Anti-hair loss composition containing stem cell exosome as well as preparation method and application of anti-hair loss composition

Abstract

The invention discloses a fusion polypeptide for preventing alopecia, a modified exosome and a composition thereof. A Wnt3a active peptide and an LGR5 targeting peptide are connected in series through a flexible connecting peptide to obtain a fusion polypeptide, and the fusion polypeptide has a specific targeting effect on hair follicle stem cells and can activate a Wnt pathway; an exosome is modified by the fusion polypeptide to prepare a fusion polypeptide-exosome (FP-Exo), and the fusion polypeptide-exosome (FP-Exo) is mixed with an anti-DKK1 monoclonal antibody to prepare the anti-hair loss composition. An in-vitro experiment shows that the composition can remarkably promote hair follicle stem cell proliferation, an in-vivo experiment proves that the composition can effectively promote hair follicle regeneration of mice with androgenetic alopecia, and compared with common exosomes and minoxidil, the composition is better in key indexes such as hair follicle density, hair follicle growth period proportion and cell proliferation, and the composition has good application prospects. And a new effective means is provided for alopecia treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an anti-hair loss composition containing stem cell exosomes, a preparation method thereof, and an application thereof in anti-hair loss products. Background Art

[0002] Hair loss is a common health problem that seriously affects people's quality of life and mental health. Its fundamental mechanism involves decreased activity of hair follicle stem cells, apoptosis of dermal papilla cells, and imbalance of the microenvironment. In the existing technology, stem cell exosomes have become a research hotspot for hair regeneration because they carry a variety of growth factors (such as VEGF, FGF, Wnt3α) and miRNA. For example, there is a patent that uses porcine adipose mesenchymal stem cell exosomes in combination with traditional Chinese medicine extracts (Polygonum multiflorum, Angelica sinensis), but this solution has many defects. On the one hand, the exosomes are not targeted enough, making it difficult to accurately act on hair follicle-related cells, resulting in low bioavailability; on the other hand, the ingredients of traditional Chinese medicine are complex and difficult to standardize, which not only increases the difficulty of quality control, but may also trigger allergic reactions, posing a potential threat to patient safety. At the same time, this solution does not synergistically regulate key pathways of hair loss (such as DKK1 / β-catenin imbalance) and fails to fundamentally solve the problem of hair loss.

[0003] Other solutions also have limitations. For example, although the microneedle delivery system can improve transdermal efficiency and promote the entry of exosomes and other drug ingredients into the deep layers of the skin, the production process is complex and costly, which limits its large-scale clinical application. Although the combined PRP and hair follicle stem cell method has the potential to promote hair regeneration in theory, it requires multiple surgical operations, causing great pain to patients, and its clinical application is limited. In addition, traditional exosome modification methods, such as liposome encapsulation or cyclodextrin complexes, although aimed at improving the stability and delivery efficiency of exosomes, can easily lead to the destruction of the exosome membrane structure, affecting the release of active ingredients, thereby reducing the therapeutic effect. Due to the shortcomings of these existing technologies, there is an urgent need to develop a more effective hair loss treatment solution. Summary of the Invention

[0004] The present invention aims to provide a fusion polypeptide, modified exosomes and a composition thereof that can effectively treat hair loss.

[0005] Therefore, the present invention discloses a fusion polypeptide in one aspect, the amino acid sequence of the fusion polypeptide is shown in SEQ ID NO.1.

[0006] In one aspect, the present invention further discloses a stem cell exosome, wherein the stem cell exosome is FP-Exo, wherein FP-Exo is a stem cell exosome with the fusion polypeptide modified on its surface.

[0007] Preferably, the stem cell exosomes of the present invention are derived from human umbilical cord mesenchymal stem cells cultured under 3% O2 conditions before modification, and 20 μM baicalin and 10 μM quercetin are added during the culture process.

[0008] In one aspect, the present invention further discloses an anti-hair loss composition, comprising an effective amount of the FP-Exo and an effective amount of an anti-DKK1 monoclonal antibody.

[0009] Preferably, the concentration of FP-Exo in the present invention is 5.0×10 10 particles / mL.

[0010] Preferably, the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-DKK1 monoclonal antibody of the present invention are shown as SEQ ID NO. 2 and SEQ ID NO. 3.

[0011] Preferably, the concentration of the anti-DKK1 monoclonal antibody of the present invention is 5.0 μg / mL.

[0012] The present invention demonstrates several significant advantages in the field of hair loss treatment: The fusion polypeptide specifically targets hair follicle stem cells, significantly enhancing their binding to hair follicle stem cells compared to individual Wnt3a active peptides and LGR5 targeting peptides, laying a solid foundation for subsequent functional development. Besides its targeting function, it also activates the Wnt pathway, promoting the physiological activity of hair follicle stem cells. The modified exosomes, incorporating these two properties, have demonstrated remarkable results in promoting hair follicle stem cell proliferation and hair follicle regeneration. In the anti-hair loss composition, the fusion polypeptide-modified exosomes synergize with the anti-DKK1 monoclonal antibody, demonstrating in vitro and in vivo efficacy compared to either single component or other control drugs in promoting hair follicle stem cell proliferation and hair follicle regeneration, providing a superior therapeutic approach. Furthermore, the anti-DKK1 monoclonal antibody, with its high affinity, strong neutralizing activity, and lack of cross-reactivity, reduces the risk of off-target effects and ensures therapeutic safety. Therefore, the present invention provides a highly effective and safe new strategy and drug for the treatment of hair loss, promising broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Wnt pathway activation experimental results (western blot). DETAILED DESCRIPTION

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0015] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0016] Example 1: Synthesis and functional verification of fusion polypeptides

[0017] I. Fusion Peptide Design and Preparation: A Wnt3a active peptide (CLQYNPQPTCDK) and an LGR5 targeting peptide (CRVLRSL) were concatenated via a flexible linker peptide (GGGGS) to achieve dual targeting of exosomes to hair follicle stem cells (activating the Wnt pathway and binding to cell surface receptors). The amino acid sequence of the designed fusion peptide is shown in SEQ ID NO. 1. The fusion peptide was synthesized at Sangon Biotech (Shanghai) Co., Ltd. to a purity of ≥95%.

[0018] Targeted Experiments

[0019] 1. Preparation of Experimental Materials

[0020] (1) Hair follicle stem cells (HFSCs): isolated and obtained from the back skin tissue of healthy adult mice. The specific steps are as follows: after the mouse is killed, the back skin is disinfected with 75% alcohol, the skin tissue of appropriate size is cut, and it is placed in PBS buffer containing double antibodies (penicillin 100U / mL, streptomycin 100μg / mL) and washed three times. The washed skin tissue is transferred to a culture dish containing 0.25% trypsin and digested at 37°C for 1-2 hours. After the epidermis is separated from the dermis, the epidermis is carefully separated with tweezers, cut into pieces, and added to DMEM / F12 culture medium containing 10% fetal bovine serum. It is gently pipetted with a pipette to make a single cell suspension. The cell suspension is transferred to a centrifuge tube, centrifuged at 1000rpm for 5 minutes, the supernatant is discarded, the cells are resuspended with DMEM / F12 culture medium containing 10% FBS, inoculated into a culture flask, and cultured in a 37°C, 5% CO2 incubator. When the cell confluence reaches 80-90%, subculture is performed.

[0021] (2) Fibroblasts: Obtained from ear skin tissue of the same strain of mice. The procedure is similar to that for HFSCs, except that 0.1% collagenase I is used as the digestion solution and digestion is carried out at 37°C for 3-4 hours. Cells are also resuspended and cultured in DMEM medium containing 10% FBS.

[0022] (3) Cy5-labeled fusion polypeptide: Label according to the instructions of the specific labeling kit. The specific operation is as follows: Take an appropriate amount of fusion polypeptide, dissolve it with an appropriate amount of DMSO, and prepare a 10mM mother solution. Mix the Cy5 dye and the fusion polypeptide mother solution in a molar ratio of 1:10, add an appropriate amount of labeling buffer, and react at room temperature in the dark for 2-3 hours. After the reaction is completed, remove the unreacted Cy5 dye by dialysis. The dialysate is PBS buffer, and the dialysate is dialyzed 3-4 times, with an interval of 4-6 hours between each change of the dialysate. Finally, the Cy5-labeled fusion polypeptide is obtained and diluted with PBS buffer to a working concentration of 10μM.

[0023] 2. Experimental Procedure

[0024] (1) Cell seeding: HFSCs and fibroblasts in the logarithmic growth phase were digested with trypsin to prepare single cell suspensions. Cells were counted using a cell counting plate and the cell density was adjusted to 1×10 6 The cell suspension was inoculated into a 24-well plate, and 1 mL of cell suspension was added to each well to make the number of cells in each well 1×10 6 The 24-well plate was placed in a 37°C, 5% CO2 incubator for 24 hours to allow the cells to adhere.

[0025] (2) Co-incubation: After the cells adhere, remove the culture medium from the 24-well plate and gently rinse the cells three times with PBS buffer. Add 1 mL of serum-free culture medium containing 10 μM Cy5-labeled fusion peptide to each well and incubate the 24-well plate in a 37°C, 5% CO2 incubator for 4 hours.

[0026] (3) Flow cytometry: After the incubation, remove the culture medium and gently rinse the cells 3 times with PBS buffer. Add 200 μL of trypsin to each well and digest at 37°C for 1-2 minutes. After the cells become round, add 800 μL of culture medium containing 10% FBS to terminate the digestion. Transfer the cell suspension to a flow cytometry tube, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Resuspend the cells with PBS buffer and adjust the cell concentration to 1×10 6 Cells / mL. Detection was performed using a BD FACSCanto II flow cytometer with an excitation wavelength of 640 nm and an emission wavelength of 670 nm, collecting data for 10,000 cells.

[0027] 3. Experimental Results As shown in Table 1, analysis of data from three experiments revealed that the binding rates of HFSCs were 78.3%, 78.8%, and 78.2%, respectively, with an average of 78.5% and a standard deviation of 3.2%. The binding rates of fibroblasts were 12.0%, 12.2%, and 12.1%, respectively, with an average of 12.1% and a standard deviation of 1.5%. It is clear that the binding rate of HFSCs to the Cy5-labeled fusion peptide was significantly higher than that of fibroblasts.

[0028] Table 1 Targeting experimental results

[0029]

[0030] 4. Experimental Summary: By co-incubating the Cy5-labeled fusion peptide with HFSCs and fibroblasts and detecting the binding efficiency by flow cytometry, the experimental results showed that the fusion peptide has a specific targeting effect on HFSCs, with a binding rate much higher than that of fibroblasts. This lays the foundation for subsequent research on HFSCs based on fusion peptides. It should be noted that the Wnt3a active peptide (CLQYNPQPTCDK) and LGR5 targeting peptide (CRVLRSL) alone have weak targeting ability to HFSCs, with binding rates of 35.3% and 30.1% for HFSCs, and 15.8% and 17.6% for fibroblasts, respectively.

[0031] 3. Wnt pathway activation experiment

[0032] 1. Preparation of Experimental Materials

[0033] (1) Cell culture: The HFSCs were cultured using the same method as in the targeted experiments until the cell confluence reached 80-90%.

[0034] (2) Antibodies: Anti-β-catenin antibody (abcam, ab32572) and anti-LEF1 antibody (abcam, ab137872). According to the instructions, dilute the antibodies to a working concentration of 1:1000 with antibody diluent.

[0035] (3) Other reagents: RIPA lysis buffer, protease inhibitors, PMSF, BCA protein quantification kit, SDS-PAGE gel preparation kit, transfer buffer, blocking solution, ECL luminescence solution, etc.

[0036] 2. Experimental Procedure

[0037] (1) Cell treatment: Cultured HFSCs were divided into two groups: a treated group and an untreated group (control group). The treated group was treated with culture medium containing 10 μM of the fusion polypeptide, while the control group was treated with an equal amount of culture medium without the fusion polypeptide. Both groups of cells were cultured in a 37°C, 5% CO2 incubator for 24 hours.

[0038] (2) Cell lysis and protein quantification: After the incubation period, the culture medium was aspirated and the cells were gently rinsed three times with pre-chilled PBS buffer. 100 μL of RIPA lysis buffer containing protease inhibitors and PMSF was added to each well and the cells were lysed on ice for 30 minutes. The lysed cell suspension was transferred to a centrifuge tube and centrifuged at 12,000 rpm for 15 minutes. The supernatant was collected. The protein concentration was determined using a BCA protein quantification kit.

[0039] (3) Western blot analysis: Based on the protein concentration, take an appropriate amount of protein sample, add 5× loading buffer, and boil at 100°C for 5 minutes to denature the protein. Perform SDS-PAGE, transfer to a membrane, incubate with antibodies, and develop with ECL. Then, take a picture and calculate the grayscale.

[0040] 3. The experimental results are shown in Table 2 and Figure 1 As shown in the figure, the grayscale values of protein bands were analyzed and corrected using GAPDH as an internal reference. The grayscale ratios of β-catenin and LEF1 relative to the internal reference were calculated. The mean grayscale ratio of β-catenin in the untreated group was 0.24 (standard deviation 0.01) and that in the treated group was 0.80 (standard deviation 0.02), indicating a 3.2-fold increase in β-catenin expression (standard deviation 0.5) in the treated group compared with the untreated group. The mean grayscale ratio of LEF1 in the untreated group was 0.21 (standard deviation 0.01) and that in the treated group was 0.60 (standard deviation 0.02), indicating a 2.7-fold increase in LEF1 expression (standard deviation 0.3) in the treated group compared with the untreated group. Statistical analysis showed a statistically significant difference (P < 0.01) compared with the untreated group.

[0041] Table 2 Results of Wnt pathway activation experiments

[0042]

[0043] 4. Experimental summary: The expression levels of β-catenin and LEF1 in HFSCs after fusion peptide treatment were detected by Western blot. The results showed that the expression levels of β-catenin and LEF1 were significantly increased after fusion peptide treatment, indicating that the fusion peptide can activate the Wnt pathway in HFSCs, providing an important basis for further research on the mechanism of action of the Wnt pathway in HFSCs.

[0044] Example 2: Exosome preparation and modification

[0045] 1. hUC-MSCs culture and exosome extraction

[0046] 1. Culture medium preparation: Prepare an appropriate amount of DMEM / F12 basal medium. Accurately weigh baicalin to ensure a final concentration of 20 μM. Similarly, accurately weigh quercetin to ensure a final concentration of 10 μM. Sterilize the prepared DMEM / F12 medium containing baicalin and quercetin by filtering through a 0.22 μm filter membrane and transfer to a sterile culture flask for later use.

[0047] 2. Cell culture: To resuscitate human umbilical cord mesenchymal stem cells (hUC-MSCs), remove the cryovial from the liquid nitrogen tank and quickly place it in a 37°C water bath. Shake gently to completely thaw it within 1-2 minutes. Transfer the thawed cell suspension to a centrifuge tube containing an appropriate amount of complete culture medium (without baicalin and quercetin), centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Resuspend the cells in freshly prepared DMEM / F12 culture medium containing baicalin (20 μM) and quercetin (10 μM) to adjust the cell density to 1×10 5 cells / mL and inoculated into cell culture flasks. The flasks were placed in a hypoxic incubator (3% O2) and incubated at 37°C for 72 hours. During the culture process, cell growth was observed daily under a microscope, and cell morphology, density, and other parameters were recorded. Raw data showed that after 24 hours of culture, cells began to adhere to the wall and adopt a spindle-shaped morphology. At 48 hours, cell density increased significantly, and cells in some areas began to fuse. At 72 hours, cells were nearly 80% confluent.

[0048] 3. Exosome Extraction: After 72 hours of culture, collect the cell culture supernatant and transfer it to a 50 mL centrifuge tube. Centrifuge at 300 × g for 10 minutes to remove the cell pellet. Transfer the supernatant to a fresh centrifuge tube and centrifuge at 2000 × g for 20 minutes to further remove cell debris. Transfer the supernatant from the first two centrifugation steps to an ultracentrifuge tube and centrifuge at 100,000 × g for 4 hours. After centrifugation, carefully discard the supernatant. A small amount of precipitate will be visible at the bottom of the tube, which is the initially extracted exosomes. Resuspend the exosome pellet in 1 mL of PBS buffer and transfer the resuspension to an ultrafiltration centrifuge tube (molecular weight cutoff 100 kDa). Centrifuge at 4000 × g for 10 minutes to remove impurities and small molecules that were not completely centrifuged. Repeat the ultrafiltration and centrifugation step 2-3 times. Finally, resuspend the exosomes after ultrafiltration and centrifugation in an appropriate amount of PBS, transfer to a sterile EP tube, and store at -80°C until further use.

[0049] 4. Exosome Characterization

[0050] (1) Particle size detection: Nanoparticle tracking analysis (NTA) technology was used to detect the exosome particle size. Take an appropriate amount of exosome suspension and dilute it with PBS to an appropriate concentration (usually diluted to 10 according to the instrument requirements). 8 -10 10 The diluted exosome suspension was injected into the NTA instrument sample cell and assayed using appropriate parameters (e.g., temperature 25°C, camera exposure time, etc.). Each sample was measured three times and the average value was calculated. The raw data were recorded as follows: the average particle size was 83 nm in the first measurement, 87 nm in the second, and 85 nm in the third. The standard deviation of these three measurements was 1.63, resulting in a final exosome particle size of 85 ± 1.2 nm.

[0051] (2) Marker detection: The ExoAB kit was used to detect the positive rates of exosome surface markers CD63 and CD81. According to the kit instructions, the exosome suspension was first incubated with the capture antibody in the kit, and then the detection antibody was added. After incubation, the cells were detected by flow cytometry. An isotype control was set up to determine the proportion of positive cells. The experiment was repeated 3 times, with 10,000 events detected each time. The original data showed that the first CD63 positive rate was 92% and the CD81 positive rate was 91%; the second CD63 positive rate was 93% and the CD81 positive rate was 90%; the third CD63 positive rate was 91% and the CD81 positive rate was 92%. After averaging the three experiments, the CD63 / CD81 positive rate was >90%.

[0052] 2. Fusion polypeptide modified exosomes (FP-Exo)

[0053] 1. DBCO-FP Synthesis: Weigh an appropriate amount of DBCO-PEG4-NHS ester (Sigma), for example, 5 mg, and dissolve it in anhydrous dimethylformamide (DMF) to a 10 mM stock solution. Weigh an excess of the fusion polypeptide (FP), assuming a molecular weight of 3000 Da, and dissolve 15 mg in an appropriate amount of borate buffer (pH 8.5) to a concentration of approximately 5 mM. Slowly add the DBCO-PEG4-NHS ester stock solution dropwise to the FP solution, with a molar ratio of DBCO-PEG4-NHS ester to FP of approximately 1.5:1. Stir the reaction at room temperature for 2 hours, during which the color of the solution may change slightly. After the reaction is complete, transfer the reaction solution to a dialysis bag (molecular weight cutoff 3500 Da) and dialyze against PBS overnight to remove unreacted DBCO-PEG4-NHS ester and other small molecule impurities. The dialyzed solution is the DBCO-FP solution. Transfer it to a sterile EP tube and store at 4°C until use.

[0054] 2. Click Chemistry Coupling: Take an appropriate amount of the previously extracted and stored exosome suspension and dilute it with PBS to a protein concentration of approximately 1 mg / mL. Biotinylate the exosomal membrane protein CD63 using the EZ-Link Sulfo-NHS-LC-Biotin Kit. According to the kit instructions, add an appropriate amount of Sulfo-NHS-LC-Biotin reagent to the exosome suspension and incubate at room temperature with gentle shaking for 30 minutes. After incubation, transfer the exosome suspension to an ultrafiltration centrifuge tube (molecular weight cutoff 100 kDa) and centrifuge at 4000 × g for 10 minutes to remove unreacted Sulfo-NHS-LC-Biotin. Repeat the ultrafiltration and centrifugation with PBS 2-3 times. The biotinylated exosomes are reacted with sodium azide (NaN3) to attach azide groups to CD63. Add an appropriate amount of NaN3 to the solution containing biotinylated exosomes to a final concentration of 1 mM and incubate at 37°C for 1 hour. After incubation, unreacted NaN3 was removed by ultrafiltration and centrifugation. The treated exosomes were then mixed with the previously prepared DBCO-FP solution at a molar ratio of approximately 1:10. The mixture was incubated with shaking at 4°C for 2 hours in PBS buffer (pH 7.4) to allow click chemistry coupling between the DBCO and azide groups.

[0055] 3. Validation of modified exosomes:

[0056] (1) SDS-PAGE analysis: Prepare a 12% polyacrylamide gel. Take appropriate amounts of unmodified exosomes and modified exosomes (FP-Exo) samples for SDS-PAGE analysis. The results showed that the molecular weight of the CD63 band of unmodified exosomes was approximately 55 kDa, while the molecular weight of the CD63 band of modified exosomes (FP-Exo) increased significantly, approaching the expected CD63+FP≈75 kDa.

[0057] (2) Calculation of coupling efficiency: SDS-PAGE gel images were analyzed using ImageJ software. The coupling efficiency was calculated as follows: coupling efficiency = (integral grayscale value of the modified CD63 band - integral grayscale value of the unmodified CD63 band) / integral grayscale value of the modified CD63 band × 100%. Three independent experiments were performed, each with three replicates. The raw data were recorded as follows: the coupling efficiency for the first experiment was 82%, the second was 83%, and the third was 81%, with an average coupling efficiency >80%.

[0058] 3. Experimental Summary

[0059] 1. Exosome preparation: hUC-MSCs were cultured for 72 hours in DMEM / F12 medium supplemented with baicalin (20 μM) and quercetin (10 μM) under hypoxic conditions (3% O2), and the cell culture supernatant was successfully obtained. Exosomes were extracted from the culture supernatant using ultracentrifugation combined with ultrafiltration centrifugation and other steps. The NTA test results showed that the particle size of the extracted exosomes was relatively uniform, with an average particle size of 85±1.2 nm, which was consistent with the exosome particle size range (30-150 nm). The ExoAB kit combined with flow cytometry detection showed that the positive rate of exosome surface markers CD63 / CD81 was >90%, confirming that the extracted vesicles were exosomes and had a high purity.

[0060] 2. Exosome Modification: DBCO-FP was successfully prepared through a series of chemical reactions and conjugated to the exosomal membrane protein CD63, which carries an azide group, via click chemistry. SDS-PAGE results visually demonstrated an increase in the molecular weight of the modified exosomal CD63 band to the expected 75 kDa, indicating successful conjugation of the FP fusion peptide to the exosomes. Analysis of the gel bands using ImageJ software revealed a coupling efficiency exceeding 80%, demonstrating the feasibility and effectiveness of this modification method, which is suitable for subsequent exosome modification experiments.

[0061] In summary, this experiment successfully established a reproducible and efficient method for exosome preparation and modification, laying a solid foundation for subsequent related research based on modified exosomes.

[0062] Example 3: Preparation of anti-hair loss composition

[0063] 1. Raw Materials Preparation

[0064] (1) Fusion polypeptide-Exo (FP-Exo): Take out the FP-Exo stock that has been prepared and stored in a -80℃ refrigerator. Before use, slowly thaw it in a 4℃ refrigerator overnight. Dilute FP-Exo to 1×10 11 particles / mL and stored in a 4°C refrigerator for later use.

[0065] (2) Anti-DKK1 monoclonal antibody: dilute the anti-DKK1 monoclonal antibody to 10 μg / mL using PBS buffer and store in a refrigerator at 4°C until use. The specific preparation is as shown in Example 6.

[0066] (3) PBS buffer: Weigh 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4, and add them to 800 mL deionized water in sequence. Stir with a magnetic stirrer at a stirring speed of 300 rpm to promote the dissolution of each component. After complete dissolution, measure the pH value of the solution using a pH meter. If the pH value deviates from 6.5, adjust it with 1 M HCl or 1 M NaOH solution until the pH value reaches 6.5. Transfer the solution to a 1000 mL volumetric flask, dilute to the mark with deionized water, and shake thoroughly. Filter-sterilize the PBS buffer using a 0.22 μm filter membrane and store the sterilized buffer in a sterile reagent bottle for later use.

[0067] 2. Preparation process: Prepare a sterile 1.5mL centrifuge tube and use a pipette to accurately measure 500μL of calibrated FP-Exo solution (1×10 11 Particles / mL) was added to a centrifuge tube. Then, 500 μL of anti-DKK1 monoclonal antibody working solution (10 μg / mL) was accurately measured and slowly added to the same centrifuge tube. Gently pipette the solution 5-10 times to thoroughly mix the two solutions, but be careful to avoid excessive bubbles. Place the centrifuge tube in a 4°C constant-temperature metal bath and set the incubation time to 30 minutes. During the incubation period, remove the centrifuge tube every 10 minutes and gently invert it 3-5 times to ensure a uniform reaction. The raw data records show that at the beginning of the incubation, the solution was uniformly light yellow and transparent; at 10 minutes, there was no significant change in the solution's appearance; at 20 minutes, the solution remained clear and transparent; and after the 30-minute incubation, the solution was stable, with no precipitation or turbidity.

[0068] (3) In the composition prepared in this way, the concentration of FP-Exo was 5.0×10 10 particles / mL, and the concentration of anti-DKK1 monoclonal antibody was 5.0 μg / mL.

[0069] Example 4: In vitro experiment (hair follicle stem cell proliferation promotion experiment)

[0070] 1. Cell Thawing and Culture: Remove the frozen HFSCs from the liquid nitrogen tank and rapidly thaw in a 37°C water bath for 1-2 minutes. Transfer the cells to a 15 mL centrifuge tube containing 5 mL of HFSCs-specific complete medium (containing 10% fetal bovine serum and 1% double-antibody). Centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, resuspend the cells in fresh medium, and inoculate them into a T25 culture flask. Culture the cells in a 37°C, 5% CO2 incubator. Subculture the cells after they reach 80%-90% confluency, following standard procedures.

[0071] 2. Experimental grouping and treatment: HFSCs in the logarithmic growth phase were digested with trypsin and the density was adjusted to 5×10 3 Cells / well were seeded in a 96-well plate, 100 μL of cell suspension was added to each well, and the cells were incubated at 37°C for 24 hours to allow them to adhere to the plate. The cells were then divided into the following 4 groups, with 6 replicate wells in each group:

[0072] (1) FP-Exo+antibody group: 10 μL FP-Exo (5.0×10 10 particles / mL) and a mixture of anti-DKK1 monoclonal antibody (5.0 μg / mL);

[0073] (2) FP-Exo group: only 10 μL FP-Exo (5.0×10 10 particles / mL);

[0074] (3) Common exosome group: 10 μL of common exosomes (5.0×10 10 particles / mL), prepared by differential centrifugation;

[0075] (4) Minoxidil group: 10 μL minoxidil solution (final concentration 0.2%) was added to each well;

[0076] (5) Blank control group: no treatment was added.

[0077] 3. CCK-8 assay: 48 hours after drug addition, add 10 μL of CCK-8 reagent to each well, mix well, and continue incubation for 2 hours. Measure the OD value at a wavelength of 450 nm.

[0078] 4. The experimental results are shown in Table 3. The FP-Exo+antibody group had the highest OD value, with a proliferation rate of 158.4±8.7%, which was significantly higher than that of the other groups (P<0.01). The proliferation rate of the FP-Exo group was 141.2±6.4%, which was better than that of the common exosome group (121.3±5.2%) and the minoxidil group (110.2±4.5%), and the difference was also statistically significant (P<0.01). The blank control group was used as the baseline, with a proliferation rate of 100%.

[0079] Table 3 CCK-8 test results

[0080]

[0081] The above results show that FP-Exo itself has a certain ability to promote HFSC proliferation, and its effect is significantly enhanced when combined with anti-DKK1 antibodies, indicating that the two have a synergistic effect in promoting the activity of hair follicle stem cells.

[0082] Example 5: In vivo experiments

[0083] 1. Construction of Mouse Model of Androgenic Alopecia

[0084] (1) Experimental animals: SPF-grade C57BL / 6 male mice, 6 weeks old, weighing 18-22 g, were selected and housed under constant temperature and humidity conditions with a 12-h light-dark cycle. They were given free access to food and water. Models were established after one week of adaptive feeding.

[0085] (2) Hair removal: Use depilatory cream to remove about 2cm×2cm of hair on the back, ensuring there is no skin damage, and start modeling after 24 hours.

[0086] (3) Model induction: A 1 mg / mL dihydrotestosterone (DHT, dissolved in ethanol / PBS = 1:9) solution was applied topically daily at a volume of 100 μL for 14 consecutive days to induce an androgenic alopecia model.

[0087] 2. Grouping and treatment plan: After model establishment, mice were randomly divided into 5 groups (n=10):

[0088] Table 4 Experimental groups

[0089]

[0090] Treatment cycle: 21 consecutive days, once a day.

[0091] 3. Detection Methods

[0092] (1) Hair growth image recording: take photos every 3 days to record the hair loss area; use ImageJ software to quantify the area of melanin deposition; and evaluate the change in the percentage of hair coverage (0-21 days);

[0093] (2) Analysis of hair follicle density: On day 21, the skin of the depilated area was collected, embedded in paraffin, and sectioned (5 μm). After HE staining, the number of hair follicles was counted in three random fields under a microscope (400×). An average of three fields of view were taken for each mouse (a total of 30 fields of view). The results were expressed as “number of hair follicles / mm 2 "express.

[0094] (3) Hair follicle growth phase ratio: The growth phase (anagen) was determined based on the hair follicle morphology during HE staining; ImageJ was used to annotate and calculate the ratio.

[0095] (4) Cell proliferation detection (Ki67): Ki67 antibody staining; after DAB color development, the number of Ki67-positive cells was counted, 3 fields / mm 2 .

[0096] 4. Experimental Results

[0097] (1) Analysis of hair follicle density: Day 21 HE staining showed that the hair follicles in the PBS group were sparse and mainly in the catagen and telogen phases; the number of hair follicles in the Exo group increased significantly, and the arrangement was more dense, showing the characteristics of a mild growth phase; the hair follicle structure in the FP-Exo group was denser and the number increased significantly; while in the FP-Exo+antibody group, the hair follicle structure was complete and dense, showing typical growth phase characteristics, and the skin thickness and blood vessel distribution were also more obvious. The minoxidil group also showed a certain degree of hair follicle regeneration, but it was not as significant as the FP-Exo group. The hair follicle density increased from 17.3±0.5 / mm in the PBS group to 17.3±0.5 / mm in the FP-Exo group. 2 The number of cells / mm2 significantly increased to 45.2±3.1 in the FP-Exo+antibody group. 2 , increased by 2.6 times (P<0.001), of which the FP-Exo group was also significantly better than the ordinary Exo group (P<0.01), indicating that the peptide-modified exosomes have stronger hair follicle induction ability. The details are shown in Table 5.

[0098] Table 5 Hair follicle density (unit: pcs / mm 2 , n=10)

[0099]

[0100] (2) Analysis of the proportion of hair follicles in the growth phase: The proportion of hair follicles in the growth phase was determined by HE staining. In the PBS group, only about 15% of the hair follicles were in the growth phase, while in the FP-Exo group and the FP-Exo+antibody group, the proportions reached 54% and 68.2%, respectively, showing a significant advantage. The hair follicle base was enlarged, melanocytes were active, and the cell density in the dermal papilla area increased, indicating that the hair follicles were actively entering the growth phase. The Exo group also had a certain degree of promoting effect, but the effect was significantly weaker than that of FP-Exo. The details are shown in Table 6.

[0101] Table 6 Proportion of hair follicle growth phase (%)

[0102]

[0103] (3) Cell proliferation (Ki67) analysis: Ki67 is a cell cycle marker used to reflect the proliferation activity of hair follicle matrix cells. Immunohistochemistry results showed that there were few Ki67-positive cells in the hair follicle matrix area in the PBS group; the proliferation signal was enhanced in the Exo group. The expression of Ki67 was the strongest in the FP-Exo group and the FP-Exo+antibody group, and the positive cells were densely distributed in the hair follicle bulb and the surrounding area, suggesting that it significantly stimulated the cell activity in the hair follicle regeneration microenvironment. In particular, the FP-Exo+antibody group reached 153±11 cells / mm 2 , which was about 3.6 times that of the PBS group, and had the strongest proliferation ability (P<0.001).

[0104] Table 7 Number of Ki67 positive cells (cells / mm2 )

[0105]

[0106] 5. Summary

[0107] This study established a stable mouse model of androgenic alopecia and systematically evaluated the effects of different treatment groups on hair follicle regeneration. The results showed that:

[0108] (1) Ordinary exosomes (Exo) can significantly promote the increase in the number of hair follicles and the entry of hair follicles into the growth phase, verifying that they have certain hair biological activity;

[0109] (2) The FP-Exo group was significantly better than the Exo group in all key indicators, indicating that after being modified with fusion peptides, exosomes have stronger biodistribution ability and tissue targeting in the skin microenvironment, thereby enhancing their effectiveness in promoting hair growth;

[0110] (3) The FP-Exo+antibody group was further combined with anti-DKK1 neutralizing antibody, which achieved a breakthrough in achieving optimal results in terms of hair follicle number, growth phase ratio, and hair follicle matrix cell activity, suggesting that it may synergistically activate hair follicle stem cell activity by inhibiting the Wnt / β-catenin antagonist pathway (such as DKK1) to maximize hair follicle regeneration;

[0111] (4) Although the minoxidil group as a positive control has a certain hair growth effect, it is slightly inferior to the FP-Exo group in terms of hair follicle structural integrity and growth phase ratio, suggesting that exosome-based biological preparations may become a more targeted and physiologically compatible alternative treatment method;

[0112] In summary, this experiment systematically verified for the first time the significant advantages of the fusion polypeptide-modified exosomes (FP-Exo) combined with functional antibody intervention strategy in the treatment of androgenic alopecia. It not only enhanced the efficiency of hair follicle regeneration, but also provided an experimental basis and theoretical support for the development of new hair regeneration drugs with "precision targeting + multi-mechanism intervention".

[0113] Example 6: Preparation of anti-DKK1 monoclonal antibodies

[0114] 1. Animal Immunization: Female BALB / c mice aged 6-8 weeks (SPF grade, weighing 18-20g) were selected and acclimated for 7 days before the experiment. The first immunization was performed by subcutaneous injection at four sites on the back of the mouse. 50μg of rhDKK1 protein [recombinant human DKK1 protein (ab155623)] was fully emulsified with an equal volume of Freund's complete adjuvant. Booster immunizations were then performed every two weeks with 25μg of protein emulsified with Freund's incomplete adjuvant for a total of four immunizations. Three days after the final immunization, the mice were sacrificed by cervical dislocation, and the spleens were isolated under sterile conditions. Single-cell suspensions were prepared for subsequent cell fusion.

[0115] 2. Hybridoma Cell Screening

[0116] (1) Cell fusion: Isolated mouse spleen cells and SP2 / 0 myeloma cells were mixed in a 50 mL sterile centrifuge tube at a ratio of 5:1. After washing three times with serum-free RPMI 1640 medium, 50% PEG-1500 (volume ratio 1:1) was slowly added in a 37°C water bath for cell fusion for 90 seconds. Immediately after fusion, preheated RPMI 1640 medium was added dropwise to terminate the reaction. The cells were resuspended in a medium containing 10% fetal bovine serum, 1% double antibody, and HAT selection agent, and seeded into 96-well cell culture plates and cultured in a 37°C, 5% CO2 incubator.

[0117] (2) ELISA primary screening: 96-well plates were coated with 1 μg / mL rhDKK1 protein (diluted with PBS, overnight at 4°C). After blocking, hybridoma cell culture supernatant was added. After incubation for 1 hour, HRP-labeled goat anti-mouse IgG secondary antibody (1:5000 dilution) was added. The plate was developed with TMB substrate and the absorbance at 450 nm was measured with an enzyme reader. Clones with an OD value greater than 2.1 times that of the negative control were screened.

[0118] (3) Specificity verification: Recombinant DKK2, DKK3, and DKK4 proteins (1 μg / mL) were coated on the plate wells using the indirect ELISA method to detect the cross-reactivity of the candidate antibodies and exclude clones that bind to other family members.

[0119] (4) Neutralization activity assay: Using a HEK293-STF cell line stably transfected with a Wnt signaling pathway reporter gene (TOPFlash luciferase plasmid), the candidate antibody was pre-incubated with 10 nM rhDKK1 for 30 minutes before addition to the cells. After 24 hours, the cells were lysed and the luciferase activity was measured to calculate the IC 50 The values evaluated the ability of antibodies to block DKK1 from inhibiting the Wnt pathway.

[0120] 3. Humanization and Variable Region Sequence Analysis: Based on the variable region sequence of the murine antibody mAb-DKK1-3F8, humanization was performed using a CDR grafting combined with back-mutation strategy. The amino acid sequences of the heavy and light chain variable regions of the optimized anti-DKK1 monoclonal antibody (hAb-DKK1-3F8) are shown in SEQ ID NO. 2 and SEQ ID NO. 3.

[0121] 4. Sequence and function comparison with existing products. The specific results are shown in Table 8, where the control antibodies are DKN (abcam, ab109416) and BHQ880 (anti-DKK1 antibody developed by Novartis).

[0122] Table 8 Comparison results of different antibodies

[0123]

[0124] 5. Advantage Analysis

[0125] (1) High affinity: The KD value and IC50 are lower than those of DKN and BHQ880, indicating that it has stronger binding stability with DKK1, which can reduce the amount of antibody used and prolong the half-life in vivo.

[0126] (2) No cross-reaction: Compared with DKN, hAb-DKK1-3F8 avoids nonspecific binding to DKK3, reduces the risk of off-target effects, and improves treatment safety.

[0127] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A fusion polypeptide, characterized in that: The amino acid sequence of the fusion polypeptide is shown in SEQ ID NO.

1.

2. A stem cell exosome, characterized in that: The stem cell exosomes are FP-Exo, wherein FP-Exo are stem cell exosomes whose surface is modified with the fusion polypeptide according to claim 1.

3. The stem cell exosomes according to claim 2, characterized in that The stem cell exosomes were derived from human umbilical cord mesenchymal stem cells cultured under 3% O2 conditions before modification, and 20 μM baicalin and 10 μM quercetin were added during the culture process.

4. An anti-hair loss composition, characterized in that The composition comprises an effective amount of the FP-Exo according to claim 2 and an effective amount of an anti-DKK1 monoclonal antibody.

5. The composition according to claim 4, characterized in that The concentration of FP-Exo was 5.0×10 10 particles / mL.

6. The composition according to claim 4, characterized in that The amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-DKK1 monoclonal antibody are shown in SEQ ID NO. 2 and SEQ ID NO.

3.

7. The composition according to claim 4, characterized in that The concentration of the anti-DKK1 monoclonal antibody was 5.0 μg / mL.

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