Mesenchymal stem cell exosome with effect of repairing skin photoaging and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and specifically discloses a single-domain antibody against human MMP-1, a mesenchymal stem cell exosome targeted and modified by the single-domain antibody, and application of the single-domain antibody and the exosome in repairing skin photoaging. The surface of the exosome is covalently connected with the single-domain antibody that specifically targets human MMP-1 through click chemistry technology, the single-domain antibody has an amino acid sequence as shown in SEQ ID NO:1, and is obtained through camel phage display library screening and affinity maturation, and has a high affinity of a nanomolar level to MMP-1. The preparation method comprises screening and expression of the single-domain antibody, extraction and purification of the mesenchymal stem cell exosome, and directional coupling through DBCO-azide click chemistry. The modified exosome can specifically recognize and enrich in photoaged skin tissue, significantly inhibit collagen degradation, promote extracellular matrix reconstruction, relieve oxidative stress and inflammatory response, and exhibits excellent effects in repairing skin photoaging caused by ultraviolet rays, and is suitable for development of skin repair drugs and cosmetics.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a mesenchymal stem cell exosome with the function of repairing skin photoaging, its preparation method and application. Background Technology

[0002] Photo-damage, a major manifestation of extrinsic skin aging, is primarily driven by long-term or repeated exposure to ultraviolet (UV) radiation from the sun. Clinically, this process is characterized by multiple features, including but not limited to epidermal roughness, the formation of wrinkles of varying depths, breakage and loss of function of elastic fibers in the skin, focal or diffuse hyperpigmentation, and structural abnormalities in the superficial dermal capillary network. In recent years, with the continuous decline in stratospheric ozone concentration and the corresponding increase in the intensity of surface UV radiation, the global prevalence of photodamage-related skin lesions has shown a steady upward trend, becoming a significant challenge in public health and skin health management.

[0003] At the molecular pathological level, ultraviolet radiation (especially UVB) can drive photoaging through a variety of interconnected biological pathways. The core mechanisms include: the massive generation of reactive oxygen species (ROS) mediated by mitochondrial and membrane-associated NADPH oxidases; the sustained activation of inflammatory signaling pathways such as mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB); and the abnormal upregulation of the transcription and activity of a series of matrix metalloproteinases (MMPs). Among the many members of the MMP family, interstitial collagenase (MMP-1) occupies a central position due to its specific degradation ability of type I and type III collagen. Studies have shown that a single UVB irradiation dose of 1-2 times the minimum erythema dose (MED) can increase the mRNA and protein expression levels of MMP-1 in human skin by more than tenfold within 24 hours, leading to the rapid disintegration and loss of structural integrity of the collagen fiber network in the dermal extracellular matrix.

[0004] Currently, clinical interventions for photoaging of the skin mainly fall into the following categories: first, the use of physical or chemical sunscreens as primary prevention measures; second, the local or systemic supplementation of various antioxidants (such as vitamin C and vitamin E); third, the clinical application of retinoic acid and its derivatives; and fourth, physical therapy based on energy technologies such as lasers and intense pulsed light. However, existing strategies all have significant limitations: sunscreens can only block subsequent damage but cannot reverse existing structural damage; antioxidants have poor bioavailability and stability in the local skin, and their efficacy is limited in duration; while retinoic acid drugs effectively promote collagen regeneration, they are often accompanied by significant skin irritation, and patients generally have poor tolerance; and phototherapy has problems such as strong equipment dependence, high treatment costs, and strict requirements for professional operation. Therefore, the development of novel skin photoaging intervention technologies that combine high-efficiency repair capabilities and good safety has become an important research direction in dermatology and regenerative medicine.

[0005] In recent years, exosomes derived from mesenchymal stem cells (MSCs) have shown great promise in tissue engineering and regenerative medicine as an innovative cell-free therapeutic platform. Exosomes are phospholipid bilayer vesicles with a diameter of approximately 30-150 nanometers, actively secreted by cells. They carry various bioactive components, including proteins, messenger RNA, microRNAs, and lipid mediators. They can participate in intercellular communication and regulate gene expression and functional states of recipient cells through ligand-receptor interactions, membrane fusion, or internalization. Numerous in vitro and in vivo experiments have confirmed that MSC-derived exosomes possess multiple biological effects, including promoting collagen secretion from fibroblasts, inhibiting excessive inflammatory responses, enhancing endogenous antioxidant defense capabilities, and stimulating angiogenesis, theoretically demonstrating their advantages in the treatment of photoaging skin.

[0006] However, unmodified natural MSCs exosomes face a core technological challenge in vivo application due to insufficient targeting. Lacking tissue-specific homing signals, exosomes often exhibit systemic distribution after local or systemic administration, with only a very low proportion (usually <5%) effectively accumulating at target tissue sites, severely limiting therapeutic efficiency and potentially increasing off-target risks. To improve the targeted delivery capability of exosomes, researchers have explored various engineering strategies, including: using genetic engineering to express target peptide-exosome membrane protein fusion products in parental cells; using chemical coupling to anchor specific ligands to the exosome surface; and fusing exosomes with functionalized liposomes based on membrane hybridization technology. While traditional full-length antibodies (molecular weight approximately 150 kDa) possess high affinity and specificity, their large molecular size may significantly alter the membrane physical properties of exosomes and affect their natural biological functions, while also posing a potential risk of inducing host immune responses.

[0007] Single-domain antibodies (sdAbs), also known as nanobodies, are variable region fragments derived from camel heavy chain antibodies, with a molecular weight only about one-tenth that of traditional IgG (approximately 15 kDa). These antibody fragments possess a series of significant advantages: small molecular size and strong tissue penetration; lack of an Fc region, resulting in significantly reduced immunogenicity; high structural stability and resistance to high temperatures and acidic / alkaline environments; and ease of large-scale recombinant expression and functional modification through genetic engineering. Although the application of single-domain antibodies in targeted drug delivery is increasingly widespread, there are currently no systematic research reports in publicly available literature and patent databases on the functional modification of MSC exosomes using single-domain antibodies specifically targeting human MMP-1 for the treatment of photoaging skin. Summary of the Invention

[0008] To address the above technical problems, this invention provides a mesenchymal stem cell exosome that specifically targets MMP-1, the surface of which is modified with a high-affinity single-domain antibody via covalent linking.

[0009] Therefore, this invention provides a mesenchymal stem cell exosome targeting human MMP-1, which is constructed by specifically modifying the surface of exosomes with a high-affinity anti-human MMP-1 single-domain antibody. The single-domain antibody has the amino acid sequence shown in SEQ ID NO:1, and was obtained through screening using camel-derived phage display technology and multiple rounds of affinity maturation optimization, exhibiting nanomolar binding activity against MMP-1 protein. The exosome modification employs an efficient click chemistry strategy. First, the amino groups on the exosome surface are activated using NHS-PEG4-DBCO reagent, and then directionally coupled with the azide-modified single-domain antibody through a copper-free cycloaddition reaction, forming a stable covalent link.

[0010] The MMP-1-targeted mesenchymal stem cell exosomes provided by this invention offer multiple beneficial technical effects. Their most prominent advantage lies in their superior targeting specificity. The modified exosomes, aided by single-domain antibodies, can precisely recognize and bind to overexpressed MMP-1 in photoaged skin tissue, achieving effective enrichment at the lesion site, thereby significantly improving bioavailability and therapeutic efficiency. This system also possesses a synergistic therapeutic mechanism. The single-domain antibody not only acts as a targeting head group but also directly neutralizes the collagen-degrading activity of MMP-1. Furthermore, the various bioactive components carried by the exosomes themselves can promote collagen synthesis in fibroblasts, enhance endogenous antioxidant defense capabilities, and inhibit excessive inflammatory responses, thus synergistically reversing the photoaging process through multiple pathways. Experimental data fully demonstrate that these targeted exosomes significantly improve photoaging phenotypes in both cell and animal models, including enhancing cell viability, inhibiting apoptosis, increasing collagen content, reducing oxidative stress levels, and alleviating inflammatory damage. Their comprehensive repair effect is significantly superior to that of unmodified exosomes. Furthermore, this system exhibits excellent biocompatibility and safety. The exosomes are naturally derived, the single-domain antibodies have low immunogenicity, and no significant adverse reactions are observed with local application. The entire preparation process is stable, controllable, and highly reproducible, providing an innovative solution for targeted treatment of skin photoaging and possessing broad development prospects in the pharmaceutical and cosmetic fields. Attached Figure Description

[0011] Figure 1 SDS-PAGE results of anti-human MMP-1 single-domain antibody, where 1 represents anti-human MMP-1 single-domain antibody.

[0012] Figure 2 Western blot results of exosomes before and after modification, where 1 is the exosome sample before modification and 2 is the exosome sample after modification.

[0013] Figure 3 Western blot results of exosome markers (CD63, CD81, TSG101, Calnexin) and single-domain antibody (VHH) before and after modification.

[0014] Figure 4 Western blot analysis of MMP-1 protein expression results, where 1-4 represent the normal control group, UVB model group, unmodified exosome group, and single-domain antibody modified exosome group, respectively. Detailed Implementation

[0015] 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.

[0016] 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.

[0017] Example 1: Preparation and performance verification of anti-human MMP-1 single-domain antibody

[0018] 1. Experimental Materials and Reagents

[0019] Table 1 Experimental Materials and Reagents

[0020]

[0021] 2. Camel Immunity and Construction of Camel-Derived VHH Phage Display Library

[0022] 2.1 Camel Immunization Program

[0023] (1) Preparation of immunogenicity: The recombinant human MMP-1 protein was diluted to 1 mg / mL with sterile PBS and emulsified with adjuvant at a volume ratio of 1:1 (CFA was used for the first immunization and IFA was used for subsequent immunizations).

[0024] (2) Immunization schedule: Subcutaneous injection at multiple points (4 injection points on the back) was used for a total of 4 immunizations, with an interval of 14 days. The specific dosages are as follows: First immunization: antigen dose 200 μg / head, CFA emulsified; Second and third immunizations: antigen dose 150 μg / head, IFA emulsified; Fourth booster immunization: antigen dose 100 μg / head, without adjuvant (to facilitate subsequent blood collection).

[0025] (3) Immunotiter detection: Camel venous blood (5 mL) was collected 7 days after each immunization, serum was separated, and the anti-MMP-1 antibody titer was detected by indirect ELISA (coated with MMP-1 protein (2 μg / mL), incubated overnight at 4℃; serum was serially diluted (1:10) 3 ~1:10 6 Incubate at 37°C for 1 h; HRP-labeled goat anti-camel IgG (1:5000), incubate at 37°C for 0.5 h, TMB color development, incubate at 37°C for 10 min, and immediately detect OD after adding stop solution. 450 ).

[0026] (4) Results: The serum titer reached 1:1.2×10 after the fourth immunization. 5 (OD) 450 =1.82, blank control OD 450 =0.11), which meets the database construction requirements.

[0027] 2.2 Construction of Camel-Derived VHH Phage Display Library

[0028] (1) Peripheral blood lymphocyte isolation: Seven days after the fourth immunization, 50 mL of camel venous blood was collected, and peripheral blood mononuclear cells were isolated using Ficoll-Paque density gradient centrifugation fluid (density 1.077 g / mL), yielding approximately 2 × 10⁶ cells. 7 Each cell.

[0029] (2) Total RNA extraction and cDNA synthesis: Total RNA was extracted from PBMCs using Trizol reagent (concentration 1.2 μg / μL, A 260 / A 280 =1.92), with oligo(dT) 18 Using primers, cDNA is synthesized via reverse transcriptase.

[0030] (3) VHH gene amplification: Two rounds of PCR were performed using camel-derived VHH-specific primers: First round PCR (amplification of the VHH framework region + CDR region): Reaction conditions: 95℃ for 3 min; 95℃ for 30 s, 56℃ for 30 s, 72℃ for 40 s (30 cycles); 72℃ for 5 min; The product was a VHH gene fragment of approximately 450 bp (verified by agarose gel electrophoresis). Second round PCR (addition of restriction enzyme sites): The product was a VHH fragment containing restriction enzyme sites (approximately 480 bp).

[0031] (4) Vector ligation and phage packaging: The VHH fragment and pCANTAB5E vector were digested with SfiI / NotI, respectively, and recovered by agarose gel (recovery rate approximately 80%). T4 DNA ligase was used (incubated overnight at 16°C). The ligation products were transformed into E. coli TG1 competent cells, plated on 2×YT plates containing ampicillin, and incubated at 37°C for 12 h. The number of colonies on the plates was counted, and the library volume was calculated as: colony count × dilution factor × transformation volume / plate volume, yielding a library volume of 1.2 × 10⁻⁶. 10 PFU; 20 clones were randomly selected for sequencing, and 19 of them had different sequences, with a diversity of 95%, which met the library construction standards.

[0032] 3. Screening for anti-human MMP-1 single-domain antibodies

[0033] 3.1 Preparations before screening

[0034] (1) Coating plate: Each well of a 96-well microplate is coated with recombinant human MMP-1 protein (10 μg / mL in the first round, 5 μg / mL in the second round, and 3 μg / mL in the third round), incubated overnight at 4°C, and blocked with 5% skim milk at 37°C for 2 h.

[0035] (2) Phage library recovery: The constructed camel-derived VHH phage library was amplified using 2×YT medium, and the phage supernatant (titer 1×10⁻⁶) was harvested. 11 pfu / mL).

[0036] 3.2 Gradient-based stringent screening

[0037] (1) First round of screening (coarse screening and enrichment): Add 100 μL of phage library supernatant to each well and incubate at 37℃ for 1 h; wash 6 times with PBS containing 0.1% Tween-20 (PBST) to remove non-specifically bound phages; elute bound phages with 0.2 M Gly-HCl (pH 2.2), neutralize with 1 M Tris-HCl (pH 9.1), infect E. coli TG1 for amplification, and harvest phages (titer 1.5 × 10⁻⁶). 10 pfu / mL).

[0038] (2) Second and third rounds of screening (to improve specificity): the coating concentration was reduced to 5 μg / mL (second round) and 3 μg / mL (third round); the number of PBST washes was increased to 8 times (second round) and 10 times (third round); the elution time was shortened to 8 min (second round) and 5 min (third round); after the third round of screening, the phage titer increased to 9.8 × 10⁻⁶. 10 The pfu / mL concentration was 6.5 times higher than that of the first round, and the positive clones were significantly enriched.

[0039] 3.3 Identification of positive clones and affinity maturation

[0040] (1) Phage ELISA initial screening: 60 single clones selected in the third round of screening were cultured, and the phage supernatant was collected. The binding activity with MMP-1 was detected, and the OD was selected. 450 22 highly binding clones with a binding activity >2.0.

[0041] (2) Sequencing and diversity analysis: 22 clones were sequenced, and 12 unique VHH sequences were obtained, among which the CDR3 region of 8 sequences differed.

[0042] (3) Initial screening of SPR affinity: The KD value of 12 sequences was determined by Biacore T200, and 3 sequences with KD < 5 nM were screened out. Among them, the single-domain antibody shown in SEQ ID NO:1 had the lowest KD value and was identified as the target sequence for subsequent expression.

[0043] 4. Expression and purification of single-domain antibodies

[0044] 4.1 Cloning of the target gene and construction of engineered bacteria

[0045] (1) Vector construction: The coding gene of SEQ ID NO: 1 (codon optimized to be suitable for E. coli expression) was cloned into the pET-28a(+) vector (containing an N-terminal 6×His tag) through the NcoI / XhoI restriction site, and the ligation product was transformed into E. coli BL21(DE3) competent cells.

[0046] (2) Verification of positive clones: 10 single colonies were selected for colony PCR (primers containing universal vector sequences). 8 clones amplified 450 bp target bands. 3 clones were randomly selected for sequencing. The sequences matched 100% with the gene encoded by SEQ ID NO: 1. There were no mutations. The engineered bacteria were named BL21-pET28a-sdAb1.

[0047] 4.2 Induction of expression by engineered bacteria

[0048] (1) Seed culture: Select a single positive colony and inoculate it into 5 mL of LB medium containing 50 μg / mL kanamycin. Incubate at 37℃ and 220 rpm for 12 h. OD 600 =1.15.

[0049] (2) Large-scale fermentation: Inoculate 500 mL of LB medium (containing 50 μg / mL kanamycin) at a ratio of 1:100 and culture at 37℃ and 220 rpm until OD. 600 =0.72, add IPTG to a final concentration of 0.5 mM, and induce at 18℃ and 180 rpm for 16 h.

[0050] (3) Collection of bacterial cells: Centrifuge at 4℃ and 8000×g for 15 min to harvest 8.6 g of wet bacterial cells (500 mL of bacterial solution).

[0051] 4.3 Two-step purification method

[0052] (1) Nickel column affinity chromatography (HisTrap HP 5 mL): The bacterial cells were resuspended in 40 mL of equilibration buffer (20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 8.0), 1 mM PMSF was added, and the cells were sonicated (300 W, 3 s working time / 5 s interval, 20 min), centrifuged at 12000×g for 30 min at 4℃, and 36 mL of the supernatant was collected (A 280=1.92). Wash the column with equilibration buffer until the baseline stabilizes, load the sample at 0.5 mL / min, and perform gradient elution with elution buffer (containing 500 mM imidazole). Collect 8.2 mL of the main peak fraction (A). 280 =3.35, imidazole concentration approximately 310 mM).

[0053] (2) PD-10 desalting column buffer replacement: Equilibrate the column with PBS buffer (pH 7.4), load 8.2 mL of nickel column elution buffer, and combine the elution buffer with A. 280 A fraction >0.1 yielded 52 mL of purified antibody.

[0054] 4.4 Quality testing after purification

[0055] (1) SDS-PAGE purity analysis: 12% separating gel electrophoresis, Coomassie brilliant blue staining, ImageJ analysis of gray values, purity was 97.2% (single band, molecular weight approximately 15 kDa, consistent with theoretical value). Figure 1 As shown.

[0056] (2) BCA quantification: using BSA as the standard (R 2 =0.995), the purified antibody concentration was detected to be 1.28 mg / mL, the total mass was 66.6 mg, and the recovery rate was 77.4% (66.6 mg / theoretical expression level 86 mg).

[0057] 5. Performance validation and advantage comparison of single-domain antibodies

[0058] 5.1 Core Performance Verification

[0059] (1) Affinity (SPR): MMP-1 was immobilized on CM5 chip (density 1250 RU), antibody gradient concentration 0.5~8 nM, fitted 1:1 binding model, KD=2.3±0.4 nM (n=3, CV=17.4%).

[0060] (2) Specificity (ELISA): Binds only to MMP-1 (OD) 450 =1.89±0.13), with no cross-reactivity with MMP-2 (0.16±0.02), MMP-9 (0.19±0.03), and BSA (0.12±0.01).

[0061] 5.2 Performance comparison with existing anti-MMP-1 antibodies is shown in Table 2.

[0062] Table 2 Comparison of partial performance of the two antibodies

[0063]

[0064] Example 2: Preparation and Click Chemical Modification of Mesenchymal Stem Cell Exosomes Targeting Human MMP-1

[0065] 1. Preparation of experimental materials and reagents

[0066] 1.1 Cells and culture media: Human umbilical cord mesenchymal stem cells (hUC-MSCs, third generation, commercially available or prepared in the laboratory); DMEM / F12 medium (Gibco); exosome-deficient fetal bovine serum (Exo-FBS, Gibco), etc.

[0067] 1.2 Extraction and purification reagents: trypsin-EDTA (0.25%); sucrose (ultrapure grade, Sigma); PBS buffer (calcium and magnesium free, Thermo); 0.22 μm PVDF filter membrane (Millipore).

[0068] 1.3 Click chemistry reagents: NHS-PEG4-DBCO (Click Chemistry Tools); Azide-modified anti-human MMP-1 single-domain antibody (prepared in Example 1); Amicon Ultra-15 ultrafiltration centrifuge tubes (100 kDa cutoff, Millipore).

[0069] 1.4 Characterization reagents: rabbit anti-human CD63 antibody, rabbit anti-human CD81 antibody, rabbit anti-human TSG101 antibody, rabbit anti-human Calnexin antibody (all Abcam); ECL chemiluminescence kit (Thermo).

[0070] 2. Culture and Expansion of Human Umbilical Cord Mesenchymal Stem Cells (hUC-MSCs)

[0071] 2.1 Cell resuscitation and seed culture: hUC-MSCs cryopreservation tubes (containing 1×10⁶ cells / tubes) were removed from the liquid nitrogen container. 6 Immediately place the cells in a 37°C water bath for rapid thawing (1-2 min). Aseptically transfer the cells to a 15 mL centrifuge tube, add 9 mL of preheated DMEM / F12 complete medium (containing 10% Exo-FBS and 1% penicillin-streptomycin), and gently pipette to mix. Centrifuge at 1000×g for 5 min at 4°C, discard the supernatant (to remove DMSO from the cryopreservation solution), resuspend the cells in 5 mL of complete medium, and seed them into T75 cell culture flasks. Incubate at 37°C, 5% CO2, and 95% saturated humidity, observing cell morphology every 24 h.

[0072] 2.2 Cell Passage and Expansion: When the cell confluence reached 80% (observed under an inverted microscope, confluence reached 82% after 48 h of culture), the cells were passaged. The old culture medium was discarded, and the cells were gently washed twice with sterile PBS (5 mL each time) to remove residual serum. 3 mL of 0.25% trypsin-EDTA was added, and the cells were incubated at 37°C for 2 min. After observing increased intercellular spaces and rounded cell shape under an inverted microscope, 5 mL of complete culture medium was immediately added to stop digestion. The cells were then gently detached by pipetting. 10 μL of the cell suspension was stained with trypan blue and counted using a hemocytometer (viability 98.5%, cell concentration 5 × 10⁻⁶). 5 Cells / mL); seeded into three T175 culture flasks at a 1:3 passage ratio (20 mL of complete culture medium per flask, approximately 3 × 10⁶ cells / mL). 6 (each vial / bottle), continue culturing until 90% confluence (approximately 48 hours), for exosome collection.

[0073] 3. Extraction and purification of mesenchymal stem cell exosomes

[0074] 3.1 Conditioned Culture Medium Collection: When the cell confluence in the T175 culture flask reached 90%, the complete culture medium was discarded, and the cells were gently washed three times (15 mL each time) with sterile PBS (preheated to 37°C) to thoroughly remove residual Exo-FBS (to avoid interference from exosomes in serum). 25 mL of serum-free DMEM / F12 medium (containing 1% penicillin and streptomycin) was added to each flask, and the cells were incubated at 37°C in a 5% CO2 incubator for 48 h. The conditioned culture medium from all flasks (a total of 3 flasks, approximately 70 mL in total volume) was collected and transferred to 50 mL centrifuge tubes.

[0075] 3.2 Differential centrifugation to remove impurities (operate at 4℃ throughout to avoid exosome degradation)

[0076] (1) First step of centrifugation (removal of live cells): Dispense 70 mL of conditioned medium into two 50 mL centrifuge tubes, centrifuge at 300×g for 10 min at 4℃, carefully aspirate the supernatant (avoid touching the precipitate at the bottom of the tube) into a new centrifuge tube, and discard the live cell precipitate at the bottom of the tube (the wet weight of the precipitate in each tube is about 2 mg).

[0077] (2) Second step centrifugation (removal of dead cells and large debris): Combine the supernatants from the first step, centrifuge at 2000×g for 20 min at 4℃, aspirate the supernatant into a new tube, and discard the dead cells and large cell debris at the bottom of the tube (the wet weight of the precipitate is about 1.5 mg).

[0078] (3) Third step centrifugation (removal of small fragments and microvesicles): Filter the supernatant from the second step with a 0.45 μm PVDF filter membrane to remove impurities with a diameter >450 nm; after filtration, transfer the supernatant to an ultracentrifuge tube, centrifuge at 4℃ and 10,000×g for 30 min, aspirate the supernatant, and discard the small fragment precipitate at the bottom of the tube (the wet weight of the precipitate is about 0.8 mg).

[0079] 3.3 Ultracentrifugation and Sucrose Bed Purification

[0080] (1) Preparation of sucrose pad: Prepare a 30% (w / v) sucrose solution using serum-free DMEM / F12 medium (weigh 3 g of sucrose and add medium to make up to 10 mL), filter it through a 0.22 μm filter membrane for sterilization; add 5 mL of 30% sucrose solution to the bottom of an ultracentrifuge tube, and then slowly stack the supernatant (about 65 mL) after the third step of centrifugation on the tube wall above the sucrose pad (avoid mixing the two layers).

[0081] (2) Ultracentrifugation: A Beckman Optima XE-90 ultracentrifuge with a Type 45 Ti rotor was used. The centrifuge was performed at 4°C and 100,000×g for 70 min (speed 9 and speed 7). After centrifugation, the upper culture medium and the middle sucrose solution were discarded. The exosome precipitate at the bottom of the tube was gently resuspended in 500 μL of sterile PBS (avoid vigorous blowing to prevent membrane rupture).

[0082] (3) Sterilization and preservation: The resuspended exosome solution was filtered through a 0.22 μm PVDF filter membrane (to remove any possible residual bacteria or large aggregates), aliquoted into 1.5 mL enzyme-free EP tubes, and stored at -80℃ (avoid repeated freeze-thaw cycles); at the same time, 10 μL was taken for BCA protein quantification (exosome protein concentration was 1.8 mg / mL, total protein amount was about 0.9 mg, that is, the yield of 70 mL conditioned medium was about 12.9 μg / mL).

[0083] 4. Click chemical coupling modification of exosomes (anti-human MMP-1 single-domain antibody)

[0084] 4.1 DBCO labeling of exosomes (NHS-PEG4-DBCO activation)

[0085] (1) Preparation of reaction system: Take 500 μL of exosome solution (concentration 1.8 mg / mL, about 900 μg of exosomes) and add it to 1.5 mL of enzyme-free EP tube. Add 1 mM NHS-PEG4-DBCO solution according to the molar ratio of "exosome surface amino group: NHS-PEG4-DBCO = 1:5" (volume calculation: the average molecular weight of exosomes is 1×10). 6Based on the amount of exosomes, 900 μg contains 0.9 nmol of amino acids, so 4.5 μL of 1 mM NHS-PEG4-DBCO needs to be added. Then, add PBS to make up to 1 mL of the total system and mix gently.

[0086] (2) Light-protected reaction: Incubate at 4℃ in the dark for 2 h, gently inverting and mixing once every 30 min to ensure adequate labeling.

[0087] 4.2 Removal of unreacted NHS-PEG4-DBCO (ultrafiltration centrifugation)

[0088] (1) Ultrafiltration preparation: Pre-equilibrate the Amicon Ultra-15 ultrafiltration centrifuge tube (100 kDa cutoff) with sterile PBS (add 5 mL PBS, centrifuge at 4℃ and 4000×g for 15 min, and discard the permeate); transfer the above 1 mL DBCO-labeled exosome solution to the ultrafiltration tube, add 4 mL PBS, and mix gently.

[0089] (2) Ultrafiltration washing: Centrifuge at 4000×g for 15 min at 4℃, discard the permeate (containing unreacted NHS-PEG4-DBCO); repeat the process of adding 5 mL PBS, centrifuging and washing 3 times, and finally retain the concentrate in the ultrafiltration tube (volume about 500 μL, BCA quantitative concentration of 1.6 mg / mL, exosome recovery rate of about 88.9%).

[0090] 4.3 Click-conjugation of Azide-single-domain antibody

[0091] (1) Antibody preparation: The single-domain antibody prepared in Example 1 was reacted with Sulfo-NHS-Azide in PBS at a molar ratio of 1:10 at 4°C for 2 hours, and then purified by desalting on a PD-10 column; the prepared Azide-modified anti-human MMP-1 single-domain antibody (concentration adjusted to 2 mg / mL) was added to 4 mL of antibody solution (containing 8000 μg antibody, matched with 500 μL of ultrafiltered exosomes (800 μg)) at a mass ratio of "exosome:antibody = 1:10", and the total system was made up to 5 mL with PBS and the pH was adjusted to 7.4.

[0092] (2) Coupling reaction: Incubate at room temperature (25℃) in the dark for 4 h, gently mixing once every 1 h during the period, and use the specific click reaction of DBCO and Azide (without catalyst) to form a stable triazole bond.

[0093] 4.4 Removal and purification of uncoupled antibodies

[0094] (1) Ultracentrifugation purification: Divide 5 mL of the coupling reaction solution into 5 equal parts (1 mL each), take one part and add 9 mL of PBS to dilute (the other 4 parts can be stored at -80℃ for later use), centrifuge at 4℃ and 100,000×g for 70 min; discard the supernatant (containing uncoupled single-domain antibody), and gently resuspend the precipitate (modified exosomes) at the bottom of the tube with 500 μL of PBS.

[0095] (2) Verification of coupling efficiency: Take 20 μL of exosome solutions before and after modification, perform 12% SDS-PAGE electrophoresis, transfer to a membrane, and then perform Western blot detection using anti-His tag antibody (e.g. Figure 2 As shown, a distinct band appeared at approximately 15 kDa in the modified exosome lane, which was not present before modification, proving that the antibody was successfully conjugated. The conjugation efficiency was calculated to be approximately 75% by analyzing the band grayscale values ​​using ImageJ.

[0096] 5. Multidimensional characterization of exosomes and modified products

[0097] 5.1 Nanoparticle Tracking Analysis (NTA): Detection of Particle Size and Concentration

[0098] (1) Sample preparation: Take 10 μL of exosome solution before and after modification, and dilute with sterile PBS to 1 mL (dilution 100 times to ensure the concentration is within the NTA detection range: 1×10⁻⁶). 8 ~1×10 11 (particles / mL).

[0099] (2) Detection parameters: ZetaView PMX-120 system, detection temperature 25℃, flow rate 50 μL / min, capture frame number 30 frames, each sample was detected 3 times.

[0100] (3) Results: Before modification, the average particle size of exosomes was 112±8 nm, the particle size distribution range was 80~150 nm, the polydispersity index (PDI) was 0.18±0.03, and the concentration was 1.2×10⁻⁶. 11 particles / mL; the average particle size of the modified exosomes was 128±10 nm (slightly increased due to the conjugated antibody, which is in line with expectations), with a particle size distribution range of 95~170 nm, PDI 0.21±0.02, and a concentration of 1.0×10⁻⁶. 11 particles / mL (recovery rate approximately 83.3%, no significant aggregation).

[0101] 5.2 Western blot: Validation of exosome markers and purity

[0102] (1) Protein sample preparation: Take 20 μL of exosome solution before and after modification, add 5 μL of 5×SDS loading buffer, denature at 95℃ for 10 min, and use as sample group; take hUC-MSCs cell lysis buffer (20 μg protein) as positive control, and take serum-free culture medium as negative control.

[0103] (2) Electrophoresis and transfer: SDS-PAGE was performed on 12% separating gel and 5% stacking gel (constant voltage 80 V stacking gel, 120 V separating gel, electrophoresis for 90 min); the protein was transferred to a PVDF membrane and transferred at a constant current of 300 mA for 90 min (ice bath).

[0104] (3) Incubation and development: 5% skim milk (prepared with TBST) was used for blocking at room temperature for 1 h; rabbit anti-human CD63 (1:1000), CD81 (1:1000), TSG101 (1:1000), and Calnexin (1:1000) antibodies were incubated overnight at 4°C in a shaker; HRP-labeled goat anti-rabbit IgG (1:5000) was incubated at room temperature for 1 h; and ECL chemiluminescence kit was used for color development.

[0105] (4) Results ( Figure 3 Before and after modification, the exosomes positively expressed the exosome characteristic markers CD63, CD81, and TSG101 (clear bands, no impurities); they negatively expressed the endoplasmic reticulum marker Calnexin (proving no cell debris contamination and high purity); after modification, the exosomes additionally positively expressed a single-domain antibody (labeled as VHH, consistent with the conjugation efficiency verification results).

[0106] Example 3: Validation Experiment on the Photoaging Repair Function of Exosomes Targeted by Human MMP-1

[0107] 1. Experimental Materials

[0108] 1.1 Cells and reagents: Human skin fibroblasts (HSF, passages 5-8); CCK-8 assay kit; Annexin V-FITC / PI apoptosis detection kit; human type I collagen ELISA kit; DCFH-DA fluorescent probe.

[0109] 1.2 Animals and consumables: BALB / c female mice (8 weeks old, weighing 18-22g); UVB lamps; H&E staining kit; Masson trichrome staining kit.

[0110] 1.3 Detection reagents: Superoxide dismutase (SOD) activity assay kit; glutathione peroxidase (GSH-Px) activity assay kit; mouse TNF-α ELISA kit; mouse IL-6 ELISA kit; qRT-PCR kit.

[0111] 2. Cellular Experiments: Validation of Repair in a UVB-Induced HSF Cell Photoaging Model

[0112] 2.1 Cell pretreatment and UVB irradiation procedures

[0113] (1) Cell seeding: HSF cells in logarithmic growth phase were taken and the concentration was adjusted to 5×10⁶ cells / year using DMEM medium containing 10% fetal bovine serum. 4 Inoculate at the following volumes into the corresponding culture plates: [Number of cells / mL]

[0114] (2) CCK-8 assay: 96-well plate, 100 μL per well (5 × 10⁻⁶ μL) 3 (cells / well)

[0115] (3) Apoptosis detection: 6-well plate, 2 mL (1×10⁻⁶) per well 5 (cells / well)

[0116] (4) Collagen / ROS detection: 6-well plate, 2 mL (1×10⁻⁶) per well 5 (cells / well)

[0117] (5) Western blot / qRT-PCR: 6-well plate, 2 mL per well (1×10⁻⁶) 5 (cells / well)

[0118] (6) Cell adhesion: Incubate at 37℃ and 5% CO2 for 24 h until the cell confluence reaches 70%~80% (observe under an inverted microscope, the confluence is 75%±3%).

[0119] (7) UVB irradiation pretreatment: Discard the culture medium, gently wash the cells twice with sterile PBS (to avoid cell shedding), and cover the cell surface with 100 μL PBS (to prevent drying).

[0120] (8) UVB irradiation parameters: Place the culture plate under the UVB irradiator and adjust the irradiation distance to 20 cm (after radiometer calibration, the UVB intensity at this distance is 1.5 mW / cm). 2 The irradiation time is calculated according to the formula "dose = intensity × time": 30 mJ / cm 2 ÷1.5 mW / cm 2 =20 s, ensuring uniform irradiation in each well (the culture plate rotates at a constant speed).

[0121] (9) Post-irradiation treatment: Discard PBS, add culture medium containing corresponding reagents according to group (no serum to avoid serum interference), continue to culture for 48 h and then detect (the detection time of each indicator is consistent to ensure data comparability).

[0122] 2.2 Group processing (4 groups, 3-6 replicates per group, n=3 independent experiments)

[0123] Table 3 Grouping and its processing method

[0124]

[0125] 2.3 Specific operating procedures and experimental results for each detection indicator

[0126] 2.3.1 Cell viability assay (CCK-8 assay)

[0127] After 48 h of culture, discard 50 μL of the old culture medium from each well, add 50 μL of CCK-8 reagent (diluted with serum-free DMEM at a ratio of 1:10), and incubate at 37℃ for 2 h. Measure the absorbance (OD value) of each well at 450 nm using a microplate reader. Cell viability (%) = (OD value of experimental group - OD value of blank group) / (OD value of normal control group - OD value of blank group) × 100%.

[0128] The results are shown in Table 4. UVB irradiation significantly reduced HSF cell viability, with the model group showing a viability of only 65.8±4.1% (vs. 100.0±3.2% in the normal control group), indicating that UVB successfully constructed a cell photoaging damage model. Compared with the model group, the viability of the unmodified exosome group increased to 83.5±3.7% (p<0.01), while the viability of the single-domain antibody-modified exosome group further recovered to 94.2±2.8% (p<0.001), which was significantly higher than that of the unmodified exosome group (difference 10.7%). This demonstrates that targeted modification can enhance the protective effect of exosomes on the viability of UVB-damaged HSF cells. It is speculated that this is because the single-domain antibody mediates the enrichment of exosomes in damaged cells with high MMP-1 expression, resulting in more efficient release of anti-damage active molecules (such as miR-21 and HGF).

[0129] Table 4. Cell viability test results (n=6, mean ± standard deviation)

[0130]

[0131] 2.3.2 Apoptosis detection (Annexin V-FITC / PI double staining, flow cytometry)

[0132] After culturing in 6-well plates for 48 h, the culture medium was discarded, the cells were washed twice with PBS, and then digested with 0.25% trypsin (EDTA-free) for 2 min. Digestion was terminated by adding serum-containing culture medium. The cells were centrifuged at 1000×g for 5 min at 4°C, and the cell pellet was collected. The cells were resuspended in 1×Binding Buffer to a concentration of 1×10⁻⁶ cells / well. 6Cells / mL were collected, and 100 μL of cell suspension was incubated with 5 μL Annexin V-FITC and 5 μL LPI at room temperature in the dark for 15 min. Then, 400 μL of 1× Binding Buffer was added, and the cells were analyzed using a BD FACSCanto II flow cytometer within 30 min. Cells labeled “Annexin V+PI-” were considered early apoptotic cells, and “Annexin V+PI+” were considered late apoptotic cells. The total apoptosis rate was calculated as the sum of the early and late apoptosis rates.

[0133] The results are shown in Table 5. UVB irradiation significantly increased the apoptosis rate of HSF cells, with the total apoptosis rate in the model group reaching 32.7±3.2% (vs. 5.3±0.8% in the normal control group). This suggests that UVB induces apoptosis through oxidative stress and DNA damage. The apoptosis rate in the unmodified exosome group decreased to 18.5±2.1% (p<0.01), while the apoptosis rate in the single-domain antibody-modified exosome group further decreased to 9.8±1.3% (p<0.001), close to the level of the normal control group, and 41.1% lower than that in the unmodified exosome group. This indicates that targeted modification of exosomes can more effectively inhibit UVB-induced apoptosis, possibly by upregulating Bcl-2 and downregulating Bax / caspase-3 expression to achieve an anti-apoptotic effect.

[0134] Table 5. Results of apoptosis detection (n=3, mean ± standard deviation)

[0135]

[0136] 2.3.3 Detection of Type I Collagen Content (ELISA Method)

[0137] Collect the culture supernatant from 6-well plates (2 mL per well), centrifuge at 12000×g for 10 min at 4℃, and keep the supernatant for later use. Extract total cell protein according to the kit instructions (RIPA lysis buffer + protease inhibitor), and quantify the protein concentration using BCA (for standardizing collagen content). Add the standard gradient (0, 15.6, 31.2, 62.5, 125, 250 ng / mL) according to the kit instructions, incubate at 37℃ for 1 h, add HRP-labeled secondary antibody (1:5000) and incubate for 30 min, develop with TMB for 15 min, terminate with 2 M H2SO4, measure the OD value at 450 nm, calculate the collagen concentration based on the standard curve, and express the result as "ng / mg protein".

[0138] The results are shown in Table 6. UVB irradiation significantly inhibited type I collagen synthesis in HSF cells. The collagen content in the model group was only 58.9±6.2 ng / mg protein (vs. 125.3±8.7 ng / mg protein in the normal control group), a decrease of 53.0%, which is consistent with the characteristics of increased collagen degradation in photoaging. The collagen content in the unmodified exosome group increased to 89.6±7.3 ng / mg protein, while the collagen content in the single-domain antibody-modified exosome group further recovered to 112.4±9.1 ng / mg protein, which was 25.4% higher than that in the unmodified exosome group and close to the level of the normal control group. This indicates that targeted modification of exosomes can more effectively promote collagen synthesis, which may be related to the single-domain antibody inhibiting MMP-1-mediated collagen degradation and the exosome delivery of COL1A1 synthesis-related miRNAs (such as miR-143).

[0139] Table 6. Results of Type I Collagen Content Detection (n=6, mean ± standard deviation)

[0140]

[0141] 2.3.4 MMP-1 protein expression detection (Western blot)

[0142] As in 2.3.3, 30 μg of total protein was subjected to SDS-PAGE (12% separating gel). The membrane was transferred at a constant voltage of 100 V for 90 min (PVDF membrane), blocked with 5% skim milk for 1 h; primary antibodies: rabbit anti-human MMP-1 (1:1000) and rabbit anti-human GAPDH (internal control, 1:5000), incubated overnight at 4℃; secondary antibody: HRP-labeled goat anti-rabbit IgG (1:5000), incubated at room temperature for 1 h. ECL chemiluminescence imaging was performed, and the grayscale ratio (relative expression level) of the MMP-1 band to the GAPDH band was calculated using ImageJ software.

[0143] The results are shown in Table 7 and Figure 4 As shown, UVB irradiation significantly upregulated MMP-1 protein expression in HSF cells. The relative gray value of the model group reached 3.85±0.32 (vs. 1.00±0.05 for the normal control group), which was 3.85 times that of the normal group, indicating that UVB activated key enzymes in collagen degradation. MMP-1 expression in the unmodified exosome group decreased to 2.13±0.21 (p<0.01), while MMP-1 expression in the single-domain antibody-modified exosome group further decreased to 1.27±0.15 (p<0.001), which was 40.4% lower than that in the unmodified exosome group and close to the level of the normal control group. Its advantage stemmed from the direct and specific binding inhibition of MMP-1 by the single-domain antibody, while the exosomes reduced MMP-1 synthesis by downregulating the activity of AP-1 transcription factor, thus doubly inhibiting MMP-1 function.

[0144] Table 7. Results of MMP-1 protein expression detection (n=3, mean ± standard deviation)

[0145]

[0146] 2.3.5 Intracellular ROS detection (DCFH-DA fluorescent probe method)

[0147] After 48 h of culture, the culture medium was discarded, the sample was washed twice with PBS, and 10 μM DCFH-DA probe (diluted with serum-free DMEM) was added. The sample was incubated at 37 °C for 30 min. After washing three times with PBS to remove unloaded probe, the sample was observed under a fluorescence microscope, and the average fluorescence intensity was quantified using ImageJ software (relative ROS level = fluorescence intensity of experimental group / fluorescence intensity of normal control group × 100%).

[0148] The results are shown in Table 8. UVB irradiation led to a large accumulation of ROS in HSF cells. The relative ROS level in the model group was 285.7±22.3% (vs. 100.0±8.5% in the normal control group), which was 2.86 times that of the normal group, a core characteristic of photoaging oxidative stress. The ROS level in the unmodified exosome group decreased to 168.4±15.7% (p<0.01), while the ROS level in the single-domain antibody modified exosome group further decreased to 118.9±12.6% (p<0.001), which was 29.4% lower than that in the unmodified exosome group and close to the level of the normal control group. This indicates that targeted modification of exosomes can clear ROS more efficiently. Due to targeted delivery, antioxidant enzymes such as SOD and GSH-Px in the exosomes are enriched at the site of injury, enhancing local antioxidant capacity.

[0149] Table 8. Results of intracellular ROS detection (n=6, mean ± standard deviation)

[0150]

[0151] 3. Animal Experiments: Validation of Repair in a UVB-Induced BALB / c Mouse Skin Photoaging Model

[0152] 3.1 Animal feeding and photoaging model construction

[0153] 3.1.1 Rearing conditions: Temperature 22±2℃, humidity 50%±5%, 12-hour light-dark cycle, free access to food and water.

[0154] 3.1.2 Back hair removal treatment: One day before the experiment, mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg), and the hair on their backs was shaved off with an electric shaver. Then, the remaining hair was removed with depilatory cream, exposing a 2 cm × 2 cm hair removal area.

[0155] 3.1.3 UVB Irradiation Scheme: Irradiation equipment: FS40T12-UVB lamp, irradiation distance 15 cm (irradiance 2mW / cm²) 2 (Radiometer calibration); Dose escalation: Initial dose 100 mJ / cm 2 (Irradiation time 50 s), increasing by 10% weekly (110 mJ / cm² in week 2, 121 mJ / cm² in week 3). 2 …), irradiate 3 times a week (Monday, Wednesday, and Friday) for a total of 8 weeks to build a stable photoaging model (model validation: 3 mice were tested at the end of the 8th week, and the epidermal thickness was ≥45 μm and the collagen density was ≤50%, which was considered a successful model).

[0156] 3.2 Group treatment (4 groups, n=8 animals per group, treatment cycle 4 weeks)

[0157] Table 9 Grouping and its processing method

[0158]

[0159] 3.3 Sample Collection and Testing Procedure

[0160] Sample collection: Four weeks after treatment, mice were euthanized by intraperitoneal injection of an excessive amount of sodium pentobarbital. Skin tissue (2 cm × 1 cm) from the hairless area on the back was immediately taken and divided into two parts. One part was fixed in 4% paraformaldehyde fixative at 4°C for 24 h for pathological staining; the other part was flash-frozen in liquid nitrogen and stored at -80°C for enzyme activity detection, ELISA and qRT-PCR.

[0161] 3.3.1 Skin histological examination (H&E staining, Masson trichrome staining)

[0162] (1) Paraffin embedding and sectioning: Tissue fixed in 4% paraformaldehyde was dehydrated in a gradient (70%→80%→90%→95%→

[0163] Clear the tissue with 100% ethanol and xylene, embed it in paraffin, cut it into 5 μm thick sections using a microtome, and mount them on a glass slide.

[0164] (2) H&E staining (epidermal thickness detection): Staining procedure: dewaxing → hydration → hematoxylin staining for 5 min → hydrochloric acid ethanol differentiation for 30 s → eosin staining for 3 min → dehydration and clearing → neutral resin mounting; Quantitative method: under an optical microscope (400× objective lens), 5 fields of view were randomly selected for each mouse, and the epidermal thickness was measured using ImageJ software, and the average value was taken.

[0165] (3) Masson's trichrome staining (collagen density detection): Staining procedure: dewaxing → hydration → hematoxylin staining for 5 min → acid fuchsin staining for 10 min → phosphomolybdic acid staining for 5 min → aniline blue staining for 5 min → dehydration and clearing → mounting; Quantitative method: under a 400× objective lens, 5 dermal fields of view were randomly selected from each mouse, and ImageJ software was used to calculate the percentage of the blue collagen area in the total dermal area (collagen density %).

[0166] (4) The experimental results are shown in Table 10: Long-term UVB irradiation caused significant epidermal hyperplasia in mice. The epidermal thickness of the model group reached 48.7±3.5 μm (vs. 22.3±1.8 μm in the normal control group), which was 1.18 times thicker, consistent with the characteristics of hyperkeratosis and acanthosis of photo-aged epidermis. The epidermal thickness of the unmodified exosome group decreased to 33.2±2.7 μm (p<0.01), while the epidermal thickness of the single-domain antibody modified exosome group further decreased to 26.5±2.1 μm (p<0.001), which was 20.2% thinner than the unmodified exosome group and close to the level of the normal control group. This indicates that targeted modification of exosomes can more effectively inhibit UVB-induced abnormal epidermal hyperplasia, possibly by downregulating keratinocyte proliferation-related protein (Ki-67). UVB irradiation caused significant degradation of dermal collagen in mice. The collagen density in the model group was only 45.3±4.2% (vs. 100.0±6.8% in the normal control group), a decrease of 54.7%, manifested as collagen fiber breakage and disordered arrangement. The collagen density in the unmodified exosome group increased to 72.8±5.9% (p<0.01), while the collagen density in the single-domain antibody-modified exosome group further recovered to 91.5±7.3% (p<0.001), which was 25.7% higher than that in the unmodified exosome group and close to the level of the normal control group. This indicates that targeted modification of exosomes can more effectively promote dermal collagen repair and regeneration, which is related to the dual effects of inhibiting MMP-1 activity and promoting collagen synthesis in fibroblasts.

[0167] Table 10. Skin histological examination results (n=8, mean ± standard deviation)

[0168]

[0169] 3.3.2 Detection of antioxidant indicators (SOD, GSH-Px activity)

[0170] (1) Preparation of tissue homogenate: Take skin tissue (about 50 mg) stored at -80℃, add 500 μL of pre-cooled physiological saline (containing protease inhibitor), homogenize with a tissue homogenizer under ice bath (3000 rpm, 30 s × 3 times), centrifuge at 4℃ and 12000 × g for 15 min, take the supernatant, and quantify the protein concentration by BCA.

[0171] (2) Enzyme activity detection: Follow the instructions of the kit. SOD activity is expressed as “U / mg protein” (the amount of enzyme that inhibits the auto-oxidation rate of SOD substrate by 50% per mg protein in 1 mL of reaction solution is 1 U); GSH-Px activity is expressed as “U / mg protein” (the amount of enzyme that catalyzes the oxidation of 1 μmol GSH to GSSG per minute per mg protein is 1 U).

[0172] (3) The experimental results are shown in Table 11: UVB irradiation significantly inhibited the SOD activity of mouse skin. The activity of the model group was only 23.8±2.1 U / mg protein (vs. 52.7±4.3 U / mg protein in the normal control group), a decrease of 54.8%, indicating that the endogenous antioxidant system was damaged. The SOD activity of the unmodified exosome group increased to 38.9±3.2 U / mg protein (p<0.01), while the SOD activity of the single-domain antibody modified exosome group further recovered to 47.6±3.8 U / mg protein (p<0.001), which was 22.4% higher than that of the unmodified exosome group and close to the level of the normal control group. This indicates that targeted modification of exosomes can more effectively activate the endogenous SOD system of the skin and enhance the ability to scavenge superoxide anion free radicals. UVB irradiation significantly inhibited GSH-Px activity in mouse skin. The activity in the model group was only 18.9±1.8 U / mg protein (vs. 45.2±3.7 U / mg protein in the normal control group), a decrease of 58.2%. The GSH-Px activity in the unmodified exosome group increased to 32.7±2.9 U / mg protein (p<0.01), while the GSH-Px activity in the single-domain antibody-modified exosome group further recovered to 40.3±3.4 U / mg protein (p<0.001), which was 23.2% higher than that in the unmodified exosome group and close to the level of the normal control group. This indicates that targeted modification of exosomes can more efficiently activate the GSH-Px system, enhance the ability to scavenge lipid peroxides such as hydrogen peroxide, and work synergistically with SOD to improve antioxidant defense.

[0173] Table 11 Results of antioxidant index detection (SOD, GSH-Px activity) (n=8, mean ± standard deviation)

[0174]

[0175] 3.3.3 Detection of inflammatory factors (TNF-α, IL-6, ELISA method)

[0176] (1) Sample preparation: Same as the tissue homogenate supernatant in 3.3.2 (protein concentration standardized).

[0177] (2) ELISA procedure: Add samples according to the instructions of the mouse TNF-α / IL-6 kit. The standard gradient is: TNF-α (0, 7.8, 15.6, 31.2, 62.5, 125 pg / mL) and IL-6 (0, 3.9, 7.8, 15.6, 31.2, 62.5 pg / mL). The detection procedure is the same as that of cell ELISA. The results are expressed as "pg / mg protein".

[0178] (3) The experimental results are shown in Table 12: UVB irradiation significantly upregulated the level of TNF-α in mouse skin. The concentration in the model group reached 45.8±4.2 pg / mg protein (vs. 12.5±1.3 pg / mg protein in the normal control group), which was 3.66 times that of the normal group, and is the core marker of chronic inflammation caused by photoaging. The TNF-α level in the unmodified exosome group decreased to 28.7±2.5 pg / mg protein (p<0.01), while the TNF-α level in the single-domain antibody modified exosome group further decreased to 17.3±1.8 pg / mg protein (p<0.001), which was 39.7% lower than that in the unmodified exosome group and close to the level of the normal control group. This indicates that targeted modification of exosomes can more effectively inhibit the release of TNF-α induced by UVB, and may achieve anti-inflammatory effects by downregulating the activity of the NF-κB pathway. UVB irradiation significantly upregulated IL-6 levels in mouse skin, with the model group reaching a concentration of 32.5±3.1 pg / mg protein (vs. 8.7±0.9 pg / mg protein in the normal control group), which was 3.74 times higher than that in the normal group. In the unmodified exosome group, IL-6 decreased to 20.3±2.2 pg / mg protein, while in the single-domain antibody-modified exosome group, IL-6 further decreased to 12.5±1.5 pg / mg protein, which was 38.4% lower than that in the unmodified exosome group and close to the level of the normal control group. This indicates that targeted modification of exosomes can more effectively inhibit the IL-6-mediated inflammatory cascade and reduce chronic inflammatory damage to the skin.

[0179] Table 12 Results of inflammatory factor detection (n=8, mean ± standard deviation)

[0180]

[0181] 3.3.4 Molecular biological detection (qRT-PCR, MMP-1, TIMP-1, COL1A1 mRNA)

[0182] (1) RNA extraction and reverse transcription: 50 mg of skin tissue was taken, and total RNA was extracted using Trizol reagent (Nanodrop detection: A 260 / A 280=1.8~2.0, concentration ≥500 ng / μL); according to the Takara qRT-PCR kit instructions, 1 μg RNA was reverse transcribed into cDNA (reaction conditions: 37℃ 15 min, 85℃ 5 s).

[0183] (2) qRT-PCR reaction: 20 μL system (cDNA 2 μL, forward and reverse primers 0.8 μL each, SYBR Green Mix 10 μL, ddH2O 6.4 μL), reaction conditions: 95℃ for 30 s; 95℃ for 5 s, 60℃ for 30 s (40 cycles); melting curve analysis (95℃ for 15 s, 60℃ for 1 min, 95℃ for 15 s).

[0184] (3) Data calculation: using 2 - The relative expression level of the target gene was calculated using the ΔΔCt method (ΔCt = Ct target gene - Ct GAPDH, ΔΔCt = ΔCt experimental group - ΔCt normal control group).

[0185] (4) The experimental results are shown in Table 13: UVB irradiation significantly upregulated the expression of MMP-1 mRNA in mouse skin. The relative expression level in the model group was 4.25±0.35 (vs. 1.00±0.08 in the normal control group), which was 4.25 times that of the normal group, indicating that UVB activates collagen degradation enzymes at the transcriptional level. The MMP-1 mRNA in the unmodified exosome group decreased to 2.38±0.22 (p<0.01), while the single-domain antibody modified exosome group further decreased to 1.35±0.15 (p<0.001), which was 43.3% lower than that in the unmodified exosome group and close to the level of the normal control group. This indicates that targeted modification of exosomes can inhibit MMP-1 expression at the transcriptional level, cooperating with the direct inhibition at the protein level and strengthening collagen protection. UVB irradiation significantly downregulated TIMP-1 (a natural inhibitor of MMP-1) mRNA expression in mouse skin. The relative expression level in the model group was only 0.48±0.06 (vs. 1.00±0.07 in the normal control group), a decrease of 52.0%. In the unmodified exosome group, TIMP-1 mRNA increased to 0.75±0.08 (p<0.01), while in the single-domain antibody-modified exosome group, it further increased to 0.92±0.09 (p<0.001), close to the level of the normal control group, which was 22.7% higher than that of the unmodified exosome group. This indicates that targeted modification of exosomes can more efficiently upregulate TIMP-1 expression by dually regulating the MMP-1 / TIMP-1 balance through "inhibiting enzyme activity + increasing inhibitor levels". UVB irradiation significantly downregulated the expression of COL1A1 (the gene encoding the type I collagen α1 chain) mRNA in mouse skin. The relative expression level in the model group was only 0.32±0.05 (vs. 1.00±0.09 in the normal control group), a decrease of 68.0%. In the unmodified exosome group, COL1A1 mRNA increased to 0.65±0.07 (p<0.01), while in the single-domain antibody-modified exosome group, it further increased to 0.88±0.08 (p<0.001), close to the level of the normal control group, which was 35.4% higher than that of the unmodified exosome group. This indicates that targeted modification of exosomes can more efficiently promote COL1A1 transcription and provide a molecular basis for collagen synthesis.

[0186] Table 13 Molecular biological detection results (n=8, mean ± standard deviation)

[0187]

[0188] 4. Summary

[0189] 4.1 Functional Validation Conclusions: Single-domain antibody-modified exosomes are significantly superior to unmodified exosomes at both the cellular and animal levels, and can effectively repair UVB-induced skin photoaging, specifically manifested in: ① protecting cell viability and inhibiting apoptosis; ② regulating collagen metabolism balance; ③ scavenging ROS and activating the antioxidant system; ④ inhibiting the release of inflammatory factors; and ⑤ improving skin tissue morphology.

[0190] 4.2 Core advantages of targeted modification: Single-domain antibodies specifically bind to MMP-1 highly expressed at photoaging sites, increasing the local concentration of exosomes by 3-5 times (flow cytometry analysis of exosome uptake rate: the modified group was 62.3% higher than the unmodified group); single-domain antibodies directly inhibit MMP-1 activity, while exosomes provide bioactive molecules (miRNAs, proteins, lipids). The two work synergistically on the key photoaging pathway of "oxidative stress-inflammation-collagen degradation," with better effects than single components; exosomes are derived from mesenchymal stem cells, have low immunogenicity (no skin redness or allergy was observed in animal experiments), and the particle size after modification is still 100-150 nm, which can easily penetrate the stratum corneum of the skin, making it suitable for local drug delivery and providing a novel targeted delivery system for photoaging treatment.

[0191] 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 antibody against human MMP-1, characterized in that, The amino acid sequence of the single-domain antibody is shown in SEQ ID NO:

1.

2. A mesenchymal stem cell exosome targeting human MMP-1, characterized in that, The exosome surface is covalently linked with the anti-human MMP-1 single-domain antibody as described in claim 1.

3. The exosomes according to claim 2, characterized in that, The anti-human MMP-1 single-domain antibody is linked to an exosome surface membrane protein via a click chemistry reaction.

4. The exosome according to claim 3, characterized in that, The click chemistry reaction is achieved through a cycloaddition reaction between DBCO and the azide group.

5. The exosomes according to claim 2, characterized in that, The mesenchymal stem cells are derived from human umbilical cord tissue, adipose tissue, or bone marrow tissue.

6. A method for preparing exosomes as described in claim 2, characterized in that, The method includes the following steps: preparation and azide modification of the anti-human MMP-1 single-domain antibody as described in claim 1, extraction and purification of mesenchymal stem cell exosomes, and conjugation of the antibody to the exosomes via click chemistry.

7. The method according to claim 6, characterized in that, The mesenchymal stem cell exosomes were extracted using differential centrifugation combined with sucrose density gradient ultracentrifugation.

8. A pharmaceutical composition, characterized in that, The composition comprises the targeted exosomes of claim 2 and a pharmaceutically acceptable carrier.

9. The use of the anti-human MMP-1 single-domain antibody according to claim 1 in the preparation of mesenchymal stem cell exosomes targeting human MMP-1.

10. Use of the mesenchymal stem cell exosomes targeting human MMP-1 as described in claim 2 in the preparation of a medicament for the prevention or treatment of photoaging of the skin.

Images