directly proves that intraluminal miR-193b-3p performs gene silencing after targeted delivery, achieving a synergistic effect of "membrane targeting (improving delivery efficiency)" and "luminal silencing (performing gene intervention)" on the same target.

[0116] Example 4: Comparison of engineered exosomes' functions in clearing senescent cells and inhibiting SASP

[0117] 4.1 Materials and Methods

[0118] 4.1.1 Cell treatment: IMR-90 cells (SA-β-Gal positivity >85%) induced by 10 Gy X-rays on day 7 were treated at a rate of 1 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells / well in 12-well plates (for staining) or 6-well plates (for supernatant collection). After cell adhesion, the medium was replaced with fresh complete medium containing the following treatments:

[0119] Young control group: young IMR-90 cells (PD 30) + PBS.

[0120] Senescent + PBS group: Senescent IMR-90 cells + PBS.

[0121] Senescent + Exo-Native group: Senescent cells + 1×10 10particles / mL Exo-Native.

[0122] Senescence + Exo-Control group: senescent cells + 1×10 10 particles / mL Exo-Control.

[0123] Senescent + Exo-VHH group: senescent cells + 1×10 10 particles / mL Exo-VHH.

[0124] Senescent + Exo^(VHH / miR) group: senescent cells + 1×10 10 particles / mL Exo^(VHH / miR).

[0125] Senescent + D + Q group: Senescent cells were treated with a combination of 100 nM dasatinib and 20 μM quercetin.

[0126] All treatment groups were configured with 6 biological replicates (n=6) and treated continuously for 72 hours at 37°C and 5% CO2 without changing the culture medium.

[0127] 4.1.2 SA-β-Gal staining and quantification

[0128] Staining: After treatment, the procedure was strictly followed according to the instructions of the senescent β-galactosidase staining kit. Cells were washed with PBS and fixed with 1× fixative at room temperature for 15 minutes. Then, staining working solution containing X-Gal (pH 6.0) was added, and the cells were incubated in a CO2-free incubator for 16 hours in the dark.

[0129] Imaging and Counting: Five non-overlapping fields of view (200x) of each sample well were randomly selected under bright field using an inverted microscope. The images were imported into ImageJ software, and the total number of cells and the number of SA-β-Gal positive (stained blue) cells in each field of view were counted using a color threshold recognition method (identifying blue precipitate) with manual correction.

[0130] Calculation: Positive rate per field of view = (Number of positive cells / Total number of cells) × 100%. The final result for each biological replicate (n=1) is the average of the positive rates of the 5 fields of view.

[0131] 4.1.3 SASP factor secretion assay (ELISA): After 72 hours of treatment, the culture supernatant of cells from each group was collected and centrifuged at 300g for 10 minutes at 4°C to remove detached cells. The supernatant was immediately aliquoted and stored at -80°C until detection. Detection was performed using a commercially available human IL-6 and MMP-3 ELISA kit. All procedures were strictly performed in accordance with the kit instructions.

[0132] 4.1.4 Statistical Analysis: All data are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 9.0 software. One-way ANOVA was used for comparisons among multiple groups. If homogeneity of variance was satisfied, Tukey's post-hoc test was used for pairwise comparisons between groups. A p-value < 0.05 was considered statistically significant.

[0133] 4.2 Results

[0134] 4.2.1 The effect of engineered exosomes on scavenging senescent cells is shown in Table 4. Basic scavenging effect: Compared with the senescent + PBS group, Exo-Native and Exo-Control treatments reduced the proportion of senescent cells to a certain extent (~20%), which may be a result of the non-specific anti-aging effect of natural exosomes.

[0135] Targeted enhancement effect: The Exo-VHH group expressing uPAR VHH showed a significantly improved clearance effect of 50.9%, which was significantly better than the two non-targeted control groups (p < 0.001), confirming the necessity of active targeting to improve clearance efficiency.

[0136] Synergistic Effect and Advantage Comparison: The Exo^(VHH / miR) group exhibited the strongest clearance ability, with the positive rate decreasing to 24.7% and the clearance rate reaching 70.7%. Its effect was significantly superior to the Exo-VHH group, which only had targeting function (p < 0.01), directly demonstrating the synergistic effect of intraluminal miRNAs. Notably, the clearance effect of Exo^(VHH / miR) was also significantly better than the commonly used preclinical senolytic positive drug combination D+Q (p < 0.001), highlighting its potential therapeutic advantages.

[0137] Table 4. Quantitative results of SA-β-Gal staining

[0138]

[0139] 4.2.2 The effect of engineered exosomes on inhibiting SASP factor secretion is shown in Table 5.

[0140] SASP inhibition trends were consistent: the inhibition trends of the two key SASP factors (pro-inflammatory cytokine IL-6 and matrix-degrading enzyme MMP-3) were consistent with the clearance of senescent cells. Senescence leads to a sharp increase in the secretion of these two factors, with slight inhibition observed in non-targeted exosomes.

[0141] Targeting and synergistic effects: Exo-VHH treatment significantly inhibited SASP secretion (IL-6 and MMP-3 decreased by approximately 45% and 47%, respectively), while Exo^(VHH / miR) showed the strongest inhibitory ability (IL-6 and MMP-3 decreased by approximately 77% and 79%, respectively), and its effect was significantly better than that of the Exo-VHH group and the D+Q positive drug group (p < 0.01).

[0142] Functional correlation: This result corroborates data showing that Exo^(VHH / miR) most effectively downregulated uPAR protein expression (Example 3) and cleared senescent cells (this example). Since the uPAR signaling pathway directly regulates SASP, synergistic inhibition of uPAR ultimately led to the most complete reversal of the SASP phenotype.

[0143] Table 5 Results of ELISA detection of IL-6 and MMP-3 concentrations in cell culture supernatant

[0144]

[0145] 4.3 In summary, this embodiment demonstrates through rigorous in vitro functional experiments that:

[0146] The engineered exosomes of this invention, particularly Exo^(VHH / miR), exhibit a strong ability to clear senescent cells and inhibit their harmful SASP secretion. This function displays a clear gradient effect: Exo^(VHH / miR) > Exo-VHH > non-targeted exosomes. This clearly validates the design logic of synergistic dual functions of "targeted delivery" and "gene silencing," and its overall effect surpasses existing senolytic strategies (D+Q). These results provide in vitro efficacy evidence for the engineered exosomes to reverse tissue aging phenotypes and treat age-related diseases in vivo.

[0147] Example 5: In vivo efficacy and safety evaluation of engineered exosomes in a naturally aging mouse model

[0148] 5.1 Materials and Methods 5.1.1 Experimental Animals and Grouping

[0149] Animals: 18-month-old naturally aging male C57BL / 6J mice (as aging model) and 3-month-old young male mice of the same strain (as young controls) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All mice were housed in an SPF-grade animal facility with a 12-hour light / dark cycle and free access to standard feed and water.

[0150] Grouping and Administration: 18-month-old mice were randomly divided into 4 groups (n=12) according to body weight: (1) aging + saline group; (2) aging + Exo-Native group; (3) aging + Exo-VHH group; (4) aging + Exo^(VHH / miR) group. A separate (5) young control group (n=12) was also included. Treatment groups received medication via tail vein injection at a dose of 5 × 10⁻⁶. 10 Particles / dose (approximately 15 mg exosome protein / kg body weight, calculated based on the specific activity of Exo-VHH in Example 2), twice weekly (e.g., Monday and Thursday) for 8 weeks, for a total of 16 doses. The aging + saline group and the young control group were injected with the same volume of sterile saline.

[0151] 5.1.2 Grip Test

[0152] Testing was conducted before treatment began (week 0 baseline) and on day 1 after all treatments were completed (week 9 endpoint).

[0153] Testing Method: A digital small animal grip strength tester was used. Before the test, the mice were allowed to become familiar with the grip strength grid. During the test, the mouse's forelimbs were gently placed on the grid bars. Once the mouse had a firm grip, its body was pulled backward at a constant horizontal speed until its forelimbs released. The maximum grip strength value displayed on the instrument (unit: gf) was recorded. Each mouse was tested 5 times consecutively, with at least 1 minute intervals between each test. The maximum value was taken as the final data for that test.

[0154] Data analysis: Using the average grip strength of the young control group at week 9 as 100%, the percentage of grip strength of each aging treatment group relative to this baseline was calculated to assess the degree of recovery of physiological function.

[0155] 5.1.3 Tissue Sample Collection and Processing 24 hours after the last administration, the mice were euthanized and dissected.

[0156] Serum collection: Blood is drawn from the heart, allowed to stand, and then centrifuged to separate the serum for biochemical analysis.

[0157] Tissue collection: Liver, back skin, heart, spleen, lungs, and kidneys were rapidly harvested. A portion of each tissue was immediately fixed with 4% paraformaldehyde (for histological purposes), and the remaining portion was frozen at -80°C.

[0158] 5.1.4 Histological Analysis

[0159] SA-β-Gal staining of liver: Fixed liver tissue was embedded in paraffin and sectioned (5 μm thickness). Staining was performed using a senescent cell histochemical staining kit, strictly following the manufacturer's instructions. Five non-overlapping 200x fields of view were randomly selected from liver sections of each mouse, and the percentage of blue-positive stained area in the total field of view was calculated using ImageJ software.

[0160] Masson staining of skin: Paraffin sections of back skin tissue were stained using the Masson trichrome staining kit (Sigma-Aldrich, HT15), making collagen fibers appear blue and muscle fibers red. Three 200x fields of view were randomly selected from the dermis of each section. The blue channel was separated using the "Color Deconvolution" plugin in ImageJ software, and the percentage of the blue (collagen) area relative to the total dermal area was calculated for semi-quantitative analysis of collagen content.

[0161] 5.1.5 Preliminary Security Assessment

[0162] General condition and weight: Observe the animal's mental state, activity, coat color, and eating and drinking habits daily, and weigh and record the weight weekly.

[0163] Serum biochemical analysis: The following indicators in serum were detected using a fully automated biochemical analyzer: alanine aminotransferase (ALT), aspartate aminotransferase (AST) (to assess liver function); creatinine (CREA), blood urea nitrogen (BUN) (to assess kidney function).

[0164] Organ pathological examination: After fixation, paraffin embedding, and H&E staining, the heart, liver, spleen, lungs, and kidneys are observed under an optical microscope to assess for the presence of drug-related pathological changes such as inflammation, necrosis, degeneration, and fibrosis.

[0165] 5.1.6 Statistical Analysis All data are expressed as mean ± standard deviation. GraphPad Prism 9.0 software was used for analysis. One-way ANOVA was used for comparisons among multiple groups. If homogeneity of variance was satisfied, Tukey's post-hoc test was used for pairwise comparisons between groups. A p-value < 0.05 was considered statistically significant.

[0166] 5.2 Results 5.2.1 Engineered exosomes improve the overall physiological function of aging mice

[0167] Before treatment, baseline grip strength was similar across all aging groups and significantly lower than that of the younger groups. After 8 weeks of treatment, the Exo-Native group showed slight improvement. The Exo-VHH group showed a clear improvement (recovering to 83.6%), while the Exo^(VHH / miR) group showed the most significant effect, with grip strength recovering to 94.2% of the younger control, significantly better than the Exo-VHH group (p < 0.05). This indicates that bifunctional exosomes have a synergistic advantage in restoring overall bodily function. See Table 6 for details.

[0168] Table 6. Grip Test Results

[0169]

[0170] 5.2.2 Engineered exosomes reduce the aging burden on multiple tissues

[0171] Clearance of senescent liver cells: Consistent with in vitro results, Exo^(VHH / miR) was the most effective at clearing senescent cells in the liver, with the lowest positive area (3.1%), significantly better than the Exo-VHH group (7.5%, p < 0.01). See Table 7.

[0172] Improving the phenotype of skin aging: Natural aging leads to significant loss of skin collagen. Exo^(VHH / miR) treatment most effectively reverses this change, increasing collagen content to levels close to those of the younger group, and the effect is significantly better than the Exo-VHH group (p <0.01), directly demonstrating its ability to improve tissue structure and repair capacity. As shown in Table 7.

[0173] Table 7 Results of histological quantitative analysis

[0174]

[0175] 5.2.3 Preliminary Security Assessment

[0176] General condition and weight: During the treatment period, all mice were active, had smooth fur, and no deaths occurred. The weight of each group showed a stable and slow growth trend, with no statistically significant differences between groups (weight change rate at week 8: aging + saline + 3.2%, Exo^(VHH / miR) group + 2.9%).

[0177] Serum biochemical indicators: After the treatment, the key liver and kidney indicators were all within the normal reference range of the laboratory (ALT: 20-55 U / L, AST: 40-120 U / L, CREA: 10-30 μmol / L, BUN: 5-12 mmol / L), and there were no statistically significant differences between the treatment groups and the aging + saline group (p > 0.05).

[0178] Organ pathology: H&E staining sections of the heart, liver, spleen, lungs and kidneys showed that, compared with the control group, the tissue structures of each treatment group were clear, and no drug-related pathological changes such as edema, necrosis, abnormal infiltration of inflammatory cells or fibrosis were observed.

[0179] 5.3 Summary: In vivo experiments confirm that:

[0180] Significantly improves overall function: Exo^(VHH / miR) can most effectively restore physiological function (grip) in naturally aging mice.

[0181] Highly effective in reducing the aging burden on multiple tissues: Exo^(VHH / miR) can synergistically clear senescent liver cells and significantly improve skin collagen loss, with better results than single-target groups.

[0182] Good preliminary safety: At the stated dose and duration of treatment, engineered exosomes did not cause any abnormal changes in body weight, blood biochemistry, or major organ structure in mice, demonstrating good tolerability.

[0183] In summary, Exo^(VHH / miR) demonstrated excellent overall anti-aging efficacy and tissue repair capabilities in natural aging models, with good safety profile, providing strong preclinical data support for its potential as a novel anti-aging therapeutic agent.

[0184] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A single-domain anti-human uPAR antibody VHH, characterized in that, The amino acid sequence of the anti-human uPAR single-domain antibody VHH is shown in SEQ ID NO:

1.

2. A VHH-PTGFRN fusion protein, characterized in that, The VHH-PTGFRN fusion protein comprises, in sequence, the anti-human uPAR single-domain antibody VHH as described in claim 1, a FLAG tag at the N-terminus of the single-domain antibody, a flexible linker peptide, and a PTGFRN protein fragment; wherein the flexible linker peptide is a GGGGS triple repeat sequence; the PTGFRN protein fragment is amino acids 448 to 730 of the protein shown in UniProt IDQ9P2B2; and the nucleotide sequence encoding the fusion protein is shown in SEQ ID NO:

2.

3. A recombinant lentiviral vector, characterized in that, The recombinant lentiviral vector contains a nucleotide sequence encoding the VHH-PTGFRN fusion protein of claim 2, wherein the lentiviral vector is pLVX-EF1α-IRES-Puro.

4. An engineered human umbilical cord mesenchymal stem cell line, characterized in that, The engineered human umbilical cord mesenchymal stem cell line stably expresses the VHH-PTGFRN fusion protein as described in claim 2.

5. An engineered exosome targeting uPAR, characterized in that, The exosomes are derived from the engineered human umbilical cord mesenchymal stem cell line of claim 4, and their surface displays the VHH-PTGFRN fusion protein of claim 2.

6. The engineered exosomes according to claim 5, characterized in that, The exosome cavity was enriched with miR-193b-3p, with a miR-193b-3p content of 2.8 ± 0.4 × 10⁻⁶. 8 The copy number per microgram of exosomal protein is named ExoVHH / miR.

7. A method for preparing engineered exosomes as described in claim 5 or 6, characterized in that, The method includes the following steps: (1) Preparation of anti-human uPAR VHH: Alpaca were immunized with recombinant human uPAR protein as antigen, a phage display antibody library was constructed, and the anti-human uPAR single-domain antibody VHH described in claim 1 was obtained by screening after three rounds of solid-phase antigen panning; (2) Construction of engineered human umbilical cord mesenchymal stem cell line: The nucleotide sequence encoding the fusion protein of claim 2 is cloned into a lentiviral vector, packaged into lentivirus and transduced into human umbilical cord mesenchymal stem cells, and then screened by puromycin and sorted by flow cytometry to obtain the engineered cell line of claim 4. (3) Induction of exosome production: The engineered cell line was seeded into Cytodex 3 microcarriers and cultured in a 125 mL stirred bioreactor using exosome-free medium under three-dimensional hypoxia conditions of 37°C, 5% CO2 and 3% O2. On the third day of culture, 1.0 mM sodium valproate was added to induce miR-193b-3p enrichment and cultured for another 48 hours. (4) Purification of exosomes: Collect cell culture supernatant, centrifuge at 300×g for 10 minutes, 2000×g for 20 minutes and 10000×g for 30 minutes to remove cells and debris, filter through a 0.22 μm filter membrane, concentrate to 5 mL using a tangential flow filtration system with a 100 kDa cutoff, and then purify by a qEV original 35 nm size exclusion chromatography column. Collect the 7th to 9th mL fraction to obtain engineered exosomes.

8. The preparation method according to claim 7, characterized in that, In step (1), the phage display antibody library has a capacity of 3.2 × 10⁻⁶. 8 CFU; the cell seeding density in step (3) is 5 × 10⁻⁶. 5 cells / mL, stirring speed 40 rpm; in step (4), the exosomes were adjusted to a protein concentration of 1.0 mg / mL by the BCA method and stored at -80°C.

9. The use of the anti-human uPAR VHH of claim 1, the VHH-PTGFRN fusion protein of claim 2, the engineered human umbilical cord mesenchymal stem cell line of claim 4, and the engineered exosome of claim 5 or 6 in the preparation of anti-aging agents or agents for treating aging-related diseases, wherein aging-related diseases include chronic inflammation, cardiovascular disease, neurodegenerative diseases, and skin aging.

10. An anti-aging preparation, characterized in that, The anti-aging formulation comprises the ExoVHH / miR engineered exosomes as described in claim 6 as the active ingredient, and further comprises a pharmaceutically acceptable carrier, wherein the formulation is an injection and the exosome protein concentration is 1.0 mg / mL.

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