Aloe exosome-like nano-vesicle capable of delaying skin photoaging as well as extraction method and application of aloe exosome-like nano-vesicle

Abstract

The invention discloses aloe exosome-like nano-vesicles capable of delaying skin photoaging and an extraction method and application thereof, and belongs to the technical field of exosome-like nano-vesicle extraction. The extraction method of the aloe exosome-like nano-vesicles is designed and optimized, the yield of the aloe exosome-like nano-vesicles is obviously improved, and the extracted aloe exosome-like nano-vesicles can obviously delay skin photoaging and have an obvious improvement effect on skin fibroblast aging damage caused by ultraviolet radiation; a potential treatment means is provided for preventing or treating actinic keratosis caused by ultraviolet radiation and further possible squamous cell carcinoma.

Description

Technical Field

[0001] The present invention belongs to the technical field of exosome-like nanovesicle extraction, and in particular relates to an aloe exosome-like nanovesicle capable of delaying skin photoaging, and an extraction method and application thereof. Background Art

[0002] Accumulation of senescent cells in prematurely aged skin caused by environmental factors such as ultraviolet (UV) radiation may lead to pathological changes in the skin and photoaging. The photoaging process is accompanied by changes in the phenotype of skin cells and structural and functional changes in extracellular matrix (ECM) components such as collagen and elastin. UV can also cause changes in dermal connective tissue and is a key reason for the appearance of skin aging characteristics. Therefore, there is an urgent need to develop new medical treatments to protect the skin from this environmental damage, especially for UVA-induced damage to dermal fibroblasts.

[0003] In recent years, mammalian exosomes have been extensively studied and found to play a vital role in regulating intercellular communication, thus affecting the development and progression of many diseases. However, the isolation of mammalian exosomes has always been costly, time-consuming, and low in yield. Compared with animals, plants do not have any zoonotic or human pathogens, suggesting that plant-derived exosomes may be more suitable for therapeutic use, showing great advantages due to their inherent low immunogenicity, low-cost production, and a wide range of production sources.

[0004] Aloe vera is a traditional Chinese medicine with a long history of medicinal use. It is rich in a variety of active substances and has many functions such as medical treatment, beauty, health care, food, and ornamental, so it is known as "all-purpose medicine", "natural beautician", "family doctor", etc. There are a few studies on the exosomes derived from aloe vera in anti-inflammatory, wound healing, and drug delivery. However, there are no reports on whether aloe vera-derived exosome-like nanovesicles can play a role in alleviating skin photoaging and damage.

[0005] Therefore, it is particularly important to provide a method for extracting aloe exosome-like nanovesicles with a high extraction yield, wherein the extracted aloe exosome-like nanovesicles can effectively alleviate the damage of dermal fibroblasts caused by ultraviolet rays. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a method for extracting aloe exosome-like nanovesicles, which can significantly improve the yield of aloe exosome-like nanovesicles.

[0007] Another object of the present invention is to provide an aloe exosome-like nanovesicle extracted by the above extraction method.

[0008] Another object of the present invention is to provide a use of the aloe exosome-like nanovesicles in the preparation of a product for preventing and / or treating damage caused by ultraviolet radiation.

[0009] Another object of the present invention is to provide a drug for preventing and / or treating damage caused by ultraviolet radiation.

[0010] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0011] The present invention provides a method for extracting aloe exosome-like nanovesicles, the method comprising the following steps:

[0012] The invention discloses a method for preparing aloe vera by washing fresh aloe vera, separating gel or cortex, squeezing the gel or cortex with a wall breaking machine, filtering and obtaining aloe vera gel juice or aloe vera cortex juice; centrifuging the aloe vera gel juice and / or the aloe vera cortex juice at 350-500×g for 12-20 min to obtain supernatant 1; centrifuging the supernatant 1 at 500-1500×g for 5-15 min to obtain supernatant 2; centrifuging the supernatant 2 at 1500-2500×g for 15-25 min to obtain supernatant 3; centrifuging the supernatant 3 at 12000-15 The supernatant 4 was centrifuged at 1500-2500×g for 25-35 min to obtain a concentrate; the concentrate was centrifuged at 100000-150000×g for 60-180 min to obtain a precipitate 1; the precipitate 1 was resuspended with PBS to obtain a suspension 1; the suspension 1 was centrifuged at 100000-150000×g for 60-180 min to obtain a precipitate 2; the precipitate 2 was resuspended with PBS to obtain aloe exosome-like nanovesicles.

[0013] Preferably, the aloe vera comprises Aloe vera.

[0014] Preferably, during the juicing, PBS ice cubes and ultrapure water are added to the gel or cortex; the mass volume ratio of the gel or cortex, PBS ice cubes and ultrapure water is 200-500g:40-100g:120-400mL

[0015] Preferably, the juicing procedure is as follows: the wall breaking machine pauses every 80 to 120 seconds, pauses for 50 to 70 seconds, and repeats this 2 to 4 times.

[0016] Preferably, the molecular weight cut-off of the ultrafiltration tube is 100 kD.

[0017] Preferably, the precipitate 2 is resuspended in a 1×PBS buffer solution containing 1.5 μg / mL protease inhibitor and 25 mM trehalose.

[0018] The present invention also provides an aloe exosome-like nanovesicle extracted by the above extraction method.

[0019] The present invention also provides an application of the aloe exosome-like nanovesicles in preparing a product for preventing and / or treating damage caused by ultraviolet radiation.

[0020] Preferably, the damage comprises aging damage of skin fibroblasts.

[0021] The present invention also provides a drug for preventing and / or treating damage caused by ultraviolet radiation, wherein the drug comprises a pharmaceutically acceptable carrier and the aloe exosome-like nanovesicles.

[0022] Beneficial effects of the present invention:

[0023] The aloe exosome-like nanovesicles extracted by the extraction method of the present invention have a particle size of 140 to 200 nm, are negatively charged, and have a zeta potential of -24.26 to -14.95 mV. They have excellent ability to cross various tissue barriers and can avoid the clearance effect of the mononuclear phagocyte system. In addition, the extraction method of the present invention has a high extraction rate, and the concentration of the exosome-like nanovesicles extracted from the aloe gel reaches 5.2×10 11 ~5.3×10 11 The concentration of exosome-like nanovesicles extracted from the aloe cortex reached 3.0×10 11 ~4.1×10 11 The number of particles / mL was significantly increased.

[0024] The present invention designs and optimizes the extraction method of exosome-like nanovesicles from aloe vera, thereby improving its yield, and the extracted aloe vera exosome-like nanovesicles can significantly delay skin photoaging, and have a significant improvement effect on aging damage of skin fibroblasts caused by ultraviolet radiation, and provides a potential treatment for preventing or treating actinic keratosis caused by ultraviolet radiation and further possible squamous cell carcinoma (SCC). BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1It is an identification diagram of aloe exosome-like nanovesicles of Experimental Example 1; wherein A is a nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM, 120.0 kV, scale = 50 nm), ζ potential (ζ-position), scanning electron microscopy (SEM, 120.0 kV, scale = 100 nm) and atomic force microscopy (AFM, scale = 2 μm) detection result diagram of exosome-like nanovesicles derived from aloe gel, and B is a NTA, TEM (120.0 kV, scale = 50 nm), ζ-position, SEM (10.0 kV, scale = 500 nm) and AFM (scale = 2 μm) detection result diagram of exosome-like nanovesicles derived from aloe cortex;

[0026] Figure 2 It is a graph showing the particle size, concentration and potential detection results of the exosome-like nanovesicles extracted by different extraction methods using NTA in Experimental Example 2; wherein G1 to G4 are respectively exosome-like nanovesicles derived from aloe gel extracted by the extraction methods of Comparative Example 1, Comparative Example 3, Example 1 and Example 4, and R1 to R4 are respectively exosome-like nanovesicles derived from aloe cortex extracted by the extraction methods of Comparative Example 2, Comparative Example 4, Example 2 and Example 5;

[0027] Figure 3 It is a data analysis chart of NTA using graphpad9.0 software in Experimental Example 2; wherein A is a bar graph of the concentration of exosome-like nanovesicles derived from aloe vera gel, B is a bar graph of the concentration of exosome-like nanovesicles derived from aloe vera cortex, C is a line graph of the concentration of exosome-like nanovesicles extracted by different extraction methods, D is a line graph of the particle size of exosome-like nanovesicles extracted by different extraction methods, E is a potential bar graph of exosome-like nanovesicles derived from aloe vera cortex, F is a potential bar graph of exosome-like nanovesicles derived from aloe vera gel, and G is a potential bar graph of exosome-like nanovesicles extracted by different extraction methods;

[0028] Figure 4 The results of laser confocal microscopy observation in Experiment 3 (1600X, 1300X, scale bar = 50 μm); A is the uptake and phagocytosis of gADNVs, and B is the uptake and phagocytosis of rADNVs;

[0029] Figure 5 is the ROS reactive oxygen level in Experimental Example 3; wherein A is the ROS fluorescent probe intensity (10×, scale = 100 μm), B is the ROS fluorescent probe intensity quantification graph, C is the flow cytometry analysis graph, and D is the flow cytometry fluorescence quantification graph;

[0030] Figure 6 β-galactosidase staining and quantification in Experimental Example 3 (10×, scale bar = 100 μm);

[0031] Figure 7 The results of CCK8 cell proliferation toxicity test and cell scratch test in Experimental Example 3;

[0032] Figure 8 This is a graph showing the results of the Western Blot immunoblotting test used in Experimental Example 3 to detect various indicators of aging, cell cycle, extracellular matrix, and DNA damage. DETAILED DESCRIPTION

[0033] The present invention provides a method for extracting aloe exosome-like nanovesicles, the method comprising the following steps:

[0034] The invention discloses a method for preparing aloe vera by washing fresh aloe vera, separating gel or cortex, squeezing the gel or cortex with a wall breaking machine, filtering and obtaining aloe vera gel juice or aloe vera cortex juice; centrifuging the aloe vera gel juice and / or the aloe vera cortex juice at 350-500×g for 12-20 min to obtain supernatant 1; centrifuging the supernatant 1 at 500-1500×g for 5-15 min to obtain supernatant 2; centrifuging the supernatant 2 at 1500-2500×g for 15-25 min to obtain supernatant 3; centrifuging the supernatant 3 at 12000-15 The supernatant 4 was centrifuged at 1500-2500×g for 25-35 min to obtain a concentrate; the concentrate was centrifuged at 100000-150000×g for 60-180 min to obtain a precipitate 1; the precipitate 1 was resuspended with PBS to obtain a suspension 1; the suspension 1 was centrifuged at 100000-150000×g for 60-180 min to obtain a precipitate 2; the precipitate 2 was resuspended with PBS to obtain aloe exosome-like nanovesicles.

[0035] In the present invention, the aloe variety can be conventionally selected according to actual needs, preferably including Aloe vera; Aloe vera has a tall plant, large and thick leaves, contains more mesophyll, has a high juice yield, is rich in soluble protein and polysaccharides, and has extremely high application value in industry and production. In the present invention, the aloe exosome-like nanovesicles include aloe gel-derived Exosom-like nanovesicles (gADNVs) and / or aloe cortex-derived Exosom-like nanovesicles (rADNVs); the aloe gel-derived exosom-like nanovesicles are aloe exosome-like nanovesicles extracted from aloe gel as raw material, and the aloe cortex-derived exosom-like nanovesicles are aloe exosome-like nanovesicles extracted from aloe cortex as raw material.

[0036] In the present invention, fresh aloe vera is washed, the gel and the cortex are separated, the gel or the cortex is squeezed with a wall-breaking machine, and filtered to obtain aloe vera gel juice or aloe vera cortex juice. The method of washing the fresh aloe vera is not particularly limited, and can be conventionally selected according to actual needs; as an optional embodiment, the washing step includes: the fresh aloe vera is washed with distilled water, PBS and ultrapure water; the number of distilled water washings is preferably 4 to 6 times, more preferably 5 times; the number of PBS washings is preferably 2 to 4 times, more preferably 3 times; the number of ultrapure water washings is preferably 2 to 4 times, more preferably 3 times. The method of separating the gel and the cortex can be conventionally selected according to actual needs. Before squeezing the juice, the gel or the cortex can be cut into small pieces, and the size of the small pieces is not particularly limited. When squeezing the juice, PBS ice cubes and ultrapure water are preferably added to the gel or cortex for squeezing the juice; the mass volume ratio of the gel or cortex, PBS ice cubes and ultrapure water is preferably 200-500g:40-100g:120-400mL; the mass volume ratio of the PBS ice cubes and ultrapure water is further preferably 1:2.5-1:4, more preferably 1:4 or 1:3-3.5 or 1:3.5-4. As an optional embodiment, when squeezing juice from the gel, the mass volume ratio of the gel, PBS ice cubes and ultrapure water is preferably 300-500g:60-100g:180-400mL, further preferably 350-450g:70-90g:210-360mL, and more preferably 380g:80g:240mL; when squeezing juice from the cortex, the mass volume ratio of the cortex, PBS ice cubes and ultrapure water is preferably 200-400g:40-80g:120-320mL, further preferably 250-350g:50-70g:150-280mL, and more preferably 280g:60g:240mL. As an implementable embodiment, the juicing procedure is preferably: the wall breaking machine pauses every 80 to 120 seconds, more preferably every 90 to 110 seconds, and more preferably every 100 seconds; pauses for 50 to 70 seconds, more preferably 55 to 65 seconds, and more preferably 60 seconds; repeats 2 to 4 times, and more preferably 3 times. After the juicing, conventional filtering can be selected to obtain aloe gel juice and / or aloe cortex juice; as an optional embodiment, gauze mesh is used for filtering; the mesh number of the filtration is preferably 72 to 100 mesh, and more preferably 100 mesh.

[0037] In the present invention, the aloe gel juice and / or the aloe cortex juice is centrifuged at 350-500×g for 12-20 min to obtain supernatant 1; the centrifugal speed is more preferably 400×g or 350-400×g or 400-450×g; the centrifugal time is more preferably 15 min or 12-15 min or 15-18 min.

[0038] In the present invention, the supernatant 1 is centrifuged at 500-1500×g for 5-15 min to obtain supernatant 2; the centrifugal speed is more preferably 1000×g or 750-1000×g or 1000-1250×g; the centrifugal time is more preferably 10 min or 7-10 min or 10-12 min.

[0039] In the present invention, the supernatant 2 is centrifuged at 1500-2500×g for 15-25 min to obtain supernatant 3; the centrifugal speed is more preferably 2000×g or 1750-2000 or 2000-2250×g; the centrifugal time is more preferably 20 min or 17-20 min or 20-22 min.

[0040] In the present invention, the supernatant 3 is centrifuged at 12000-15000×g for 42-50 min to obtain supernatant 4; the centrifugal speed is more preferably 15000×g or 13000-15000 or 14000-15000×g; the centrifugal time is more preferably 45 min or 43-45 min or 45-48 min.

[0041] In the present invention, the supernatant 4 is placed in an ultrafiltration tube and centrifuged at 1500-2500×g for 25-35 min to obtain a concentrated solution; the centrifugal speed is more preferably 2000×g or 1700-2000 or 2000-2200×g; the centrifugal time is more preferably 30 min or 27-30 min or 30-32 min. In the present invention, the molecular weight cutoff of the ultrafiltration tube is preferably 100 kD.

[0042] In the present invention, the concentrate is centrifuged at 100,000 to 150,000 × g for 60 to 180 min to obtain precipitate 1. The centrifugal speed is more preferably 150,000 × g, 100,000 to 120,000, 120,000 to 140,000, or 140,000 to 150,000 × g; the centrifugal time is more preferably 120 min, 90 to 120 min, or 120 to 150 min.

[0043] In the present invention, PBS is used to resuspend the precipitate 1 to obtain a suspension 1; washing the precipitate 1 with PBS can reduce the impurity content and improve the purity of the exosomes; the amount of the PBS can be conventionally selected according to actual needs.

[0044] In the present invention, the suspension 1 is centrifuged at 100,000-150,000×g for 60-180 min to obtain a precipitate 2; the centrifugal speed is more preferably 150,000×g or 100,000-120,000 or 120,000-140,000 or 140,000-150,000×g; the centrifugal time is more preferably 120 min or 90-120 min or 120-150 min.

[0045] In the present invention, PBS is used to resuspend the precipitate 2 to obtain aloe exosome-like nanovesicles; preferably, a 1×PBS buffer solution containing 1.5 μg / mL protease inhibitor and 25 mM trehalose is used to resuspend the precipitate 2. The 1×PBS buffer solution containing 1.5 μg / mL protease inhibitor and 25 mM trehalose can be purchased through conventional channels or prepared by conventional methods. The exosome-like vesicles resuspended in this buffer solution have good dispersibility. In the present invention, the amount of the PBS can be conventionally selected according to actual needs.

[0046] The present invention also provides an aloe exosome-like nanovesicle extracted by the above extraction method.

[0047] The aloe exosome-like nanovesicles extracted by the extraction method of the present invention have a particle size of 140 to 200 nm, are negatively charged, and have a zeta potential of -24.26 to -14.95 mV. They have excellent ability to cross various tissue barriers and can avoid the clearance effect of the mononuclear phagocyte system. In addition, the extraction method of the present invention has a high extraction rate, and the concentration of the exosome-like nanovesicles extracted from the aloe gel reaches 5.2×10 11 ~5.3×10 11 The concentration of exosome-like nanovesicles extracted from the aloe cortex reached 3.0×10 11 ~4.1×10 11 The number of particles / mL is greatly improved compared with the existing technology.

[0048] The present invention also provides an application of the aloe exosome-like nanovesicles in preparing a product for preventing and / or treating damage caused by ultraviolet radiation.

[0049] In the present invention, the damage preferably includes skin fibroblast aging damage. The aloe exosome-like nanovesicles extracted by the present invention can be taken up by fibroblasts, can reduce the increase in reactive oxygen levels caused by ultraviolet radiation, can significantly reduce the production of β-galactosidase, can promote the migration of fibroblasts after ultraviolet radiation, promote their proliferation, and can also effectively protect the collagen degradation, nuclear lamina protein dissolution, and inhibition of the increase of key factors P21 and P16 in cell cycle regulation during ultraviolet-induced skin aging, reduce the expression level of γ-H2AX, and alleviate skin aging caused by DNA damage. The aloe exosome-like nanovesicles extracted by the present invention can significantly delay skin photoaging, have a significant improvement effect on skin fibroblast aging damage caused by ultraviolet radiation, and can be used to prevent and / or treat actinic keratosis caused by ultraviolet radiation and further possible disease squamous cell carcinoma (SCC).

[0050] In the present invention, there is no special limitation on the types of the products, including but not limited to medicines, skin care products, etc.; the dosage form of the products can be conventionally selected according to actual needs.

[0051] The present invention also provides a drug for preventing and / or treating damage caused by ultraviolet radiation, wherein the drug comprises a pharmaceutically acceptable carrier and the aloe exosome-like nanovesicles.

[0052] In the present invention, there is no special limitation on the dosage form of the drug, and it can be conventionally selected according to actual needs. The drug of the present invention can be administered in various forms suitable for the selected route of administration, such as oral, injection, respiratory, skin and the like. Therefore, the adapted drug dosage form can be selected according to the selected route of administration. Examples of drug dosage forms for oral administration include, but are not limited to, powders, tablets, granules, capsules, solutions, emulsions or suspensions, etc.; examples of drug dosage forms for administration by injection include, but are not limited to, liquid injections, powders for injection or tablets for injection, etc.; examples of drug dosage forms for administration through the respiratory tract include, but are not limited to, sprays, aerosols or powder mists, etc.; examples of drug dosage forms for administration through the skin include, but are not limited to, solutions, lotions, liniments, ointments, plasters, pastes or patches, etc. The carrier can be conventionally selected according to the drug dosage form and actual needs.

[0053] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0054] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0055] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0056] In the following examples, the 1×PBS containing 1.5 μg / mL protease inhibitor and 25 mM trehalose was purchased from Shandong Qidu Biopharmaceutical Co., Ltd. with the catalog number QB1001.

[0057] Example 1

[0058] A method for extracting aloe exosome-like nanovesicles:

[0059] 1. Preparation of fresh aloe vera gel juice: After fresh aloe vera is fully washed with distilled water for 5 times, it is washed with PBS and ultrapure water for 3 times respectively to separate the aloe vera gel and cortex; the gel is cut into small pieces, 380g of gel is weighed and placed in a wall-breaking machine; 80g of prepared PBS ice cubes and 320mL of pre-cooled ultrapure water are added, and the mixture is put into the wall-breaking machine and crushed, pausing once every 100s for 60s, for a total of 480s. The fresh aloe vera gel juice is obtained, filtered through a 100-mesh funnel gauze net, and placed at 4°C overnight for use.

[0060] 2. Preparation of aloe exosome-like vesicles: take out the aloe gel juice placed at 4°C, centrifuge it at 400×g for 15 minutes, discard the precipitate to obtain supernatant A1, centrifuge supernatant A1 at 1000×g for 10 minutes to obtain supernatant A2; supernatant A2 is further centrifuged at 2000×g for 20 minutes to obtain supernatant A3; supernatant A3 is centrifuged at 15000×g for 45 minutes to obtain supernatant A4; supernatant A4 is placed in a 100kD ultrafiltration tube and centrifuged at 2000g for 30 minutes, the vesicles are ultrafiltered and concentrated to obtain concentrate A1; concentrate A1 is ultracentrifuged at 150000×g for 120 minutes to obtain precipitate A1.

[0061] 3. Resuspend the precipitate A1 with PBS to obtain suspension A1; centrifuge the suspension A1 again at 150,000×g for 120 min to obtain precipitate A2; resuspend the precipitate A2 with 400 μL PBS (1×PBS containing 1.5 μg / mL protease inhibitor and 25 mM trehalose) to obtain exosome-like nanovesicles derived from aloe vera gel.

[0062] Example 2

[0063] A method for extracting aloe exosome-like nanovesicles:

[0064] 1. Prepare fresh aloe vera cortex juice: After fresh aloe vera is fully washed with distilled water for 5 times, it is washed with PBS and ultrapure water for 3 times respectively to separate the aloe gel and cortex; the cortex is cut into small pieces, 280g is weighed, and placed in a wall-breaking machine; 60g of prepared PBS ice cubes and 240mL of pre-cooled ultrapure water are added, and the mixture is put into the wall-breaking machine and crushed, pausing once every 100s for 60s, for a total of 480s. The fresh aloe vera cortex juice is obtained, filtered through a 100-mesh funnel gauze net, and placed at 4°C overnight for use.

[0065] 2. Preparation of aloe exosome-like vesicles: Take out the aloe cortex juice placed at 4°C, centrifuge it at 400×g for 15 minutes, discard the precipitate, and obtain supernatant B1. Centrifuge supernatant B1 at 1000×g for 10 minutes to obtain supernatant B2; supernatant B2 is further centrifuged at 2000×g for 20 minutes to obtain supernatant B3; supernatant B3 is centrifuged at 15000×g for 45 minutes to obtain supernatant B4; supernatant B4 is placed in a 100kD ultrafiltration tube and centrifuged at 2000g for 30 minutes, and the vesicles are ultrafiltered and concentrated to obtain concentrate B1; concentrate B1 is ultracentrifuged at 150000×g for 120 minutes to obtain precipitate B1.

[0066] 3. Resuspend the precipitate B1 with PBS to obtain suspension B1; centrifuge the suspension B1 again at 150,000×g for 120 min to obtain precipitate B2; resuspend the precipitate B2 with 300 μL PBS (1×PBS containing 1.5 μg / mL protease inhibitor and 25 mM trehalose) to obtain exosome-like nanovesicles derived from the aloe cortex.

[0067] Example 3

[0068] A method for extracting aloe exosome-like nanovesicles:

[0069] Fresh Aloe vera gel juice and cortex juice were prepared by the methods of Example 1 and Example 2, respectively. In step 2, a mixture of Aloe vera gel juice and Aloe vera cortex juice was used in a mass ratio of 1:1, and the remaining steps were the same as in Example 1.

[0070] Example 4

[0071] A method for extracting aloe exosome-like nanovesicles:

[0072] The difference from Example 1 is that in step 2, the concentrated solution A1 is ultracentrifuged at 100,000×g for 120 min to obtain a precipitate A1; in step 3, the suspension A1 is centrifuged again at 100,000×g for 120 min to obtain a precipitate A2.

[0073] Example 5

[0074] A method for extracting aloe exosome-like nanovesicles:

[0075] The difference from Example 2 is that in step 2, the concentrated solution B1 is ultracentrifuged at 100,000×g for 120 min to obtain a precipitate B1; in step 3, the suspension B1 is centrifuged again at 100,000×g for 120 min to obtain a precipitate B2.

[0076] Example 6

[0077] A method for extracting aloe exosome-like nanovesicles:

[0078] The difference from Example 4 is that in step 2, a mixture of aloe gel juice and aloe cortex juice is used to prepare aloe exosome-like vesicles, and the mass ratio of the two is 1:1.

[0079] Example 7

[0080] A method for extracting aloe exosome-like nanovesicles:

[0081] 1. Preparation of fresh aloe vera gel juice: After fresh aloe vera is fully washed with distilled water for 4 times, it is washed with PBS and ultrapure water for 4 times respectively to separate the aloe gel and cortex; the gel is cut into small pieces, 500g is weighed, and placed in a wall-breaking machine; 100g of prepared PBS ice cubes and 350mL pre-cooled ultrapure water are added, and the mixture is put into the wall-breaking machine and crushed, pausing once every 80s, pausing for 50s, for a total of 520s. The fresh aloe vera gel juice is obtained, filtered through a 72-mesh funnel gauze net, and placed at 4°C overnight for use.

[0082] 2. Preparation of aloe exosome-like vesicles: The aloe gel juice placed at 4°C was taken out, centrifuged at 350×g for 20 min, the precipitate was discarded, and supernatant A1 was obtained. The supernatant A1 was centrifuged at 500×g for 15 min to obtain supernatant A2; supernatant A2 was centrifuged at 1500×g for 25 min to obtain supernatant A3; supernatant A3 was centrifuged at 12000×g for 50 min to obtain supernatant A4; supernatant A4 was placed in a 100kD ultrafiltration tube and centrifuged at 1500g for 35 min, the vesicles were ultrafiltered and concentrated to obtain concentrate A1; concentrate A1 was ultracentrifuged at 100000×g for 180 min to obtain precipitate A1.

[0083] 3. Resuspend the precipitate A1 with PBS to obtain suspension A1; centrifuge the suspension A1 again at 100,000×g for 180 min to obtain precipitate A2; resuspend the precipitate A2 with 450 μL PBS (1×PBS containing 1.5 μg / mL protease inhibitor and 25 mM trehalose) to obtain exosome-like nanovesicles derived from aloe vera gel.

[0084] Example 8

[0085] A method for extracting aloe exosome-like nanovesicles:

[0086] 1. Preparation of fresh aloe vera gel and cortex juice: After fresh aloe vera was fully washed with distilled water for 4 times, it was washed with PBS and ultrapure water for 4 times respectively to separate the aloe vera gel and cortex; the cortex was cut into small pieces, 400 g was weighed, and placed in a wall-breaking machine; 80 g of prepared PBS ice cubes and 280 mL of pre-cooled ultrapure water were added, and the mixture was put into the wall-breaking machine and crushed, pausing once every 80 seconds, pausing for 50 seconds, and a total of 520 seconds. The fresh aloe vera cortex juice was obtained, filtered through a 72-mesh funnel gauze net, and placed at 4°C overnight for use.

[0087] 2. Preparation of aloe exosome-like vesicles: Take out the aloe cortex juice placed at 4°C, centrifuge it at 350×g for 20 min, discard the precipitate, and obtain supernatant B1. Centrifuge supernatant B1 at 500×g for 15 min to obtain supernatant B2; supernatant B2 is further centrifuged at 1500×g for 25 min to obtain supernatant B3; supernatant B3 is centrifuged at 12000×g for 50 min to obtain supernatant B4; supernatant B4 is placed in a 100kD ultrafiltration tube and centrifuged at 1500g for 35 min, and the vesicles are ultrafiltered and concentrated to obtain concentrate B1; the concentrate is ultracentrifuged at 100000×g for 180 min to obtain precipitate B1.

[0088] 3. Resuspend the precipitate B1 with PBS to obtain suspension B1; centrifuge the suspension B1 again at 100,000×g for 180 min to obtain precipitate B2; resuspend the precipitate B2 with 350 μL PBS (1×PBS containing 1.5 μg / mL protease inhibitor and 25 mM trehalose) to obtain exosome-like nanovesicles derived from the aloe cortex.

[0089] Example 9

[0090] A method for extracting aloe exosome-like nanovesicles:

[0091] Fresh Aloe vera gel juice and Aloe cortex juice were prepared by the methods of Example 7 and Example 8, respectively. In step 2, a mixture of Aloe vera gel juice and Aloe cortex juice was used in a mass ratio of 2:1. The remaining steps were the same as in Example 7.

[0092] Example 10

[0093] A method for extracting aloe exosome-like nanovesicles:

[0094] 1. Preparation of fresh aloe vera gel juice: After fresh aloe vera was fully washed with distilled water for 6 times, it was washed with PBS and ultrapure water for 2 times respectively to separate the aloe vera gel and cortex; the gel was cut into small pieces, 300 g was weighed, and placed in a wall-breaking machine; 60 g of prepared PBS ice cubes and 180 mL of pre-cooled ultrapure water were added, and the mixture was put into the wall-breaking machine and crushed, pausing once every 120 seconds, pausing for 70 seconds, for a total of 380 seconds. The fresh aloe vera gel juice was obtained, filtered through an 80-mesh funnel gauze net, and placed at 4°C overnight for use.

[0095] 2. Preparation of aloe exosome-like vesicles: take out the aloe gel juice placed at 4°C, centrifuge it at 500×g for 12 minutes, discard the precipitate, and obtain supernatant A1. Centrifuge supernatant A1 at 1500×g for 5 minutes to obtain supernatant A2; supernatant A2 is further centrifuged at 2500×g for 15 minutes to obtain supernatant A3; supernatant A3 is centrifuged at 14000×g for 42 minutes to obtain supernatant A4; supernatant A4 is placed in a 100kD ultrafiltration tube and centrifuged at 2500g for 25 minutes, and the vesicles are ultrafiltered and concentrated to obtain concentrate A1; concentrate A1 is ultracentrifuged at 140000×g for 60 minutes to obtain precipitate A1.

[0096] 3. Resuspend the precipitate A1 with PBS to obtain suspension A1; centrifuge the suspension A1 again at 140,000×g for 60 min to obtain precipitate A2; resuspend the precipitate A2 with 350 μL PBS (1×PBS containing 1.5 μg / mL protease inhibitor and 25 mM trehalose) to obtain exosome-like nanovesicles derived from aloe vera gel.

[0097] Embodiment 11

[0098] A method for extracting aloe exosome-like nanovesicles:

[0099] 1. Preparation of fresh aloe vera cortex juice: After fresh aloe vera was fully washed with distilled water for 6 times, it was washed with PBS and ultrapure water for 2 times respectively to separate the aloe gel and cortex; the cortex was cut into small pieces, 200 g was weighed, and placed in a wall-breaking machine; 40 g of prepared PBS ice cubes and 120 mL of pre-cooled ultrapure water were added, and the mixture was put into the wall-breaking machine and crushed, pausing once every 120 seconds, pausing for 70 seconds, for a total of 380 seconds. The fresh aloe vera cortex juice was obtained, filtered through an 80-mesh funnel gauze net, and placed at 4°C overnight for use.

[0100] 2. Preparation of aloe exosome-like vesicles: take out the aloe cortex juice placed at 4°C, centrifuge it at 500×g for 12 minutes, discard the precipitate, and obtain supernatant B1. Centrifuge supernatant B1 at 1500×g for 5 minutes to obtain supernatant B2; supernatant B2 is further centrifuged at 2500×g for 15 minutes to obtain supernatant B3; supernatant B3 is centrifuged at 14000×g for 42 minutes to obtain supernatant B4; supernatant B4 is placed in a 100kD ultrafiltration tube and centrifuged at 2500g for 25 minutes, and the vesicles are ultrafiltered and concentrated to obtain concentrate B1; concentrate B1 is ultracentrifuged at 140000×g for 60 minutes to obtain precipitate B1.

[0101] 3. Resuspend the precipitate B1 with PBS to obtain suspension B1; centrifuge the suspension B1 again at 140,000×g for 60 min to obtain precipitate B2; resuspend the precipitate B2 with 250 μL PBS (1×PBS containing 1.5 μg / mL protease inhibitor and 25 mM trehalose) to obtain exosome-like nanovesicles derived from the aloe cortex.

[0102] Example 12

[0103] A method for extracting aloe exosome-like nanovesicles:

[0104] Fresh Aloe vera gel juice and Aloe cortex juice were prepared by the methods of Example 10 and Example 11, respectively. In step 2, a mixture of Aloe vera gel juice and Aloe cortex juice was used in a mass ratio of 1:2. The remaining steps were the same as in Example 10.

[0105] Comparative Example 1

[0106] A method for extracting aloe exosome-like nanovesicles (refer to the extraction method of patent CN111905107A):

[0107] Step a. Aloe vera gel juice: After fresh aloe vera is fully washed with distilled water for 5 times, it is washed with PBS and ultrapure water for 3 times respectively to separate the aloe vera gel and the cortex; the gel is cut into small pieces and weighed 380g, and 190g of prepared PBS ice cubes and 1520mL pre-cooled PBS are added, and the pieces are put into a wall-breaking machine and crushed, pausing every 30s, with an interval of 10s, for a total of 240s. The fresh aloe vera gel juice is obtained, filtered through 100-mesh gauze, and placed at 4°C for use;

[0108] Step b. Preparation of aloe vera gel exosome-like vesicles:

[0109] Fresh aloe vera gel juice was centrifuged at 300×g for 10 min, and the precipitate was discarded to obtain supernatant A1; then supernatant A1 was centrifuged at 1000×g for 10 min to obtain supernatant B1; supernatant B1 was centrifuged at 3000×g for 20 min to obtain supernatant C1; supernatant C1 was centrifuged at 11000×g for 40 min to obtain supernatant D1, and finally supernatant D1 was filtered through a 0.22μm filter membrane and centrifuged at 50000×g for 90 min to obtain precipitate E1, which was resuspended in PBS to obtain suspension F1, and suspension F1 was centrifuged at 50000×g for 90 min to obtain precipitate G1, which was resuspended in 400μL PBS (1×PBS containing 1.5μg / mL protease inhibitor and 25mM trehalose) to obtain exosome-like nanovesicles derived from aloe vera gel.

[0110] Comparative Example 2

[0111] A method for extracting aloe exosome-like nanovesicles (refer to the extraction method of patent CN111905107A):

[0112] Step a. Aloe peel juice: After fresh aloe vera is fully washed with distilled water for 5 times, it is washed with PBS and ultrapure water for 3 times respectively to separate the aloe gel and cortex; the cortex is cut into small pieces and weighed 280g, and 140g of prepared PBS ice cubes and 1120mL pre-cooled PBS are added, and the pieces are put into a wall-breaking machine and crushed, pausing every 30s, with an interval of 10s, for a total of 240s. The fresh aloe peel juice is obtained, filtered through 100-mesh gauze, and placed at 4°C for use;

[0113] Step b. Preparation of aloe vera peel exosome-like vesicles:

[0114] Fresh aloe peel juice was centrifuged at 300×g for 10 min, and the precipitate was discarded to obtain supernatant A2; then supernatant A2 was centrifuged at 1000×g for 10 min to obtain supernatant B2; supernatant B2 was centrifuged at 3000×g for 20 min to obtain supernatant C2; supernatant C2 was centrifuged at 11000×g for 40 min to obtain supernatant D2, and finally supernatant D2 was filtered through a 0.22μm filter membrane and centrifuged at 50000×g for 90 min to obtain precipitate E2, which was resuspended in PBS to obtain suspension F2, and suspension F2 was centrifuged at 50000×g for 90 min to obtain precipitate G2, which was resuspended in 300μL PBS (1×PBS containing 1.5μg / mL protease inhibitor and 25mM trehalose) to obtain exosome-like nanovesicles derived from the aloe cortex.

[0115] Comparative Example 3

[0116] A method for extracting aloe exosome-like nanovesicles:

[0117] Fresh aloe vera was cleaned in the same manner as in Example 1, and 380 g of gel cubes were taken. The extraction method in the article Zhou et al. Aloe-derived vesicles enable macrophage reprogramming to regulate the inflammatory immune environment. Front. Bioeng Biotechnol. 11: 1339941. doi: 10.3389 / fbioe.2023.1339941 was referred to for differential centrifugation. The obtained aloe exosome-like vesicle particles were resuspended in 400 μL PBS (1×PBS containing 1.5 μg / mL protease inhibitor and 25 mM trehalose) to obtain exosome-like nanovesicles derived from aloe gel.

[0118] Comparative Example 4

[0119] A method for extracting aloe exosome-like nanovesicles:

[0120] Fresh aloe vera was cleaned in the same manner as in Example 1, and 280 g of cortex pieces were taken. The extraction method in the article Zhou et al. Aloe-derived vesicles enable macrophage reprogramming to regulate the inflammatory immune environment. Front. Bioeng Biotechnol. 11: 1339941. doi: 10.3389 / fbioe.2023.1339941 was referred to for differential centrifugation. The obtained aloe exosome-like vesicle particles were resuspended in 300 μL PBS (1×PBS containing 1.5 μg / mL protease inhibitors and 25 mM trehalose) to obtain exosome-like nanovesicles derived from the aloe cortex.

[0121] Test Example 1

[0122] The exosome-like nanovesicles from aloe vera gel were extracted by the extraction method of Example 1, and the exosome-like nanovesicles from aloe vera cortex were extracted by the extraction method of Example 2. The extracted exosome-like nanovesicles were observed and characterized by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM, 120.0 kV), ζ-position, scanning electron microscopy (SEM, 120.0 kV), and atomic force microscopy (AFM). Figure 1 shown.

[0123] Depend on Figure 1 It can be seen that the aloe gel and cortex-derived exosome-like nanovesicles extracted by the extraction methods of Examples 1 and 2 have uniform particle sizes and high enrichment levels; ζ potential measurements show that the vesicle structure is stable and not easy to break. TEM is one of the gold standard methods for identifying the morphology of exosomes. The extracted exosomes can be clearly observed to have a typical cup-shaped or saucer-shaped structure by transmission electron microscopy TEM counterstaining technology. The surface morphology of the exosomes and the vesicle-like structure extracted were further confirmed by scanning electron microscopy. Atomic force microscopy AFM observations showed the surface topology of the extracted exosomes, such as height, adhesion, etc., which further characterized the exosomes extracted in Examples 1 and 2.

[0124] Test Example 2

[0125] The extraction method of Example 1 was used to extract exosome-like nanovesicles derived from aloe gel (denoted as Gev-3), the extraction method of Example 2 was used to extract exosome-like nanovesicles derived from aloe cortex (denoted as Rev-3), the extraction method of Example 4 was used to extract exosome-like nanovesicles derived from aloe gel (denoted as Gev-4), the extraction method of Example 5 was used to extract exosome-like nanovesicles derived from aloe cortex (denoted as Rev-4), the extraction method of Comparative Example 1 was used to extract exosome-like nanovesicles derived from aloe gel (denoted as Gev-1), the extraction method of Comparative Example 2 was used to extract exosome-like nanovesicles derived from aloe cortex (denoted as Rev-1), the extraction method of Comparative Example 3 was used to extract exosome-like nanovesicles derived from aloe gel (denoted as Gev-2), and the extraction method of Comparative Example 4 was used to extract exosome-like nanovesicles derived from aloe cortex (denoted as Rev-2).

[0126] Nanoparticle tracking analyzer (NTA) was used to compare and analyze the particle size, potential, and concentration of exosome-like nanovesicles extracted by different extraction methods, and graphpad9.0 software was used to analyze and plot the data obtained by NTA. The results are shown in Figure 2-3 shown.

[0127] Depend on Figure 2 and Figure 3 The results show that, for particle size, the particle size of the aloe exosome-like nanovesicles extracted in Examples 1, 2, 4, and 5 of the present invention is between 140 and 200 nm, which is not much different from the comparative example. For potential, the aloe exosome-like nanovesicles extracted in Examples 1, 2, 4, and 5 of the present invention are all negatively charged, with a zeta potential between -24.26 and -14.95 mV, which is not much different from the comparative example. For concentration, the concentrations of the aloe gel-derived exosome-like nanovesicles (Gev-3 and Gev-4) extracted in Examples 1 and 4 of the present invention reached 5.3×10 11 Number of particles / mL, 5.2×10 11The number of particles / mL was increased by 76.67% and 73.33% respectively compared with comparative example 1 (Gev-1), and by 140.90% and 136.36% respectively compared with comparative example 3 (Gev-2); the concentrations of exosome-like nanovesicles (Rev-3 and Rev-4) from the aloe cortex extracted in Examples 2 and 5 of the present invention reached 4.1×10 11 Number of particles / mL, 3.0×10 11 The number of particles / mL was increased by 215.38% and 130.77% respectively compared with Comparative Example 2 (Rev-1), and by 355.56% and 233.33% respectively compared with Comparative Example 4 (Rev-2). By statistically comparing different schemes, it can be found that the extraction method of the present invention can significantly increase the concentration of aloe exosome-like nanovesicles.

[0128] Test Example 3

[0129] The extraction method of Example 1 was used to extract exosome-like nanovesicles (denoted as gADNVs) derived from aloe gel, and the extraction method of Example 2 was used to extract exosome-like nanovesicles (denoted as rADNVs) derived from aloe cortex.

[0130] 1. Uptake of two vesicles by fibroblasts

[0131] Methods: SD rat skin fibroblasts were cultured to P3 and then subcultured to P4. At the same time, the cells were inoculated into fluorescent slides in 12-well plates. When the cells proliferated to 70-80%, 1×10 10 The exosomes of gADNVs or rADNVs with the number of particles were co-incubated with cells. After 24 hours, the cell slides were fixed and membrane-permeated. After phalloidin and Hochest staining, the uptake and phagocytosis of gADNVs and rADNVs vesicles by fibroblasts were observed using a laser confocal microscope. The results are as follows: Figure 4 shown.

[0132] Depend on Figure 4 It can be seen that rat skin fibroblasts can take up two different vesicles, indicating that the two exosome-like nanovesicles extracted by the extraction methods of Examples 1 and 2 can be effectively taken up by the target cells, which is the premise for it to play all damage repair and anti-aging functions. It also shows that it has the typical characteristics of exosomes crossing cell barriers and cell membranes, proving the reliability of the extracted exosomes.

[0133] 2. Determination of ROS Levels

[0134] Methods: SD rat skin fibroblasts were cultured to P3 and then subcultured to P4. The cells were seeded into 6-well plates and 1×10 10 The exosomes of gADNVs or rADNVs were added with 1000 gADNVs particles. No exosomes were added to the CTR group and UV group. After 24 hours, the UV, gADNVs, and rADNVs groups were irradiated under ultraviolet light for 3 days with a daily irradiation dose of 8 J / cm 2 After irradiation, ROS fluorescent probe was added and incubated at 37°C for 20 minutes, observed under a fluorescence microscope, and ROS reactive oxygen levels were detected using ROS flow cytometry. Figure 5 shown.

[0135] Depend on Figure 5 It can be seen that compared with the CTR group, the level of reactive oxygen species induced by ultraviolet rays in the UV group was significantly increased, while both gADNVs and rADNVs treatments could alleviate the increase in reactive oxygen species levels induced by ultraviolet rays.

[0136] 3. Intervention effects of two vesicles on β-galactosidase staining

[0137] Methods: The experimental group settings and treatment methods were the same as those in the above “ROS reactive oxygen species level determination”. After UV irradiation, the culture medium was aspirated from each well of the plate and washed with PBS three times, 3 minutes each time. The β-galactosidase produced by senescent cells was determined by colorimetric analysis using the Yisheng Bio β-galactosidase staining kit and observed under an upright fluorescence microscope. Results Figure 6 shown.

[0138] Depend on Figure 6 It can be seen that compared with the CTR group, the production of β-galactosidase in the UV group increased significantly, while both vesicle treatments could significantly reduce the production of β-galactosidase.

[0139] 4. Evaluation of the effects of the two vesicles on fibroblast proliferation and migration

[0140] Methods: The setting and treatment methods of each experimental group were the same as those in the above “ROS reactive oxygen species level determination”. After UV irradiation, the culture medium was aspirated from each well of the plate and washed with PBS three times, each time for 3 minutes. CCK8 cell proliferation toxicity test and cell scratch test were performed respectively, and the absorbance was tested by cell imaging microplate detector and photographed by upright fluorescence microscope. The results are shown in Figure 7 shown.

[0141] Depend on Figure 7 It can be seen that both vesicles can promote the migration of fibroblasts after ultraviolet irradiation and promote their proliferation. This shows that both vesicles can activate the proliferation and migration activity of senescent cells and have positive functions on the proliferation and migration of senescent cells.

[0142] 5. Effects of the two vesicles on cell cycle indicators and extracellular matrix degradation, which are closely related to aging

[0143] The reduction of collagen COL1a1 is an important manifestation of extracellular matrix degradation in aging cells. Lamin B1 (LaminB1) is highly degraded and reduced in aging cells. P21 and P16 are indicators related to cell cycle arrest associated with aging, and they increase during aging. γ-H2AX is a key indicator of the aging pathway caused by ultraviolet-induced DNA damage, and it increases significantly during aging.

[0144] Methods: The experimental group settings and treatment methods were the same as those in the above “ROS reactive oxygen species level determination”. After UV irradiation, the culture medium was removed from each well of the plate and washed with PBS three times, each time for 3 minutes. Western Blot was used to detect senescence, cell cycle, extracellular matrix, and DNA damage. Figure 8 shown.

[0145] Depend on Figure 8 It can be seen that the two vesicles can effectively protect collagen degradation and laminin dissolution during UV-induced skin aging, inhibit the increase of P21 and P16, reduce the expression level of γ-H2AX, and alleviate skin aging caused by DNA damage.

[0146] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for extracting aloe exosome-like nanovesicles, characterized in that: The method comprises the following steps: Wash fresh aloe vera, separate the gel or cortex, squeeze the gel or cortex with a wall-breaking machine, filter, and obtain aloe vera gel juice or aloe vera cortex juice; centrifuge the aloe vera gel juice and / or the aloe vera cortex juice at 350-500×g for 12-20 minutes to obtain supernatant 1; centrifuge the supernatant 1 at 500-1500×g for 5-15 minutes to obtain supernatant 2; centrifuge the supernatant 2 at 1500-2500×g for 15-25 minutes to obtain supernatant 3; centrifuge the supernatant 3 at 12000-15000×g for 42-50 minutes to obtain supernatant 4; The supernatant 4 is placed in an ultrafiltration tube and centrifuged at 1500-2500×g for 25-35 min to obtain a concentrate; the concentrate is centrifuged at 100000-150000×g for 60-180 min to obtain a precipitate 1; The precipitate 1 is resuspended with PBS to obtain a suspension 1; the suspension 1 is centrifuged at 100,000-150,000×g for 60-180 min to obtain a precipitate 2; the precipitate 2 is resuspended with PBS to obtain aloe exosome-like nanovesicles.

2. The extraction method according to claim 1, characterized in that The aloe vera includes Aloe vera.

3. The extraction method according to claim 1, characterized in that When squeezing the juice, PBS ice cubes and ultrapure water are added to the gel or cortex; the mass volume ratio of the gel or cortex, PBS ice cubes and ultrapure water is 200-500g:40-100g:120-400mL.

4. The extraction method according to claim 1, characterized in that The procedure of juicing is as follows: the wall breaking machine pauses every 80 to 120 seconds, pauses for 50 to 70 seconds, and repeats 2 to 4 times.

5. The extraction method according to claim 1, characterized in that The molecular weight cut-off of the ultrafiltration tube is 100 kD.

6. The extraction method according to claim 1, characterized in that The precipitate 2 was resuspended in a 1×PBS buffer solution containing 1.5 μg / mL protease inhibitor and 25 mM trehalose.

7. Aloe exosome-like nanovesicles extracted by the extraction method according to any one of claims 1 to 6.

8. Use of the aloe exosome-like nanovesicles according to claim 7 in the preparation of a product for preventing and / or treating damage caused by ultraviolet radiation.

9. The use according to claim 8, characterized in that: The damage includes aging damage of skin fibroblasts.

10. A drug for preventing and / or treating damage caused by ultraviolet radiation, characterized in that: The drug comprises a pharmaceutically acceptable carrier and the aloe exosome-like nanovesicles according to claim 7.

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