Active peptide, stem cell exosome and application of active peptide and stem cell exosome in improvement of skin aging

Abstract

The invention discloses an active peptide, the full-length sequence of the active peptide is (GPO) 5-K (R-Dopa) GD-[GHK (Cu < 2 + >)] 2-(TPP) 3-TAT-NH2, Dopa is 3, 4-dihydroxyphenylalanine, TPP is a triphenylphosphine cation, and GPO is Gly-Pro-Hyp. The invention further discloses an active peptide and stem cell exosome composition with a remarkable anti-aging effect. Through the synergistic effect of the active peptide and the stem cell exosome, the skin aging can be improved from the cellular level, and the active peptide composition has wide application prospects and is suitable for the fields of skin beautifying, anti-aging treatment and the like.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering, and relates to a composition of an active peptide and stem cell exosomes and its application in skin aging. Background Art

[0002] With the increase of age, skin aging gradually becomes an inevitable physiological process. During the aging process, obvious skin relaxation, wrinkles, age spots, dryness and other phenomena occur, which are closely related to the reduction of the metabolic function of skin cells, the weakening of antioxidant capacity and the decrease of important components such as collagen and hyaluronic acid. At present, there are already some anti-aging skin care products on the market, but most of these products only improve the skin appearance through surface repair and lack the regulation of deep skin physiological mechanisms.

[0003] In recent years, stem cell exosomes have become a research hotspot in the fields of regenerative medicine and anti-aging due to their unique biological activities. Stem cell exosomes are rich in various growth factors, cytokines and miRNAs, and can potentially have anti-aging effects on the skin by promoting skin cell proliferation, antioxidant activity and repairing damaged tissues. As a molecule with special biological activity, active peptides can activate the self-repair function of skin cells and enhance the elasticity and firmness of the skin during the repair and regeneration process of skin cells.

[0004] However, there is no relevant application of combining active peptides with stem cell exosomes in the existing technology. Therefore, the present invention aims to solve this technical gap and proposes a new type of composition that uses the synergistic effect of active peptides and stem cell exosomes to improve skin aging. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for synergistically improving skin aging by combining active peptides with stem cell exosomes and its application in beauty and medicine.

[0006] Therefore, on the one hand, the present invention discloses an active peptide, and the full-length sequence of the active peptide is (GPO) 5 -K(R-Dopa)GD-[GHK(Cu 2+ )] 2 -(TPP) 3 -TAT-NH 2 , where Dopa = 3,4-dihydroxyphenylalanine, TPP = triphenylphosphonium cation, and GPO = Gly-Pro-Hyp.

[0007] Preferably, the molecular weight of the active peptide of the present invention is 4523.6 Da.

[0008] In one aspect, the present invention also discloses a stem cell exosome, which is a TD-1 / active peptide double-modified exosome, and the active peptide is the aforementioned active peptide.

[0009] Preferably, the amino acid sequence of TD-1 in the present invention is ACSSSPSKHCG.

[0010] Preferably, the particle size distribution of the exosome in the present invention is 90±5nm, the surface potential is -25±2mV, and the modification efficiency is TD-1≥82% and active peptide≥75%.

[0011] In one aspect, the present invention also discloses a composition of an active peptide and a stem cell exosome, which composition comprises the aforementioned active peptide and exosome, wherein the content of the active peptide is 5mg / mL and the content of the exosome is 1×10 10 particles / mL.

[0012] In one aspect, the present invention also discloses the use of the aforementioned active peptide in the preparation of a composition of an active peptide and a stem cell exosome.

[0013] In one aspect, the present invention also discloses the use of the aforementioned stem cell exosome in the preparation of a drug for improving skin aging.

[0014] The composition of the active peptide and the stem cell exosome of the present invention can significantly promote the proliferation of skin cells, enhance the anti-aging ability of the skin, increase the synthesis of collagen and hyaluronic acid, and improve the elasticity and firmness of the skin. Specifically, it includes the following advantages:

[0015] (1) Synergistic effect: The combination of the active peptide and the stem cell exosome can complement each other and enhance the skin repair, regeneration and anti-aging effects.

[0016] (2) Deep repair: This composition can delay the process of skin aging through multiple effects such as promoting skin cell proliferation, improving antioxidant capacity, and promoting collagen synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 TEM image of exosome (100nm). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this invention belongs. The terms used in the specification of this invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0019] 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.

[0020] Example 1: Design, preparation and testing of active peptides

[0021] 1. Design of active peptides

[0022] 1. Functional modular design

[0023] (1) Triple helical domain (GPO) 5 :The characteristic repeating unit of collagen (Gly-XY, X is usually Pro, Y is usually Hyp), in which Hyp (hydroxyproline) strengthens the hydrogen bond network through the hydroxyl group and stabilizes the triple helical conformation. 5 Replace with (GPO) 5 , the thermal stability was improved by introducing Hyp (Tm value increased from 42℃ to 58℃).

[0024] (2) Dual-mode targeting domain KRGD: in K 7 D-arginine (D-Arg) was introduced into the site to enhance the affinity for integrin αvβ3 (Kd=3.2nM vs 15nM of L-Arg). 3,4-dihydroxyphenylalanine (Dopa) was inserted before the RGD sequence. In the acidic environment of the wound (pH 5.5), Dopa was oxidized to form a quinone group, which covalently bound to the thiol group on the surface of fibroblasts (targeting efficiency increased by 40%).

[0025] (3) Repairing functional domain GHK-Cu 2+ Double copy: GHK (Gly-His-Lys) chelates Cu through the imidazole ring of His 2+ , forming a stable complex (stability constant logK = 16.5), achieving sustained release (72-hour release rate <30%). 2+ / Zn 2+ The dual ion design activates SOD1 enzyme activity (EC 1.15.1.1) and removes ROS with an efficiency of 92.4%. 2+ The efficiency is 78%.

[0026] (4) Mitochondrial delivery system: via PEG 4 The spacer arm is connected to three triphenylphosphine (TPP) cations, and the mitochondrial membrane potential (ΔΨm = -180mV) is used to drive the enrichment, and the targeting efficiency is increased by 3.2 times compared with single TPP. HIV-TAT (sequence: YGRKKRRQRRR) is fused to the C-terminus, and the site-specific coupling is carried out by thiol-maleimide reaction. The cell penetration rate detected by flow cytometry is >90%.

[0027] 2. Dynamic response design

[0028] (1) Photo-crosslinking control: Insert azidophenylalanine (AzF) at the K 7 site, and ultraviolet light (365 nm, 50 mW / cm 2 , 30 s) triggers click chemical reaction to form intermolecular crosslinking (crosslinking degree > 80%), enhancing the wound retention time (half-life extended from 6 h to 24 h).

[0029] (2) Enzyme / pH dual response: Insert the Val-Cit-PABC linker into the peptide chain, which is cleaved only in the wound microenvironment with overexpressed MMP-2 (> 10 nM) and pH < 6.0 to release the active ingredient.

[0030] 3. Active peptide sequence: After the above design, the full-length sequence of the final active peptide is:

[0031] (GPO) 5 -K(R-Dopa)GD-[GHK(Cu 2+ )] 2 -(TPP) 3 -TAT-NH 2 , where Dopa = 3,4-dihydroxyphenylalanine, TPP = triphenylphosphine cation, and GPO = Gly-Pro-Hyp.

[0032] II. Preparation of active peptide

[0033] 1. Raw materials and equipment

[0034] (1) Resin: RinkAmide MBHA resin (loading 0.6 mmol / g, 100 - 200 mesh).

[0035] (2) Reagents: Fmoc-AzF-OH (azidophenylalanine); Fmoc-Dopa(Boc) 2 -OH (Boc-protected Dopa); TPP-PEG 4 -COOH (triphenylphosphine-PEG4-carboxylic acid).

[0036] 2. Synthesis steps, and the key process parameters are shown in Table 1.

[0037] (1) Resin swelling: Place the resin in the reaction column and swell it with DCM for 30 minutes (volume 5 mL / g resin).

[0038] (2) Fmoc deprotection: Deprotect with 20% piperidine / DMF solution (v / v) twice (5 minutes + 10 minutes), and wash with DMF 5 times.

[0039] (3) Amino acid coupling: Fmoc-AA (4 eq), HOBt (4 eq), and DIC (4 eq) were dissolved in DMF and activated for 5 minutes. The reaction was carried out for 60 minutes under nitrogen (K 7 The extension time at the AzF site was extended to 90 minutes), and the coupling efficiency was monitored by the ninhydrin test to ensure successful coupling.

[0040] (4) Selective deprotection of Alloc: At the K 7 site: Pd(PPh 3 )(0.1 eq) and phenylsilane (10 eq) were reacted in DCM for 2 hours to remove the Alloc protecting group. 4 (0.1 eq) and phenylsilane (10 eq) were reacted in DCM for 2 hours to remove the Alloc protecting group.

[0041] (5) Connection of the TPP-TAT module: TPP-PEG 4 -COOH (3 eq) was coupled with the deprotected amino group of the Lys side chain (activated by HATU / DIPEA and reacted at room temperature for 12 hours).

[0042] (6) Cleavage and purification: TFA / TIPS / H 2 O (94:3:3, v / v), reacted at room temperature for 3 hours; precipitated with cold ether, centrifuged (10000 rpm, 10 minutes), repeated 3 times; semi-preparative HPLC (C18 column, mobile phase A: 0.1% TFA / H 2 O, B: 0.1% TFA / acetonitrile, gradient 15% → 45% B, 30 minutes).

[0043] Table 1 Key process parameters

[0044] Step Parameter Quality control point Amino acid coupling Reaction temperature 25 ± 1 °C Ninhydrin detection (>99% coupling) TPP linkage HATU / DIPEA molar ratio 1:2 MALDI-TOF verification of molecular weight Cleavage TFA concentration ≥ 95% Free amino group detection (<0.1%)

[0045] III. Inspection of the bioactive peptide

[0046] 1. Mass spectrometry analysis: The measured molecular weight by MALDI-TOF MS was 4523.6 Da (theoretical value 4523.2 Da, error < 0.01%).

[0047] 2. Function verification

[0048] (1) Targeting efficiency: The DiO-labeled peptide was incubated with fibroblasts for 1 hour, and the fluorescence intensity detected by flow cytometry was 4.8 times that of the control group. Among them, the MFI of the bioactive peptide of the present invention was 2850, and the MFI of the traditional peptide (tripeptide-1 (GHK-Cu)) was 593.

[0049] (2) Mitochondrial enrichment: Detected by confocal microscopy, the co-localization rate of this bioactive peptide with MitoTracker was 89.3%, and that of the traditional peptide (tripeptide-1 (GHK-Cu)) was 19.2%.

[0050] 3. Stability test: Samples were placed at 40 °C / 75% RH for 3 months. The purity detected by HPLC remained at 98.7%, and the retention rate of the triple helix structure detected by CD was >95%.

[0051] 4. Biological activity

[0052] (1) The scratch assay showed that the healing rate of the combined exosome group at 24 hours was 92.4%, while that of the control EGF group was 68.3%.

[0053] (2) Antioxidant capacity: The IC50 of the DPPH free radical scavenging rate was 28.5 μM, while that of the control vitamin C was 42.7 μM.

[0054] Example 2: Preparation and testing of stem cell exosomes

[0055] I. Exosome preparation

[0056] 1. Cell culture and pretreatment

[0057] (1) Materials: Human iPSC-derived mesenchymal stem cells (iPSC-MSC); exosome DMEM / F12 + 10% exosome-free FBS + 20 ng / mL TGF-β3.

[0058] (2) Procedures

[0059] 1) Amplification culture: Inoculate 5×10 6 iPSC-MSC in a T75 culture flask and culture at 37 °C, 5% CO 2 until 80% confluence (about 48 - 72 hours).

[0060] 2) Exosome induction: To promote the differentiation of iPSC into MSC, add 20 ng / mL of TGF-β3 to the medium and culture in a hypoxic (5% O 2 ) environment for 72 hours. Use a microscope to observe whether the cells show a typical spindle cell morphology, and regularly change the medium to ensure the best cell growth environment. When the cells reach 80% - 90% confluence, digest them with 0.25% trypsin, count the cells, and ensure that the inoculated cell density is in the best state. The cells cultured at this time can significantly upregulate miR-21-5p in exosomes (the expression level was verified by qPCR to increase 12.3-fold).

[0061] 3) Exosome collection: After culturing the cells for 48 hours, change to exosome-free serum medium (exosome-free FBS) and continue to culture at 37 °C, 5% CO 2Culture for 48 hours, and collect the exosomes secreted by the cells into the culture medium. Remove cells and cell debris at 300×g for 10 minutes; remove larger cell residues at 2,000×g for 10 minutes. Further remove cell residues and larger particles using a 0.22μm PES filter.

[0062] 2. Exosome Isolation and Purification

[0063] (1) Coarse separation: First, remove larger impurities (such as dead cells, small debris) at 10,000×g for 30 minutes; then purify exosomes at 100,000×g for 70 minutes; then resuspend with PBS for later use.

[0064] (2) Fine purification: Use a microfluidic chip separation system ( Benchtop) for purification, where the control parameters of the microfluidics are: flow rate ratio (aqueous phase: oil phase) is 3:1, the chip pore size is 100nm, and the temperature is 2 - 8°C. The particle size of the exosomes after this purification is 80 - 120nm.

[0065] The effects of the two-step purification and separation are shown in Table 2.

[0066] Table 2 Comparison Results of Purification Efficiency at Each Step

[0067]

[0068] (3) Concentration: Use an ultrafiltration centrifugal tube (100kDa MWCO), 4,000×g, to concentrate the above-purified exosomes to 5×10^11 particles / mL.

[0069] II. Exosome Modification, and the quality control is shown in Table 3.

[0070] 1. Penetrating peptide TD-1 modification (chemical coupling): Mix the above-prepared exosomes (1×10 10 particles) + TD-1-SH (molar ratio 1:50) + Traut's Reagent (2mM), react with shaking at 4°C for 2 hours, and remove free peptides by ultrafiltration to obtain TD-1-modified exosomes, which are stored at -80°C for later use. Among them, TD-1-SH (C-terminal thiol modification, sequence: ACSSSPSKHCG).

[0071] 2. Active peptide coupling (DBCO-Azide)

[0072] (1) Exosome pre-modification: Dissolve 1mg / mL of DSPE-PEG 2000 -DBCO in an appropriate amount of solvent (PBS) to ensure complete dissolution. Then dissolve the DSPE-PEG 2000-Mix the DBCO with the exosomes prepared above and incubate at 37 °C for 1 hour to allow the PEG-DBCO molecules to undergo a chimeric reaction with the phospholipid bilayer structure of the exosome membrane. This step can be promoted by gently shaking or stirring (incubate at 37 °C for 1 hour), and after completion, purification can be carried out to remove the unreacted DSPE-PEG 2000 -DBCO.

[0073] (2) Active peptide modification: At the C-terminus of the active peptide prepared in Example 1, an Azide group is introduced through the Fmoc-Lys(N 3 )-OH synthesis method. The brief steps are as follows: First, react Fmoc-lysine (Fmoc-Lys-OH) with sodium azide (NaN 3 ) in anhydrous dichloromethane to introduce the azide group and generate Fmoc-Lys(N 3 )-OH; then, use diethylamine (DIPEA) to remove the Fmoc protecting group to obtain the target product. After the reaction, it is purified by column chromatography and characterized by mass spectrometry, NMR, and HPLC to finally obtain high-purity Fmoc-Lys(N 3 )-OH.

[0074] (3) Coupling reaction: Mix the modified exosome DBCO and the modified active peptide in a molar ratio of 1:10, and react at 25 °C for 4 hours; purify by size exclusion chromatography (Superose6 Increase 10 / 300GL) to obtain the active peptide-modified exosomes, and store them at -80 °C for later use.

[0075] 3. Based on the TD-1 modified exosomes obtained in Step 1, couple the active peptide according to the method in Step 2 to obtain the TD-1 / active peptide double-modified exosomes, and store them at -80 °C for later use.

[0076] Table 3 Quality control standards, methods, and test results

[0077]

[0078] III. Exosome function verification

[0079] 1. Transdermal absorption efficiency experiment and results

[0080] (1) Experimental materials

[0081] Skin samples: Porcine skin (thickness 500 μm), taken from slaughtered pigs, immediately frozen and stored, thawed and trimmed to the required size before use.

[0082] Exosome samples: Include three groups: unmodified exosomes, TD-1 modified exosomes, and TD-1 / active peptide double-modified exosomes.

[0083] (2) Franz diffusion cell experimental setup

[0084] Experiments were conducted using a Franz diffusion cell (with appropriately sized receptor and donor chambers), ensuring that the porcine skin was in a horizontal position, and an appropriate amount of exosome solution (concentration: 1×10 10 particles / mL) was added to the donor chamber.

[0085] Skin sample preparation: The porcine skin was placed in the diffusion cell, with the epidermal side facing the donor chamber and the dermal side facing the receptor chamber.

[0086] Experimental conditions: Constant temperature at 37 °C, maintaining appropriate humidity (PBS solution) in the donor and receptor chambers, and setting an appropriate flow rate to simulate the transdermal process.

[0087] (3) Experimental operations

[0088] Samples of exosomes in each group (unmodified exosomes, TD-1 modified exosomes, TD-1 / active peptide double-modified exosomes) were separately added to the donor chamber. Samples were taken every 2 hours to detect the exosome concentration in the receptor chamber and quantified using microplate spectroscopy. After 24 hours, the permeated liquid in the receptor chamber was collected, and the transdermal penetration amount was measured. After the experiment, the skin sample was dissected, the epidermal layer was extracted, and the amount of exosomes retained in the epidermal layer was quantified using the same method.

[0089] (4) Data analysis

[0090] Transdermal penetration amount: Calculated by measuring the cumulative penetration amount over 24 hours.

[0091] Retention rate in the epidermal layer: Calculate the retention rate of exosomes in the epidermal layer.

[0092] (5) Experimental results, as shown in Table 4 specifically.

[0093] Unmodified exosome group: The cumulative penetration amount within 24 hours was 12.1 ± 2.3 μg / cm 2 , and the retention rate in the epidermal layer was 18.5%, indicating that the transdermal absorption ability of exosomes was low, and most of them were retained on the skin surface.

[0094] TD-1 modified exosome group: The transdermal penetration amount increased significantly to 82.3 ± 7.6 μg / cm 2 , and the retention rate in the epidermal layer increased to 63.2%, indicating that the penetrating peptide (TD-1) significantly enhanced the transdermal absorption ability of exosomes and increased their retention in the epidermal layer.

[0095] TD-1 / active peptide double-modified exosome group: Further increased the transdermal penetration amount to 95.6 ± 6.1 μg / cm 2, the retention rate in the epidermal layer was 70.4%, indicating that the transdermal absorption efficiency of the double-modified exosomes was optimal, and the retention effect on the skin surface was further enhanced.

[0096] The results of this experiment showed that TD-1 modified exosomes had a significant improvement in transdermal absorption efficiency, while TD-1 / active peptide double-modified exosomes showed the best transdermal absorption effect and a relatively high retention rate in the epidermal layer, demonstrating their potential excellent skin delivery ability.

[0097] Table 4 Detection results of transdermal absorption efficiency

[0098]

[0099] (Compared with the unmodified group, **p<0.01)

[0100] 2. Targeting and cell uptake experiments and results

[0101] (1) Experimental materials:

[0102] Cell line: HaCaT cells (human keratinocytes)

[0103] Exosome samples: including three groups of unmodified exosomes, TD-1 modified exosomes, and TD-1 / active peptide double-modified exosomes, and the concentration of all exosomes was 1×10 10 particles / mL.

[0104] Cell culture medium: DMEM / F12 + 10% FBS, 37°C, 5% CO 2 culture conditions.

[0105] Mitochondrial dye: MitoTracker Deep Red, used to label cell mitochondria.

[0106] (2) Cell culture: Inoculate HaCaT cells in a 6-well plate at an inoculation density of 5×10 4 cells / well and culture until 60%-70% confluence. Twenty-four hours before the experiment, change the culture medium to serum-free medium to ensure that it does not contain exosomes.

[0107] (3) Exosome treatment: Add each group of exosomes (unmodified, TD-1 modified, TD-1 / active peptide double-modified) to cell culture, with a final concentration of 1×10 10 particles / mL, and continue to incubate for 4 hours.

[0108] (4) Cell uptake experiment: After incubation for 4 hours, the culture medium was removed and the cells were washed 3 times with PBS to remove the exosomes that had not been taken up by the cells. Imaging was performed using a confocal microscope. The excitation wavelength was 488 nm, and the detection channels were set to green fluorescence (for exosome labeling) and red fluorescence (for mitochondrial labeling).

[0109] (5) Mitochondrial co-localization experiment: One hour after treatment with exosomes, MitoTracker Deep Red was added to label the mitochondria, and incubation was continued for 30 minutes. Imaging was also performed using a confocal microscope, and the co-localization of exosomes and mitochondria was evaluated by superimposing the images of exosomes and mitochondria.

[0110] (6) Data analysis:

[0111] Cell uptake rate: From the images obtained by confocal microscopy, the average intracellular fluorescence intensity was calculated to further analyze the uptake efficiency of exosomes.

[0112] Mitochondrial co-localization rate: The degree of overlap of red and green fluorescence in the confocal microscope was calculated to evaluate whether exosomes targeted mitochondria.

[0113] (7) Experimental results are shown in Table 5 specifically.

[0114] 1) Cell uptake rate

[0115] Unmodified exosome group: The cell uptake rate was 27.6 ± 4.5%, indicating that unmodified exosomes were difficult to be taken up by HaCaT cells.

[0116] TD-1 modified exosome group: The cell uptake rate was significantly increased to 75.8 ± 6.2%, indicating that TD-1 modification significantly enhanced the cell uptake ability of exosomes.

[0117] TD-1 / active peptide double-modified exosome group: The cell uptake rate reached 92.4 ± 3.1%, which was the highest among the three groups, indicating that simultaneous modification with TD-1 and active peptide could significantly enhance the cell uptake efficiency of exosomes.

[0118] 2) Mitochondrial co-localization rate

[0119] Unmodified exosome group: The mitochondrial co-localization rate was 24.2 ± 4.1%, indicating that unmodified exosomes had almost no specific targeting.

[0120] TD-1 modified exosome group: The mitochondrial co-localization rate was 63.5 ± 5.3%, indicating that TD-1 modified exosomes had certain targeting ability and could partially target mitochondria.

[0121] TD-1 / Active Peptide Dual-Modified Exosome Group: The mitochondrial co-localization rate was 88.7 ± 4.0%, which was the highest among the three groups, indicating that the dual-modified exosomes had significantly enhanced targeting ability and could highly specifically target mitochondria.

[0122] The results of this experiment showed that TD-1-modified exosomes significantly increased the cellular uptake rate and enhanced the targeting ability to mitochondria. Further, the dual modification of TD-1 and active peptide could significantly enhance the cellular uptake rate and mitochondrial targeting ability of exosomes, showing great application potential.

[0123] Table 5 Results of Targeting and Cellular Uptake Experiments

[0124]

[0125] (Compared with the unmodified group, **p<0.01)

[0126] 3. Efficacy Experiment and Results on Diabetic Ulcer Animal Model

[0127] (1) Experimental animals: Sprague-Dawley (SD) rats, 6 - 8 weeks old, weighing 180 - 220 g, were divided into 5 groups with 10 rats in each group.

[0128] (2) Animal grouping:

[0129] Combined treatment group: TD-1 / Active Peptide Dual-Modified Exosome Group;

[0130] Single exosome group: Unmodified Exosome Group;

[0131] Positive control group (EGF group): Epidermal Growth Factor (EGF) treatment group;

[0132] TD-1 Modified Exosome Group: TD-1 Modified Exosome Group;

[0133] Negative control group: No treatment group.

[0134] (3) Establishment of diabetic ulcer model:

[0135] Animal feeding conditions: Ambient temperature 22 ± 2°C, humidity 55 ± 10%, 12-hour light-dark cycle.

[0136] Diabetes induction: Streptozotocin (STZ) was injected intraperitoneally at a dose of 15 mg / kg for 5 consecutive days, and individuals with fasting blood glucose > 16.7 mmol / L were observed for ulcer model establishment.

[0137] Ulcer model establishment: A standard ulcer (size approximately 10 mm × 10 mm) was formed by cutting the skin on the back of the rats with a scalpel. Treatment started 24 hours after the operation.

[0138] (4) Treatment plan:

[0139] Combined treatment group (TD-1 / active peptide dual-modified exosome group): Each rat was locally injected with TD-1 / active peptide dual-modified exosomes (1×10 10 particles / mL), once every 2 days for 14 days.

[0140] Single exosome group: Injected with unmodified exosomes, once every 2 days for 14 days.

[0141] Positive control group (EGF group): Injected with epidermal growth factor (EGF, 100 ng / mL), once every 2 days for 14 days.

[0142] TD-1 modified exosome group: Injected with TD-1 modified exosomes (1×10 10 particles / mL), once every 2 days for 14 days.

[0143] Negative control group: No treatment.

[0144] (5) Wound healing assessment:

[0145] Healing rate: The ulcer area was photographed every 3 days using a camera, and the wound healing area was calculated using image analysis software (such as ImageJ). The calculation formula is:

[0146]

[0147] Collagen density: At 14 days after the end of the experiment, the collagen deposition in the wound tissue was evaluated by histological sections (H&E staining) and Masson's trichrome staining. Images were taken using a microscope and the collagen density was measured using image analysis software.

[0148] (6) Experimental results are shown in Table 6 specifically.

[0149] 1) Healing rate: The wound healing rate of the combined treatment group (TD-1 / active peptide dual-modified exosome group) was 78.4±5.2% at 7 days, significantly higher than that of other groups (the unmodified exosome group was 53.1±6.7%, and the EGF group was 61.3±4.9%). This indicates that TD-1 and active peptide dual-modified exosomes significantly promoted wound healing. The healing rates of the single exosome group and the positive control group (EGF group) were lower, but still higher than that of the negative control group.

[0150] 2) Collagen density: The collagen density of the combined treatment group was 35.7±2.1 mg / cm 3, significantly higher than other groups, indicating that the dual-modified exosomes can effectively promote collagen deposition, thus accelerating wound repair. The collagen density in the single exosome group was 24.6 ± 1.8 mg / cm 3 , lower than the combination treatment group but higher than the positive control group (28.9 ± 2.3 mg / cm 3 ), indicating that the use of exosomes alone has a certain effect on promoting collagen deposition. The positive control group (EGF group) had a relatively high collagen density, further confirming the role of EGF in wound repair, but it was not as good as the combination treatment group.

[0151] The above results indicate that TD-1 / active peptide dual-modified exosomes significantly increased the wound healing rate and collagen deposition density in the diabetic ulcer model, indicating their potential in promoting the repair of diabetic ulcers. This effect was better than that of the single-modified exosome group and the EGF treatment group, probably because the dual modification enhanced the targeting and biological activity of exosomes. Therefore, TD-1 / active peptide dual-modified exosomes have good application prospects in the field of wound repair.

[0152] Table 6 Therapeutic efficacy experiment and results of the diabetic ulcer animal model

[0153]

[0154] (Compared with other groups, **p < 0.01)

[0155] Example 3: Skin cell culture and experiment

[0156] 1. Cell culture

[0157] (1) Cell type: Human dermal fibroblasts (HDFs);

[0158] (2) Culture medium: DMEM (containing 10% fetal bovine serum and 1% penicillin / streptomycin);

[0159] (3) Culture conditions: 37 °C, 5% CO 2 ;

[0160] (4) Cell density: 1 × 10 6 cells / mL.

[0161] 2. Experimental groups, the experiment was divided into four groups:

[0162] (1) Control group: Cells were not treated (i.e., standard culture medium).

[0163] (2) Exosome group: Added exosomes prepared in Example 2 at a concentration of 1 × 10 10 particles / mL.

[0164] (3) Active peptide group: Add the active peptide prepared in Example 1 at a concentration of 5 mg / mL.

[0165] (4) TD-1 / active peptide double-modified exosome group: Add the TD-1 / active peptide double-modified exosomes prepared and modified in Example 2 at a concentration of 1×10 10 particles / mL.

[0166] (5) Mixed treatment group: Add the exosomes prepared in Example 2 at a concentration of 1×10 10 particles / mL and the active peptide prepared in Example 1 at a concentration of 5 mg / mL.

[0167] 3. Cell proliferation detection (CCK-8 method): Samples of cells in each group were taken at 24 hours, 48 hours, and 72 hours, and CCK-8 reagent was added. The OD values at each time point were measured by an enzyme-linked immunosorbent assay (ELISA) reader (450 nm), and the cell proliferation rate was calculated.

[0168] 4. Western Blot detection of the expression of collagen type I (COL-I) and hyaluronan synthase (HAS2):

[0169] Total cellular proteins were extracted and subjected to SDS-PAGE electrophoresis. After transferring the membrane, it was incubated with primary antibodies against COL-I and HAS2, and detected with an HRP-labeled secondary antibody. The relative expression levels were calculated and analyzed using ImageJ software.

[0170] 5. RT-PCR detection of the expression levels of antioxidant-related genes (SOD, CAT, etc.): Cellular RNA was extracted and cDNA was synthesized. RT-PCR was performed using specific primers, with β-actin as an internal reference. The relative gene expression levels were calculated by the ΔΔCt method.

[0171] 6. Experimental results

[0172] (1) Cell proliferation: CCK-8 assays were performed at 24 hours, 48 hours, and 72 hours. The results are shown in Table 7: The cell proliferation rates of the TD-1 / active peptide double-modified exosome group and the mixed treatment group were significantly faster than those of the other three groups (P<0.05), and the TD-1 / active peptide double-modified exosome group was better than the mixed treatment group.

[0173] Table 7 Results of cell proliferation detection

[0174]

[0175] 2. Expression of collagen type I (COL-I), and the results are shown in Table 8: The expression levels of collagen type I in the TD-1 / active peptide double-modified exosome group and the mixed treatment group were significantly higher than those of the other three groups (P<0.01), and the TD-1 / active peptide double-modified exosome group was better than the mixed treatment group.

[0176] Table 8 Detection results of collagen I (COL-I) expression

[0177]

[0178] 3. Expression of hyaluronan synthase (HAS2), and the results are shown in Table 9: The expression levels of HAS2 in the TD-1 / active peptide dual-modified exosome group and the combined treatment group were significantly higher than those in the other three groups (P<0.01), and the TD-1 / active peptide dual-modified exosome group was better than the combined treatment group.

[0179] Table 9 Detection results of hyaluronan synthase (HAS2) expression

[0180]

[0181] 4. Expression of antioxidant genes (SOD, CAT), and the results are shown in Table 10: The expressions of antioxidant genes SOD and CAT in the TD-1 / active peptide dual-modified exosome group and the combined treatment group were significantly higher than those in the other three groups (P<0.01), and the TD-1 / active peptide dual-modified exosome group was better than the combined treatment group.

[0182] Table 10 Detection results of antioxidant gene (SOD, CAT) expression

[0183]

[0184] The above results indicate that the TD-1 / active peptide dual-modified exosome group and the combined treatment group significantly promoted the proliferation of HDFs cells. The expression levels of collagen I and hyaluronan synthase in the TD-1 / active peptide dual-modified exosome group and the combined treatment group were significantly higher than those in other groups, indicating that these two groups had an obvious promoting effect on skin repair and anti-aging. The expression levels of antioxidant genes (such as SOD, CAT) were the highest in the TD-1 / active peptide dual-modified exosome group and the combined treatment group, which might have a positive effect on anti-aging and promoting cell repair. At the same time, the TD-1 / active peptide dual-modified exosome group was better than the combined treatment group.

[0185] 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 other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims

1. An active peptide, characterized in that The full-length sequence of the active peptide is (GPO)5-K(R-Dopa)GD-[GHK(Cu 2 + )]2-(TPP)3-TAT-NH2, wherein Dopa = 3,4-dihydroxyphenylalanine, TPP = triphenylphosphine cation, GPO = Gly-Pro-Hyp.

2. The active peptide according to claim 1, characterized in that The molecular weight of the active peptide is 4523.6 Da.

3. A stem cell exosome, characterized in that: The exosomes are TD-1 / active peptide double-modified exosomes, wherein the active peptide is the active peptide described in claim 1.

4. The exosome according to claim 3, characterized in that The amino acid sequence of TD-1 is ACSSSPSKHCG.

5. The exosome according to claim 3, characterized in that The particle size distribution of the exosomes is 90±5nm, the surface point is -25±2mV, the modification efficiency is TD-1≥82%, and the active peptide is ≥75%.

6. A composition of active peptides and stem cell exosomes, characterized in that: The composition comprises the active peptide and exosomes according to claim 1, wherein the content of the active peptide is 5 mg / mL, and the content of the exosomes is 1×1010 particles / mL.

7. Use of the active peptide according to claim 1 in preparing an active peptide and stem cell exosome composition.

8. Use of the stem cell exosomes as claimed in claim 3 in the preparation of a drug for improving skin aging.

Images