Fusion exosome-like vesicle based on stem cell exosome and application of fusion exosome-like vesicle

Abstract

The invention relates to a fused exosome-like vesicle based on stem cell exosomes and application thereof, and the fused exosome-like vesicle is prepared by a method comprising the following steps: mixing stem cell exosomes and aloe exosome-like vesicles under an ultrasonic condition, and incubating to obtain the fused exosome-like vesicle. The fused exosome-like vesicle can effectively promote percutaneous penetration of stem cell exosome, and can effectively improve cell damage caused by ultraviolet rays, regenerate and repair skin damage barriers such as basilar membranes and the like, resist inflammation and oxidation, effectively remove melanin, and improve the skin quality. The composition has good repair potentials for chloasma and skin damage, aging and melanin deposition related to chloasma. Sufficient data support and technical support are provided for research and development of stem cell exosome innovative drugs, and clinical application of the stem cell exosome innovative drugs is accelerated.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a fusion exosome-like vesicle based on stem cell exosomes and an application thereof. Background Art

[0002] Melasma is a chronic, acquired facial hyperpigmentation skin disease, clinically manifested as light brown or dark brown patches with different depths and unclear boundaries symmetrically distributed on the cheeks, forehead and mandible. The pathogenesis of melasma is complex, and there is currently no ideal treatment method. It has a serious negative impact on the patient's life, psychology and spirit, and is known as the "king of spots". The consensus of experts in the diagnosis and treatment of melasma shows that the onset of melasma is related to factors such as genetic susceptibility, sunlight exposure, changes in sex hormone levels, increased melanin synthesis, inflammatory response, and damaged skin barrier. The latest research has found that there is basement membrane damage in the lesions of melasma. Corresponding histopathological findings show that melanocytes break through the basement membrane zone and hang down on the papillary dermis. At the same time, mast cells under the basement membrane act on epidermal melanocytes to produce more melanin, which aggravates the difficulty of treating melasma. In the past, the treatment of melasma mainly focused on inhibiting melanin formation and enhancing melanin metabolism. The discovery of chloasma's pathological histology has brought new breakthroughs in the clinical treatment of chloasma.

[0003] Stem cell exosomes (SC-Exos) are lipid bilayer vesicles of uniform size and molecular diameter of 30-150 nanometers that are formed by stem cells through the regulatory process of "endocytosis-fusion-extromission" and actively secreted to the extracellular space. SC-Exos have most of the functions of the stem cells from which they are derived. Because they have no nucleus, they are safer and easier to store and transport, making clinical transformation more convenient. In the field of plastic surgery, SC-Exos have the effects of improving tissue regeneration and repair potential, resisting skin photoaging, anti-inflammatory and antioxidant, inhibiting tyrosinase activity, and inhibiting melanin production, so they have broad research and application prospects in the field of skin beauty and anti-aging.

[0004] However, due to the barrier function of the skin, SC-Exos cannot penetrate the skin well through the skin. In particular, SC-Exos are obtained in small quantities and are relatively precious, and the barrier function of the skin will cause unnecessary waste. Currently, most animal models use local injections of SC-Exos to treat melasma, which has problems such as high pain, poor diffusion of exosomes, and a long recovery period. While the commonly used chemical penetration enhancement or pharmaceutical penetration enhancement methods help drugs overcome the skin barrier to achieve effective penetration, the possibility of skin irritation or damage or affecting the properties of the drug has also become a major problem. Summary of the invention

[0005] In view of the deficiencies of the prior art, the object of the present invention is to provide a fusion exosome-like vesicle based on stem cell exosomes and its application.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a fused exosome-like vesicle based on stem cell exosomes, wherein the fused exosome-like vesicle is prepared by a method comprising the following steps:

[0008] The stem cell exosomes and the aloe exosome-like vesicles are mixed and incubated under ultrasonic conditions to obtain the exosomes.

[0009] The technical problem to be solved by the present invention is to overcome the problems that stem cell exosomes are difficult to break through the skin barrier and have poor absorption effect during the current transdermal administration of stem cell exosomes, and to get rid of the problems of high pain, inconvenience in use, and impact on drug performance of existing penetration-enhancing means. Fusion exosome-like vesicles based on stem cell exosomes are prepared to achieve direct smear-type administration to promote transdermal penetration of stem cell exosomes, improve the therapeutic performance of stem cell exosomes, and achieve comfortable skin care at the living level and at home.

[0010] Preferably, the temperature of the ultrasound is 0-4°C, the time is 25-35 min, and the power is 180-220W.

[0011] The temperature can be selected as 0℃, 0.5℃, 1℃, 1.5℃, 2℃, 2.5℃, 3℃, 3.5℃, 4℃, etc. The time can be selected as 25min, 26min, 27min, 28min, 29min, 30min, 31min, 32min, 33min, 34min, 35min, etc. The power can be selected as 180W, 185W, 190W, 195W, 200W, 205W, 210W, 215W, 220W, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be elaborated here.

[0012] Preferably, the incubation temperature is 35-40°C and the incubation time is 55-65 min.

[0013] The temperature can be selected as 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, etc. The time can be selected as 55min, 56min, 57min, 58min, 59min, 60min, 61min, 62min, 63min, 64min, 65min, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be elaborated here.

[0014] Preferably, the stem cell exosomes are mesenchymal stem cell-derived exosomes.

[0015] Preferably, the ratio of the number of stem cell exosomes to the number of aloe exosome-like vesicles is (2-4):1.

[0016] The specific point values ​​in (2-4) can be selected as 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, etc. Other specific point values ​​within the above numerical range can be selected and will not be listed here one by one.

[0017] Preferably, the stem cell exosomes are prepared by a method comprising the following steps:

[0018] (1) centrifuging the mesenchymal stem cell culture medium and removing the precipitate to obtain a cell supernatant;

[0019] (2) The cell supernatant is centrifuged and collected. After filtering, the filtrate is subjected to ultracentrifugation and the precipitate is resuspended to obtain the product.

[0020] Preferably, the centrifugation speed in step (1) is 300-500 xg, the time is 8-12 min, and the temperature is 0-4°C.

[0021] The speed can be selected as 300xg, 320xg, 350xg, 380xg, 400xg, 420xg, 450xg, 480xg, 500xg, etc. The time can be selected as 8min, 8.5min, 9min, 9.5min, 10min, 10.5min, 11min, 11.5min, 12min, etc. The temperature can be selected as 0℃, 0.5℃, 1℃, 1.5℃, 2℃, 2.5℃, 3℃, 3.5℃, 4℃, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be elaborated here.

[0022] Preferably, the centrifugation and collection of the supernatant in step (2) are performed twice, the first centrifugation speed is 2000-3000 xg, the time is 8-12 min; the second centrifugation speed is 10000-15000 xg, the time is 25-35 min.

[0023] The speed of the first centrifugation can be selected from 2000xg, 2100xg, 2200xg, 2300xg, 2400xg, 2500xg, 2600xg, 2700xg, 2800xg, 2900xg, 3000xg, etc., and the time can be selected from 8min, 8.5min, 9min, 9.5min, 10min, 10.5min, 11min, 11.5min, 12min, etc.; the speed of the second centrifugation can be selected from 10000xg, 105 00xg, 11000xg, 11500xg, 12000xg, 12500xg, 13000xg, 13500xg, 14000xg, 14500xg, 15000xg, etc. The time can be selected as 25min, 26min, 27min, 28min, 29min, 30min, 31min, 32min, 33min, 34min, 35min, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be described here one by one.

[0024] Preferably, the speed of ultracentrifugation is 100000-150000 xg, and the time is 60-80 min.

[0025] The rotation speed can be selected as 100000xg, 105000xg, 110000xg, 115000xg, 120000xg, 125000xg, 130000xg, 135000xg, 140000xg, 145000xg, 150000xg, etc., and the time can be selected as 60min, 62min, 65min, 68min, 70min, 72min, 75min, 78min, 80min, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be elaborated here.

[0026] Preferably, the aloe exosome-like vesicles are prepared by a method comprising the following steps:

[0027] The aloe vera juice is centrifuged and the supernatant is collected, the supernatant is subjected to ultracentrifugation, and the obtained precipitate is resuspended to obtain the product.

[0028] Preferably, the centrifugation and collection of the supernatant are performed four times in total, the first centrifugation speed is 300-500xg, the time is 10-20min; the second centrifugation speed is 2000-3000xg, the time is 15-25min; the third centrifugation speed is 10000-15000xg, the time is 50-70min; the fourth centrifugation speed is 40000-50000xg, the time is 50-70min.

[0029] The speed of the first centrifugation can be selected from 300xg, 320xg, 350xg, 380xg, 400xg, 420xg, 450xg, 480xg, 500xg, etc., and the time can be selected from 10min, 12min, 14min, 16min, 18min, 20min, etc.; the speed of the second centrifugation can be selected from 2000xg, 2100xg, 2200xg, 2300xg, 2400xg, 2500xg, 2600xg, 2700xg, 2800xg, 2900xg, 3000xg, etc., and the time can be selected from 15min, 18min, 20min, 22min, 25min, etc.; the speed of the third centrifugation can be selected from 10000xg, 10500xg, 11000xg, 11500xg, 12000xg, 12500 The speed of the fourth centrifugation can be selected as 40000xg, 41000xg, 42000xg, 43000xg, 44000xg, 45000xg, 46000xg, 47000xg, 48000xg, 49000xg, 50000xg, etc., and the time can be selected as 50min, 52min, 55min, 58min, 60min, 62min, 65min, 68min, 70min, etc. Other specific point values ​​within the above numerical range can be selected, which will not be repeated here.

[0030] Preferably, the speed of the ultracentrifugation is 100000-150000 xg, and the time is 1.5-2.5 h.

[0031] The speed can be selected as 100000xg, 110000xg, 115000xg, 120000xg, 125000xg, 130000xg, 135000xg, 140000xg, 145000xg, 150000xg, etc., and the time can be selected as 1.5h, 1.8h, 2h, 2.2h, 2.5h, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be elaborated here.

[0032] In a second aspect, the present invention provides a use of the fused exosome-like vesicles based on stem cell exosomes according to the first aspect in the preparation of a drug for improving / treating chloasma.

[0033] The fused exosome-like vesicles obtained by the present invention can overcome the problems of poor treatment effect, easy recurrence, low patient compliance, and low satisfaction in existing chloasma treatment methods. In the past, the treatment of chloasma mainly focused on inhibiting melanin formation and enhancing melanin metabolism. The vesicles prepared by the present application can also regenerate and repair damaged skin barriers such as basement membranes, resist inflammation and oxidation, and strengthen the skin, solving the problem of chloasma treatment from the root. After treatment with this fused exosome-like vesicle based on stem cell exosomes, it can more effectively promote collagen regeneration, remove melanin particles, inhibit the expression of inflammatory factors, repair DNA damage, and promote basement membrane repair, and the therapeutic effect is significant, which is better than simple stem cell exosomes, aloe exosome-like vesicles and traditional tranexamic acid treatment.

[0034] In a third aspect, the present invention provides a use of the fused exosome-like vesicles based on stem cell exosomes according to the first aspect in the preparation of a drug for improving / treating skin barrier damage, aging or melanin deposition.

[0035] Preferably, the dosage form of the drug includes powder, tablet or granule.

[0036] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0037] In a fourth aspect, the present invention provides a use of the fused exosome-like vesicles based on stem cell exosomes according to the first aspect in the preparation of a cellular melanin inhibitor.

[0038] According to the research results of the present invention, the fused exosome-like vesicles of stem cell exosomes can inhibit the production of melanin at the cellular level (in vitro level) and clear melanin, that is, the fused exosome-like vesicles of stem cell exosomes can be made into a simple experimental preparation for exploring the metabolic process of cells. The cell melanin inhibitor claimed for protection by the present invention is not used to eliminate the cause or lesion, that is, it is an application in the preparation of a cell melanin inhibitor for non-therapeutic purposes.

[0039] In a fifth aspect, the present invention provides a use of the fused exosome-like vesicles based on stem cell exosomes according to the first aspect in the preparation of a ROS generation inhibitor.

[0040] According to the research results of the present invention, the fused exosome-like vesicles of stem cell exosomes can inhibit the generation of ROS at the cellular level (in vitro level), that is, the fused exosome-like vesicles of stem cell exosomes can be made into a simple experimental preparation for exploring the metabolic process of cells. The ROS generation inhibitor claimed by the present invention is not used to eliminate the cause or lesion, that is, it is an application in the preparation of ROS generation inhibitors for non-therapeutic purposes.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] The fusion exosome-like vesicles based on stem cell exosomes of the present invention can effectively promote the percutaneous penetration of stem cell exosomes, improve the biological activity of stem cell exosomes, and show good potential in the treatment of transdermal skin diseases such as chloasma. It can be used to resist skin oxidative stress, anti-inflammatory, and inhibit melanin production. More importantly, it can effectively exert the regeneration and repair ability of stem cell exosomes, promote skin collagen regeneration, and repair damaged basement membranes of the skin, bringing new treatment approaches such as high comfort and low recurrence rate to refractory skin diseases. It is expected to accelerate the clinical application of innovative stem cell exosome drugs, and is conducive to the customized development of "innovative stem cell exosome drugs" that are easy to use, reduce anti-blackening and recurrence, and improve the quality of treatment of chloasma and related skin damage, aging, melanin deposition and other diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Schematic diagram of the preparation process of fusion vesicles.

[0044] Figure 2 The verification results of fusion vesicles, where (a) is the fluorescence imaging of each group of vesicles in a super-resolution laser scanning confocal microscope, (b) is a schematic diagram of fluorescence resonance energy transfer of fusion vesicles, (c) is the ultraviolet absorption spectrum characterization of DiO / gAELVs, DiI / Exos and gAEs, and (d) is the fluorescence spectrum characterization of DiO / gAELVs, DiI / Exos and gAEs.

[0045] Figure 3 Figure 1 is the characterization result of fusion vesicles, where Figure a is the potential characterization of each group of vesicles, Figure b is the particle size analysis of each group of vesicles, Figure c is the morphology structure of each group of vesicles characterized by transmission electron microscopy, and Figure d is the characterization of CD63 and TSG101 in the vesicles.

[0046] Figure 4 The penetration depth of fusion vesicles with different ratios was observed using laser confocal microscopy.

[0047] Figure 5 Laser confocal microscopy was used to characterize the uptake of three types of vesicles by HaCaT.

[0048] Figure 6 The graphs show the antioxidant activity results of each vesicle, where Figure a shows the changes in fluorescence spectra after gAELVs, Exos, and gAEs as antioxidants react with AAPH and FL, and Figure b shows the recovery of the fluorescence signal of FL after the action of vesicles of different concentrations.

[0049] Figure 7 Analysis of the scavenging effect of each group of vesicles on intracellular ROS.

[0050] Figure 8These are the effect diagrams of the repair ability of each vesicle on human keratinocytes damaged by ultraviolet light, where Figure a shows the morphological observation of cells damaged by ultraviolet irradiation under the intervention of each group of vesicles under an optical microscope, Figure b shows the repair effect of each group of vesicles on cells damaged by ultraviolet light by Calcein-AM / PI staining, Figure c shows the cell apoptosis rate fitting of cell live-dead staining, and Figure d shows the MTT method analysis of the repair effect of each group of vesicles on cells damaged by ultraviolet light.

[0051] Fig. 9 These are the effects of various vesicles on the migration performance of human keratinocytes, where Figure a shows the cell migration under the action of each group of vesicles, and Figure b shows the migration rate of each group of cells fitted by Image J.

[0052] Fig.10 Figure 2 is the inhibitory effect of each vesicle on the melanin of melanoma cells B16F10, where Figure a shows the melanin clearance in cells under the action of each group of vesicles, and Figure b shows the tyrosinase activity analysis in cells under the action of each group of vesicles.

[0053] Fig.11 These are graphs showing the effects of each group of vesicles on the expression of type IV collagen in human keratinocytes.

[0054] Fig.12 Figure 3 is the effect diagram of each group of vesicles on zebrafish melanin clearance, reactive oxygen species clearance and zebrafish uptake, among which Figure a is the analysis of zebrafish uptake of each group of vesicles, Figure b is the in vivo imaging of zebrafish after the action of each group of vesicles, Figure c is the change of melanin content in zebrafish of each group, Figure d is the ROS fluorescence imaging of zebrafish under the action of each group of drugs, and Figure e is the fitting of ROS clearance in zebrafish under the action of each group of drugs.

[0055] Fig.13 This is a diagram showing the effect of each group of vesicles on the repair of skin tissue in chloasma model mice after HE staining analysis. Figure a is the HE staining result, and Figure b is the imageJ fitting of the epidermal thickness of each group of skin.

[0056] Fig.14 Masson and PAS staining were used to analyze the effects of each group of vesicles on promoting collagen regeneration and basement membrane repair in the melasma mouse model. Figure a is the result of Masson staining analysis, and Figure b is the result of PAS staining analysis.

[0057] Fig.15 These are the effects of each group of vesicles in clearing melanin particles in the chloasma mouse model, where Figure a is the melanin staining result, and Figure b is the analysis of melanin content in the skin tissue of each group.

[0058] Fig.16These are the effects of various vesicles on the DNA damage repair of the chloasma mouse model, where Figure a is the result of p53 immunofluorescence staining, and Figure b is the p53 fluorescence fitting analysis in the skin tissues of each group.

[0059] Fig.17 These are the effects of various vesicles on the repair of inflammatory damage in the melasma mouse model, where Figure a is the CD45 staining result, Figure b is the TNF-α staining result, Figure c is the IL-1β staining result, Figure d is the CD45 signal in the skin tissue fitted by Image J, Figure e is the TNF-α signal in the skin tissue fitted by Image J, and Figure f is the IL-1β signal in the skin tissue fitted by Image J. DETAILED DESCRIPTION

[0060] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0061] The reagents and raw materials used in the present invention are commercially available.

[0062] Preparation Example 1

[0063] This preparation example provides a method for preparing mesenchymal stem cell-derived exosomes (hUCMSC-Exos), the preparation method comprising:

[0064] When the adherent cells fill 70%-80% of the bottom area of ​​the culture flask, wash with PBS and replace the culture medium with DMEM culture medium without fetal bovine serum for 48 hours. After 48 hours, collect the cell supernatant in the culture flask into a 50mL centrifuge tube and perform the following centrifugation operation:

[0065] (1) Centrifuge the cell culture medium at 4°C at 400 × g for 10 min to remove the precipitate;

[0066] (2) Take the supernatant obtained in step (1) and centrifuge it at 4°C, the speed of the centrifuge is 2500×g, and the time is 10 min; take the supernatant and centrifuge it at 4°C, the speed of the centrifuge is 13000×g, and the time is 30 min; take the supernatant and filter it through a 0.22μm filter to remove impurities, and place the filtrate in an ultraspeed refrigerated centrifuge for centrifugation; the temperature of the centrifugation is 4°C, the speed is 120000×g, and the time is 70 min; the supernatant is completely discarded, the precipitate is taken, resuspended with PBS, and placed in an ultraspeed refrigerated centrifuge for centrifugation again, the temperature of the centrifugation is 4°C, the speed is 120000×g, and the time is 70 min, the precipitate is resuspended three times with sterile PBS, the resuspension is collected in a 1.5mL EP tube, and stored in a -80°C refrigerator for subsequent experiments.

[0067] Preparation Example 2

[0068] This preparation example provides a method for preparing aloe exosome-like vesicles, the preparation method comprising:

[0069] Peel the fresh aloe vera and homogenize in a high-speed blender at 4°C for 1 minute. Collect the juice and filter through a paper filter. Centrifuge the filtrate at 400xg for 15 minutes, collect the supernatant and centrifuge at 2500xg for 20 minutes, collect the supernatant and centrifuge at 12000xg for 1 hour, collect the supernatant and centrifuge at 45000xg for 1 hour, collect the supernatant and perform ultracentrifugation at a temperature of 4°C, a speed of 120000xg, and a time of 2 hours. The isolated aloe exosome-like vesicles are further purified in a sucrose gradient (8%, 30%, 45%, 60% sucrose in 20mM Tri-Cl, pH 7.2).

[0070] Example 1

[0071] Preparation of fusogenic vesicles

[0072] Exos (mesenchymal stem cell derived exosomes) and gAELVs (aloe exosome-like vesicles) were broken and recombined at low temperature by ultrasound, the ultrasound temperature was 4°C, the time was 30 minutes, the power was 200W; and then incubated at 37°C for 60 minutes. The vesicles were better fused to obtain fused exosome-like vesicles. The results are as follows Figure 1 shown.

[0073] Example 2

[0074] Validation and characterization of fusogenic vesicles

[0075] Vesicles were labeled with two membrane dyes, DiO and DiI, respectively, to obtain DiO / gAELVs and DiI / Exos. Fusion vesicles were prepared using the method described in Example 1, and their fusion was observed under a super-resolution laser scanning confocal microscope. The results are as follows: Figure 2 As shown in Figure a, after the two vesicles fuse, the fluorescence they carry can be effectively integrated and superimposed to form yellow fluorescence. In addition, due to the unique luminescence characteristics between the two fluorescent molecules DiO and DiI, when the Forster radius between the two molecules is less than 10nm, the resonance energy transfer effect is easy to occur, such as Figure 2 As shown in Figure b. When the dye concentration of each vesicle is the same, the fluorescence spectrum is characterized. It is found that when the two vesicles fuse, the fluorescence signal of DiO inside them decreases significantly, while the fluorescence signal of DiI increases. This indicates that under the action of the excitation light, due to the shortened distance, the energy of DiO is transferred to the DiI molecule, as shown in Figure 2. Figure 2 As shown in Figures c and d. The above results show that this method can effectively promote the fusion of two vesicles, providing an effective guarantee for subsequent research and application.

[0076] In order to clarify the properties of the prepared fusion vesicles (gAEs), the particle size, potential distribution and morphology of the gAEs were characterized by laser particle size analyzer and transmission electron microscope. Figure 3 As shown in Figure a, the potential of gAELVs is -4.6mV, compared with -11.5mV for Exos. After the two vesicles fuse, the potential of gAEs is -7.5mV, which effectively combines the electronegativity of the two. The particle size after fusion is 116.6nm, which is not significantly different from gAELVs (110.7nm) and Exos (124.2nm). Figure 3 Figure b). Under a transmission electron microscope, it can be observed that all three vesicles have a complete lipid bilayer structure, indicating that the fusion of the vesicles did not cause significant damage to their structure ( Figure 3 (c in Figure ).

[0077] Western blotting was used to characterize the exosome marker proteins CD63 and TSG101 confirmed in the guidelines of the International Extracellular Vesicle Association. The results showed that Exos were rich in the above two markers, while gAELVs had no obvious protein bands due to different species. The gAEs obtained by fusing the two vesicles effectively carried CD63 and TSG101 ( Figure 3 This further indicates the successful fusion of the two vesicles.

[0078] In summary, under super-resolution fluorescence microscopy, the two fused vesicles showed obvious fluorescence co-localization. The fused vesicles expressed the proteins CD63 and TSG101 expressed by stem cell exosomes. The particle size of the fused vesicles was about 116.6 nm. Under electron microscopy, it showed the saucer-like structure of exosomes with a complete lipid layer, which was consistent with the structural characteristics of exosome-like vesicles.

[0079] Example 3

[0080] Transdermal permeation study of each vesicle

[0081] To explore the percutaneous permeability of Exos and the changes after fusion with gAELVs, the vesicles were labeled with DiO and applied to the skin surface of depilated mice at a drop size of 1×10 9 The penetration of vesicles with different fusion ratios in the skin was observed using a laser confocal microscope when the drop-coating time was equal. Figure 4 As shown in the figure, the skin penetration depth of Exos is about 100 μm, penetrating only into the cortex. As the fusion ratio with gAELVs increases, the penetration depth gradually deepens. When the ratio of gAELVs to Exos particles is 1:3, the penetration depth is nearly 400 μm, reaching the dermis, indicating that the fused vesicles can increase the penetration effect.

[0082] Example 4

[0083] Analysis of the uptake of various vesicles by human keratinocytes

[0084] Cellular uptake is the key to drug therapy. In order to investigate whether gAEs can be taken up by human keratinocytes, they were labeled with the green fluorescent dye DiO. DiO / gAELVs, DiO / Exos and DiO / gAEs were prepared and co-incubated with human epidermal cells (HaCaT) to observe their uptake.

[0085] The results are as follows Figure 5 As shown, after each group of vesicles was incubated with cells for 12 hours, obvious green fluorescent spots were visible in the cytoplasm. DiO, as a commonly used cell membrane dye, is usually bound to the cell membrane in its free state and does not enter the cytoplasm (Park S, Kim H K. Development of skin-permeable flexible liposomes using ergosterol esters containing unsaturated fatty acids [J]. Chemistry and Physics of Lipids, 2023, 250: 105-270.). Therefore, it can be inferred that the DiO green fluorescent spots in the cytoplasm are mainly carried and transported to the cytoplasm by each group of vesicles as carriers. This result shows that gAELVs, as plant-derived nanovesicles, can be taken up by epidermal cells like Exos, and have a good cross-species effect. The fusion of the two does not affect its internalization by cells. The above results provide an effective guarantee for its use as a therapeutic agent for subsequent damaged cell treatment.

[0086] Example 5

[0087] Study on the antioxidant activity of each vesicle

[0088] Cells also produce oxidative stress under ultraviolet irradiation, producing a large amount of reactive oxygen, which in turn damages organelles and genetic material, leading to cell apoptosis. In addition to being rich in a variety of lipids that maintain the stability of the vesicles, gAELVs also contain a variety of antioxidant active ingredients such as aloe-emodin, aloesin, and β-sitosterol, which may give gAEs excellent antioxidant activity after fusion with Exos. To this end, the antioxidant activity was analyzed using the oxygen radical absorbance capacity method (ORAC) (Xiao J, Feng S, Wang X, et al. Identification of exosome-like nanoparticle-derived microRNAs from 11edible fruits and vegetables [J]. Peer J, 2018, 6: e5186.). AAPH, as a peroxyl radical generator, can oxidize sodium fluorescein (FL) and quench its fluorescence. When antioxidants are present, they can competitively bind to peroxyl radicals, thereby inhibiting the fluorescence quenching phenomenon caused by FL oxidation. Figure 6 As shown in Figure a, FL emits obvious fluorescence signals under 480nm excitation. When AAPH is added, its fluorescence is rapidly quenched. In the presence of gAELVs, Exos and gAEs, the fluorescence signal of FL recovers to varying degrees. The antioxidant activity of gAEs is significantly higher than that of Exos. As the concentration of vesicles increases, the fluorescence signal of FL gradually recovers. At 1×10 9 At a concentration of 100 / mL, the efficiency of Exos in promoting fluorescence recovery was only 60%, while the fluorescence recovery rates of gAELVs and gAEs groups were 100% and 90% ( Figure 6 (b) This shows that after fusion with gAELVs, gAEs have better peroxyl radical scavenging performance and can play a good antioxidant role.

[0089] Example 6

[0090] Study on the anti-reactive oxygen generation of each vesicle

[0091] Based on the premise that gAEs can scavenge oxygen free radicals in vitro, we further explored their ability to scavenge reactive oxygen species (ROS) produced by oxidative stress in human keratinocytes after UV irradiation. The DCFH-DA fluorescent probe was used to characterize the ROS in the cells.

[0092] Test method: Each group of human keratinocytes was irradiated with UV for 15 minutes, and then gAELVs, Exos, and gAEs were administered at a dosage of 1×10 9particles / mL, and after co-cultured with human keratinocytes for 24 h, each group of cells was given 1 mL of 5 μM DCFH-DA solution and incubated in the dark for 30 min, and the results were observed under an inverted fluorescence microscope.

[0093] The results are as follows Figure 7 As shown in the figure, a large amount of ROS is generated in the cells under the action of ultraviolet light, which oxidizes the intracellular DCFH into DCF, showing obvious green fluorescence (+UV) under 488nm laser. After ultraviolet irradiation, the intracellular ROS level of cells incubated with vesicles in each group decreased significantly, and the fluorescence signal of DCFH oxidation caused by ROS was lower than that of the +UV group, and showed a certain concentration dependence. The above results show that gAEs exert good antioxidant activity in cells, can effectively inhibit the generation of intracellular ROS caused by ultraviolet-induced oxidative stress, and are beneficial to ultraviolet-damaged human keratinocytes, thereby avoiding the proliferation of melanocytes and the aggravation of inflammatory response caused by oxidative stress transmission.

[0094] Example 7

[0095] Study on the repair ability of various vesicles on human keratinocytes damaged by ultraviolet rays

[0096] Oxidative stress induced by UV damage may further damage human keratinocytes, leading to cell apoptosis, and thus affecting skin homeostasis. To study the repair effect of gAEs on damaged cells, the effects of UV irradiation (+UV) and the administration of gAELVs, Exos, and gAEs on cells after UV irradiation were investigated, and compared with tranexamic acid (TXA), a commonly used clinical drug for the treatment of melasma.

[0097] The results showed that compared with the blank group (Blank) cells, the +UV group cells changed from an adherent state to a nearly round shape, with more vacuoles, which may be a precursor to cell apoptosis. Under the action of drugs, this phenomenon was significantly reduced, and more cells in the gAEs group recovered to an adherent state ( Figure 8 To further verify whether this phenomenon is cell apoptosis under UV irradiation, Calcein-AM / PI staining was used for evaluation. Figure 8 In Figure b, obvious green fluorescence (live cell staining) can be seen in the cells of the blank treatment group (Blank) without ultraviolet intervention. In contrast, the cells in the +UV group showed obvious red fluorescence (dead cell staining), and the number of live cells was significantly reduced. Under the intervention of other drug groups, the proportion of red fluorescence gradually decreased, indicating that more damaged cells survived under the action of the drug. The apoptosis rate of cells in each group was fitted, and it was found that the apoptosis rate of cells in the gAEs treatment group was the lowest (<10%), which was not significantly different from that of the blank group, indicating that it has a good repair effect. In addition, the MTT method was used to further verify its repair performance. Figure 8As shown in Figure c, compared with the Blank group, the cell survival rate in the +UV group was only 50%, and under the action of each drug group, different degrees of repair were shown. The repair effect of each group of vesicles was higher than that of TXA, and gAEs had the strongest repair effect on damaged cells, restoring cell viability to more than 90%, which was basically consistent with the results of the live-dead cell staining experiment, further indicating that gAEs has excellent repair potential for UV-damaged cells.

[0098] Example 8

[0099] Study on the effect of various vesicles on the migration performance of human keratinocytes

[0100] The migration ability of keratinocytes is an effective indicator of their functional activity. In order to investigate the changes in the migration ability of UV-damaged cells under the action of each drug group, the cell scratch method was used to evaluate the migration of cells after treatment. Fig. 9 As shown in Figure a, cells in the Blank group (cultured in RPMI medium without drug intervention) have obvious migration behavior, and the scratches gradually close over time. The migration ability of cells in the +UV group is significantly inhibited. Under the action of TXA and vesicles in each group, the migration ability of UV-damaged cells is restored. Among them, gAEs has the best repair effect on the migration of damaged cells. The scratch area of ​​each drug group at each time point was fitted, and the closure rate was calculated. Fig. 9 As shown in Figure b, after 48 hours, the cell migration rate of the gAEs group exceeded 90%, which was significantly different from the other groups. The above studies show that under the repair effect of gAEs, the migration ability of epidermal cells damaged by UV is significantly restored, which is of great significance for repairing UV-damaged skin.

[0101] Example 9

[0102] Inhibitory effects of various vesicles on melanin production in melanoma cells B16F10

[0103] In addition to cell damage caused by oxidative stress, melanin deposition is also one of the main manifestations of melasma. Effectively removing melanin and inhibiting its production are important for the treatment of melasma. In order to explore the scavenging effect of each group of drugs on melanin, B16F10 cells that are prone to melanin production were used as a model and incubated with TXA, Exos, gAELVs, and gAEs for 48 hours to monitor the changes in melanin content. Fig.10As shown in Figure a, the cell extracts of the untreated group contained a large amount of melanin, while under the action of each group of drugs, the melanin in the cells was significantly reduced. The tyrosinase activity in the cells was measured and it was found that the decrease in melanin in the cells was closely related to its tyrosinase activity. In the cells intervened by TXA, gAELVs and Exos, the tyrosinase activity decreased by 50%, and gAEs showed a more obvious tyrosinase activity inhibition, with an inhibition rate of 88% ( Fig.10 The above results show that gAEs have good melanin scavenging performance and are expected to efficiently remove melanin in chloasma skin and inhibit the formation of melanin, which is of great significance for inhibiting the recurrence of chloasma.

[0104] Example 10

[0105] Effects of various vesicles on the expression of type IV collagen in human epidermal cells

[0106] The destruction of the basement membrane is one of the typical histological features of melasma. Effective repair of the basement membrane is an effective means to treat and inhibit the recurrence of melasma. The basement membrane contains abundant anchoring proteins, which can increase the cohesion between the dermis and the epidermis. Among them, collagen VII (COL4A1) is the most important structural component of the basement membrane, which largely determines the mechanical properties of the basement membrane. Therefore, this study took COL4A1 as the research object and used protein immunoblotting technology to analyze its expression in cells after ultraviolet irradiation.

[0107] like Fig.11 As shown in the results, after ultraviolet irradiation, the intracellular COL4A1 content decreased significantly, and its relative expression level increased under the action of each drug-treated group, among which the effect of gAEs was particularly significant. Combined with the above research results, it shows that its repair effect on ultraviolet-damaged cells plays an important role in maintaining the expression level of COL4A1 in cells, which provides a strong guarantee for its subsequent use in the repair of damaged basement membrane in chloasma skin.

[0108] Embodiment 11

[0109] Study on the effects of various vesicles on zebrafish melanin removal, reactive oxygen species removal and zebrafish uptake

[0110] As a new model organism, zebrafish has a genetic similarity of more than 85% with humans. Its melanocytes are mostly found in the basal layer of the epidermis, similar to human skin, and can be used as a good in vivo melanin clearance model. To further explore the melanin clearance effect of gAEs, each group of vesicles was fluorescently labeled with DiI and added to zebrafish culture medium to observe whether it can be taken up by zebrafish and exert its therapeutic effect. Fig.12As shown in Figure a, zebrafish can take in each group of vesicles through feeding and swallowing, showing obvious red fluorescence. On this basis, each group of vesicles was co-cultured with zebrafish, and after 4 days of administration, the melanin on the back of the zebrafish was observed. Fig.12 As shown in Figures b and c, the back of wild-type zebrafish has obvious melanin. Under the intervention of each group of drugs, the melanin content in the zebrafish is significantly reduced, among which the scavenging effect of gAEs on melanin is particularly significant, indicating that after taking gAEs, zebrafish can effectively remove melanin and inhibit the production of melanin in their bodies. In addition, an oxidative stress model was constructed by ultraviolet irradiation of zebrafish to evaluate the scavenging effect of each drug group on ROS in vivo. Fig.12 As shown in Figures d and e, a large amount of ROS was produced in the zebrafish in the UV-treated group, and the scavenging performance of gAEs on ROS was more significant compared with the other treatment groups. The above results show that gAEs have good potential in scavenging melanin and reactive oxygen species, and are expected to become an efficient and mild therapeutic agent for melasma.

[0111] Example 12

[0112] HE staining analysis of the effects of various vesicles on the repair of skin tissue in chloasma model mice

[0113] This study used ultraviolet light and hormone intervention in mice to construct a mouse chloasma skin model and explore the repair effect of gAEs on chloasma in living mice. H&E staining of skin tissue is a common pathological section staining method that can reveal changes in skin tissue structure [Song H, Meng M, Cheng X, et al. The effect of collagen hydrolysates from silver carp (Hypophthalmichthys molitrix) skin on UV-induced photoaging in mice: molecular weight affects skin repair [J]. Food & Function, 2017, 8 (4): 1538-46.]. H&E staining was used to observe changes in mouse chloasma skin tissue. Fig.13As shown, the stratum corneum of normal skin tissue is intact and the hair follicles in the dermis are arranged relatively regularly, while the model group showed different states after being treated with PBS, TXA, Exos, gAELVs and gAEs respectively. Among them, the epidermal thickness of the PBS-treated group increased significantly, abnormal elastic tissue accumulation was shown in the dermis, and highly curled and fragmented elastic fibers were observed. It is reported that 93% of melasma patients' skin tissue is prone to moderate or severe solar elastic fiber degeneration [Kwon SH, Hwang YJ, Lee SK, et al. Heterogeneous pathology of melasma and its clinical implications [J]. International journal of molecular sciences, 2016, 17 (6): 824.], and ultraviolet radiation promotes excessive proliferation of keratinocytes, thereby showing abnormal thickening of the epidermis [Brent AA, Mancuso E, Smith E. Considerations in geriatric dermatopathology [J]. International Journal of Dermatology, 2023, 62 (12): 1458-74.]. Transdermal administration of TXA had no obvious repairing effect on the typical characteristics of melasma skin, and the epidermal thickness had no significant difference compared with the PBS group. However, Exos, gAELVs, and gAEs showed different degrees of therapeutic effects. Among them, gAEs could effectively reduce the epidermal thickening caused by excessive proliferation of keratinocytes and reduce the degeneration of skin solar elastic fibers, indicating that it has excellent potential for treating melasma by transdermal administration.

[0114] Embodiment 13

[0115] Masson and PAS staining analysis of the effects of various vesicles on promoting collagen regeneration and basement membrane repair in the chloasma mouse model

[0116] In order to evaluate the regeneration and repair of skin collagen and basement membrane damage in each treatment group, skin collagen and basement membrane were stained using Masson and PAS, respectively. Fig.14 As shown in Figure a, Masson staining results show that normal skin tissue collagen is arranged in an orderly manner, and the basement membrane zone is obvious and complete. However, after PBS and TXA administration in the model group, the collagen is still relatively disordered and fragmented, and the basement membrane zone is blurred or broken. Exos, gAELVs and gAEs can effectively repair the orderliness of collagen arrangement and repair broken basement membranes. At the same time, PAS staining can easily observe that gAEs have the most significant effect on the repair of basement membranes ( Fig.14Figure b in [reference], the above results indicate that gAEs can more effectively repair the damaged basement membrane of chloasma skin during transdermal drug delivery, which plays an important role in preventing the deposition of melanin into the dermis.

[0117] Example 14

[0118] The situation of each vesicle clearing melanin granules in the chloasma mouse model

[0119] To observe the pigmentation in the chloasma skin tissue, the melanin in it was stained, and the results are as Fig.15 shown. There is no obvious melanin in the skin of normal mice, while a large amount of melanin is contained in both the epidermis and dermis of the skin in the model group treated with PBS. Among them, the accumulation of melanin in the dermis may be due to the damage of the basement membrane, which conforms to the typical characteristics of chloasma skin. The melanin clearance effect of the remaining drug administration groups shows a trend of TXA < gAELVs < Exos < gAEs. By fitting the melanin content in the tissue, it is found that there is a significant difference in the melanin content in the skin of the gAEs group compared with the PBS group, and there is no difference from normal skin, indicating that through transdermal penetration, gAEs can effectively combine the excellent characteristics of the two vesicles and play a good role in melanin clearance.

[0120] Example 15

[0121] The situation of each vesicle repairing DNA damage in the chloasma mouse model

[0122] Characterizing the repair effect of drugs on DNA damage in chloasma skin is crucial. For this reason, in this study, immunofluorescence staining was used to label p53 in the skin tissue (red fluorescence) to observe the DNA damage in each group. As Fig.16 shown, there is no obvious DNA damage in normal skin tissue, while a large amount of p53 fluorescence is presented in the PBS treatment group. The p53 fluorescence signals in the remaining drug administration groups show a trend of TXA < gAELVs < Exos < gAEs, indicating that with the intervention of drugs, the DNA damage in the skin tissue is repaired to varying degrees. Among them, the gAEs treatment group significantly reduces the expression of p53, indicating that it has a significant repair effect on DNA damage in chloasma skin, which is of great significance for enhancing cell activity, repairing the skin barrier, and reducing melanin synthesis.

[0123] Example 16

[0124] The situation of each vesicle repairing inflammatory damage in the chloasma mouse model

[0125] Under the action of ultraviolet rays, chloasma skin is also prone to inflammatory reactions, which promote the increase of inflammatory cells or the secretion of inflammatory factors, further weakening the stability of the skin basement membrane and stimulating melanin production. Therefore, this study conducted immunofluorescence staining of inflammatory factors CD45, TNF-α, and IL-1β to observe the inflammatory changes in skin tissue under the action of each drug group. Fig.17 Figure a), TNF-α( Fig.17 (b), IL-1β ( Fig.17 c) in the model group, while the model group still showed a large number of CD45, TNF-α, and IL-1β inflammatory factor signals after PBS intervention, indicating that a significant inflammatory response occurred in the chloasma skin and that it could not self-repair in the short term without drug intervention. In the skin tissue of the TXA, gAELVs, Exos, and gAEs administration groups, the expression of inflammatory factors was significantly reduced. The inflammatory factor signals in the skin of each group were fitted, and it was found that gAEs had the most significant inhibitory effect on inflammatory factors, indicating that it effectively inhibited the inflammatory response in the skin tissue while repairing damaged cells. This plays an important role in reducing a series of injuries caused by inflammation during the treatment of chloasma, and is expected to repair the damaged barrier of the chloasma skin during the treatment process and reduce the recurrence rate of chloasma.

[0126] The applicant declares that the present invention illustrates a fusion exosome-like vesicle based on stem cell exosomes and its application through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0127] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0128] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A fusion exosome-like vesicle based on stem cell exosomes, characterized in that: The fused exosome-like vesicles are prepared by a method comprising the following steps: The stem cell exosomes and the aloe exosome-like vesicles are mixed and incubated under ultrasonic conditions to obtain the exosomes.

2. The fusion exosome-like vesicle based on stem cell exosomes according to claim 1, characterized in that: The temperature of the ultrasound is 0-4°C, the time is 25-35min, and the power is 180-220W; Preferably, the incubation temperature is 35-40°C and the incubation time is 55-65 min.

3. The fusion exosome-like vesicle based on stem cell exosomes according to claim 1 or 2, characterized in that: The stem cell exosomes are exosomes derived from mesenchymal stem cells; Preferably, the ratio of the number of stem cell exosomes to the number of aloe exosome-like vesicles is (2-4):

1.

4. The fusion exosome-like vesicle based on stem cell exosomes according to any one of claims 1 to 3, characterized in that: The stem cell exosomes are prepared by a method comprising the following steps: (1) centrifuging the mesenchymal stem cell culture medium and removing the precipitate to obtain a cell supernatant; (2) centrifuging the cell supernatant and collecting the supernatant, filtering, ultracentrifuging the filtrate, and resuspending the precipitate to obtain; Preferably, the centrifugation speed in step (1) is 300-500 x g, the time is 8-12 min, and the temperature is 0-4° C.; Preferably, the centrifugation and collection of the supernatant in step (2) is performed twice, the first centrifugation speed is 2000-3000 xg, the time is 8-12 min; the second centrifugation speed is 10000-15000 xg, the time is 25-35 min; Preferably, the speed of ultracentrifugation is 100000-150000 xg, and the time is 60-80 min.

5. The fusion exosome-like vesicle based on stem cell exosomes according to any one of claims 1 to 4, characterized in that: The aloe exosome-like vesicles are prepared by a method comprising the following steps: The aloe vera juice is centrifuged and the supernatant is collected, the supernatant is subjected to ultracentrifugation, and the obtained precipitate is resuspended to obtain; Preferably, the centrifugation and collection of the supernatant are performed four times in total, the first centrifugation is performed at a speed of 300-500 xg for 10-20 min; the second centrifugation is performed at a speed of 2000-3000 xg for 15-25 min; the third centrifugation is performed at a speed of 10000-15000 xg for 50-70 min, and the fourth centrifugation is performed at a speed of 40000-50000 xg for 50-70 min; Preferably, the speed of the ultracentrifugation is 100000-150000 xg, and the time is 1.5-2.5 h.

6. Use of the fused exosome-like vesicles based on stem cell exosomes according to any one of claims 1 to 5 in the preparation of a drug for improving / treating chloasma.

7. Use of the fused exosome-like vesicles based on stem cell exosomes according to any one of claims 1 to 5 in the preparation of a drug for improving / treating skin barrier damage, aging or melanin deposition.

8. The use according to claim 6 or 7, characterized in that: The dosage form of the drug includes powder, tablet or granule; Preferably, the drug further comprises pharmaceutically acceptable excipients.

9. Use of the fused exosome-like vesicles based on stem cell exosomes according to any one of claims 1 to 5 in the preparation of a cellular melanin inhibitor.

10. Use of the fused exosome-like vesicles based on stem cell exosomes according to any one of claims 1 to 5 in the preparation of a ROS generation inhibitor.

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