Avocado-derived exosome-like nanovesicles, and preparation method and application thereof

Abstract

The application provides avocado-derived exosome-like nanovesicles, a preparation method and application thereof; the application obtains a preparation method of avocado-derived exosome-like nanovesicles through condition optimization and screening, the preparation method is more optimal for the extraction effect of the avocado-derived exosome-like nanovesicles, can significantly improve the yield of the vesicles, and is more suitable for large-scale preparation; the avocado-derived exosome-like nanovesicles extracted by the method can significantly delay skin aging, can obviously improve the aging damage of skin fibroblasts caused by D-galactose, and also shows a significant improvement effect on the skin aging damage of ICR mice induced by D-galactose, and has a good application prospect in preventing or delaying skin aging.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to an avocado-derived exosome-like nanovesicle, its preparation method, and its application. Background Technology

[0002] As the largest organ and outermost defense barrier in the human body, the aging of the skin is a complex physiological degenerative process driven by both endogenous and exogenous factors. Among them, glycosylation has attracted attention as one of the important drivers of endogenous aging.

[0003] Recent studies have revealed that skin aging is not an isolated local phenomenon, but is closely related to systemic aging throughout the body. On the one hand, the skin's own neuroendocrine system can respond to environmental stress and transmit signals to the central nervous system, affecting the body's overall function. On the other hand, senescent cells accumulated in aging skin can induce secondary aging in adjacent and even distant healthy tissues by secreting aging-associated secretory phenotypes (SASPs), such as inflammatory factors, chemokines, matrix metalloproteinases (MMPs), and extracellular vesicles, thereby accelerating the body's overall aging process.

[0004] The above findings indicate that the skin is not only a "mirror" of systemic aging but also an active participant driving systemic aging. The accumulation of senescent cells and the continuous secretion of SASPs in localized areas of the skin can create a chronic, low-grade inflammatory microenvironment, gradually damaging the integrity of skin structure and affecting the whole body. Therefore, effective intervention targeting localized skin aging is not only necessary to improve skin appearance and function but may also become a crucial breakthrough in delaying systemic aging and related diseases.

[0005] D-galactose (D-gal) is a classic inducer of skin aging models. When excessively supplied in the body, it leads to the abnormal metabolism of galactitol and oxidative stress, resulting in the generation and accumulation of advanced glycation end products (AGEs). These products gradually deposit in skin tissue, ultimately accelerating the skin aging process by disrupting normal cellular function and inducing oxidative stress damage. Based on this mechanism, D-galactose has been widely used to construct models simulating endogenous aging and glycation damage, becoming an important research tool for evaluating the efficacy of anti-aging drugs and active ingredients.

[0006] In recent years, mammalian-derived exosomes have been extensively studied and found to play a crucial role in regulating intercellular communication. However, the isolation of mammalian-derived exosomes has consistently faced challenges such as high cost, time consumption, and low yield. Meanwhile, plant-derived exosomes, due to their inherently low immunogenicity, low-cost production, and widespread availability, have demonstrated significant advantages and attracted considerable attention.

[0007] Avocado (Persea americana, also known as alligator pear) is a highly regarded natural skincare product. Its flesh is rich in monounsaturated fatty acids, vitamin E, polyphenols, and other active substances, and it is recognized for its nourishing, repairing, antioxidant, moisturizing, and health-promoting properties. Currently, there is limited research on the anti-inflammatory, wound-healing, and drug delivery effects of plant-derived exosome-like nanovesicles; however, there are no reports on whether avocado-derived exosome-like nanovesicles can play a role in improving skin aging.

[0008] Therefore, establishing a convenient and high-yield method for preparing avocado exosome-like nanovesicles and verifying their efficacy in improving skin aging is of significant research value and practical importance. Summary of the Invention

[0009] To address some shortcomings in existing technologies, this invention provides avocado-derived exosome-like nanovesicles (AELNs), their preparation method, and applications. Through condition optimization and screening, this invention obtains a method for preparing avocado-derived exosome-like nanovesicles. This method exhibits superior extraction efficiency, significantly increases vesicle yield, and is more suitable for large-scale preparation. The avocado-derived exosome-like nanovesicles extracted by this method can significantly delay skin aging. They can significantly improve D-galactose-induced fibroblast aging damage in the skin, and also show significant improvement effects on D-galactose-induced skin aging damage in ICR mice. Therefore, this invention has promising application prospects in preventing or delaying skin aging.

[0010] To achieve the above-mentioned technical objectives, the present invention employs the following technical means:

[0011] This invention first provides a method for preparing avocado-derived exosome-like nanovesicles, the method comprising:

[0012] (1) Cut the washed, peeled and pitted fresh avocado into chunks, add buffer solution and juice, then filter with 100 mesh gauze to obtain avocado juice;

[0013] (2) The avocado juice was subjected to differential-ultracentrifugation to obtain the supernatant. Then, the obtained supernatant was ultracentrifuged to obtain the precipitate. The precipitate was dissolved in buffer overnight and resuspended to obtain the resuspended solution.

[0014] (3) The resuspension was filtered in a sterile filter to obtain sterile avocado-derived exosome-like nanovesicles.

[0015] Preferably, in step (1), the avocado is a Hass avocado (Persea americana 'Hass').

[0016] The buffer solution includes PBS, and the mass-to-volume ratio of avocado to PBS during juicing is 200g:400mL;

[0017] The juicing process is as follows: pause the blender every 90 seconds, pause for 30 seconds, and repeat twice.

[0018] Preferably, in step (2), the differential-ultracentrifugation step includes:

[0019] S1. Centrifuge the avocado juice at 4°C at 5000×g for 30 minutes, 8000×g for 30 minutes, and 12000×g for 30 minutes in sequence. After each centrifugation, discard the precipitate and collect the supernatant.

[0020] S2. Repeat step 2 to 5 of centrifugation at 12000×g until there is no visible precipitate at the bottom of the centrifuge tube;

[0021] S3. Combine all supernatants, dispense into ultracentrifuge tubes, centrifuge at 100,000×g for 1.5 hours at 4°C, and discard the supernatant;

[0022] S4. After resuspending the precipitate with phosphate buffer, wash it again by centrifugation at 100,000×g for 1.5 hours at 4°C; discard the supernatant, add an appropriate amount of phosphate buffer to the precipitate, and let it stand overnight at 4°C to dissolve.

[0023] Preferably, in step (3), the filtration is performed using a sterile filter with a diameter of 0.22 μm.

[0024] The present invention also provides avocado-derived exosome-like nanovesicles obtained by the above method. The avocado-derived exosome-like nanovesicles are saucer-like, have a phospholipid bilayer structure, an average particle size of 164.8 nm, and an average zeta potential of -31.6 ± 0.43 mV.

[0025] This invention also provides the application of the above-mentioned avocado-derived exosome-like nanovesicles in the preparation of anti-aging products.

[0026] Preferably, the products include medical devices, tissue-engineered products, and cosmetics.

[0027] The present invention also provides an anti-aging product comprising avocado-derived exosome-like nanovesicles.

[0028] The present invention also provides the application of the above-mentioned avocado-derived exosome-like nanovesicles in the preparation of products for the prevention and / or treatment of skin damage.

[0029] Preferably, the skin lesions are caused by D-galactose;

[0030] The products include medical devices, tissue engineering products, and cosmetics.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0032] The avocado-derived exosome-like nanovesicles extracted by the method of this invention exhibit a saucer-like structure with a phospholipid bilayer. The average particle size is 164.8 nm, and the average zeta potential is -31.6 ± 0.43 mV, indicating negative charge. This allows them to effectively cross various tissue barriers while avoiding the clearance effect of mononuclear phagocytes. Furthermore, the extraction method of this invention achieves a high extraction rate, with the extracted avocado-derived exosome-like nanovesicles reaching a concentration of 5 × 10⁻⁶. 11 ~7×10 11 The yield was significantly improved by increasing the number of particles per mL.

[0033] This invention optimizes the extraction method of avocado-derived exosome-like nanovesicles, improving their yield. The extracted avocado-derived exosome-like nanovesicles can significantly delay skin aging, significantly improve D-galactose-induced dermal fibroblast aging damage, and also show significant improvement on skin aging in ICR mice. It has good application prospects in preventing or delaying skin aging. Attached Figure Description

[0034] Figure 1 Figure A shows the identification and characterization of avocado-derived exosome-like nanovesicles; Figure A is a transmission electron microscope image (scale bar = 100 nm); Figure B shows the results of nanoparticle tracking analysis, where the left image in Figure B shows the average particle size distribution and the right image shows the average zeta potential.

[0035] Figure 2 This study describes the biological characteristics of dermal fibroblasts (DFs) and the construction of a D-galactose-induced dermal fibroblast senescence model. In the figure, A represents CK10 detected by immunofluorescence (scale bar = 100 μm); B represents α-SMA detected by immunofluorescence (scale bar = 100 μm); C represents Vimentin detected by immunofluorescence (scale bar = 100 μm); D represents cell viability of DF cells after treatment with different concentrations of D-gal for different time periods, as detected by CCK-8 assay; and E represents the expression levels of COL1A1 and P21 proteins of cells treated with D-gal for 24 h by Western blotting.

[0036] Figure 3This study evaluates the anti-aging effects and safety of avocado-derived exosome-like nanovesicles in vitro. In the figure, A shows cell viability after co-incubation with AELNs and DFs for different times, as detected by CCK-8 assay; B shows the effect of AELN pretreatment on D-galactose-induced cell viability, as detected by CCK-8 assay; C shows the β-galactosidase staining image (C1) and its semi-quantitative analysis (C2) (scale bar = 200 μm); D shows the cellular reactive oxygen species level (D1) and its semi-quantitative analysis (D2) (scale bar = 2). E is the cell immunofluorescence detection of COL1A1 content (E1) and its semi-quantitative analysis (E2) (scale bar = 100μm); F is the cell immunofluorescence detection of Ki67 content (F1) and semi-quantitative analysis (F2) (scale bar = 100μm); G is the cell immunofluorescence detection of γ-H2AX content (G1) and semi-quantitative analysis (G2) (scale bar = 100μm); H is the Western blot detection of the protein expression levels of COL1A1, P21 (H1), LaminB1, TGF-β (H2), and BAX (H3) after AELNs treatment; I is the real-time PCR detection of the mRNA expression levels of P16 (I1), TGF-β (I2), and IL-8 (I3) in cells after AELNs treatment.

[0037] Figure 4 The particle size distribution of avocado-derived exosome-like nanovesicles obtained under different centrifugation parameters is compared. In the figure, A shows the particle size distribution of vesicles obtained by differential centrifugation (30 minutes each at 1000×g, 2000×g, and 10000×g) combined with ultracentrifugation (100,000×g, 1.5 hours); B shows the particle size distribution of vesicles obtained by differential centrifugation (30 minutes each at 5000×g, 8000×g, and 12000×g, with the 12000×g centrifugation step repeated 2-5 times) combined with ultracentrifugation (120,000×g, 2 hours); C shows the particle size distribution of vesicles obtained by differential centrifugation (30 minutes each at 5000×g, 8000×g, and 12000×g, with the 12000×g centrifugation step repeated 2-5 times) combined with ultracentrifugation (150,000×g, 2 hours).

[0038] Figure 5 To evaluate the in vivo anti-aging effects of avocado-derived exosome-like nanovesicles; Figure A is a schematic diagram of Masson staining of skin tissue (scale bar = 100 μm); Figure B is a schematic diagram of HE staining of skin tissue (scale bar = 50 μm). Detailed Implementation

[0039] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto. The technical solutions of the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Unless otherwise defined, the technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0041] Example 1:

[0042] (1) Preparation of avocado juice:

[0043] Take fresh avocado fruit, wash the surface thoroughly with distilled water, and remove the peel and pit with a peeler. Accurately weigh 200 g of pulp, cut it into small pieces of about 1 cm³, transfer it to a juicer, and add 400 mL of pre-cooled PBS. Use an intermittent homogenization mode (90 seconds on, 30 seconds off, repeated twice) for juicing. Filter the resulting homogenate thoroughly through 100-mesh nylon gauze, collect the filtrate, and aliquot it into 50 mL centrifuge tubes for temporary storage at 4°C.

[0044] (2) Extraction of avocado-derived exosome-like nanovesicles:

[0045] The avocado juice obtained above was subjected to differential-ultracentrifugation in the following order: First, centrifuged at 5000×g for 30 min at 4°C, discarded the precipitate, and collected the supernatant; then centrifuged at 8000×g for 30 min, discarded the precipitate, and collected the supernatant; then centrifuged at 12000×g for 30 min, discarded the precipitate, and collected the supernatant; this 12000×g centrifugation step was repeated 2 to 5 times until no visible precipitate was found at the bottom of the centrifuge tube. The resulting supernatants were combined, aliquoted into ultracentrifuge tubes, precisely balanced, and ultracentrifuged at 100,000×g for 1.5 h at 4°C. The supernatant was discarded, the precipitate was resuspended in PBS, and washed again by ultracentrifugation at 100,000×g for 1.5 h at 4°C. The supernatant was discarded, and the precipitate was gently resuspended in 400 μL of PBS to obtain a resuspended solution, which was then allowed to stand overnight at 4°C to dissolve.

[0046] In this invention, PBS is used to resuspend the precipitate; washing the precipitate with PBS can reduce the impurity content and improve the purity of avocado-derived exosome-like nanovesicles; the amount of PBS used can be conventionally selected according to actual needs.

[0047] (3) Filtering treatment:

[0048] In a clean bench, the obtained resuspension was drawn up using a 1 mL sterile syringe and filtered through a 0.22 μm sterile needle filter for sterilization. The resulting filtrate was sterile avocado-derived exosome-like nanovesicles (AELNs), which were aliquoted into 1.5 mL centrifuge tubes and stored at -80°C for later use.

[0049] Figure 1 A is a transmission electron microscope image of AELNs. As shown in the figure, the vesicles exhibit a typical exosome-like "cabinet-like" structure with clear and intact membrane boundaries. Figure 1 Image B shows the particle size distribution and zeta potential of AELNs detected by NTA. As can be seen from the image, the average particle size of AELNs is 164.8 nm, and the particle size distribution is concentrated. The average zeta potential is -31.6 ± 0.43 mV, which is relatively high in absolute value, indicating that the electrostatic repulsion between vesicles is strong, the dispersion system is stable, and the vesicle membrane structure is intact.

[0050] Example 2:

[0051] To determine the optimal combination of centrifugation parameters, this embodiment systematically optimized the differential centrifugation speed and ultracentrifugation conditions. All experiments used the particle size distribution measured by nanoparticle tracking analysis technology as the main evaluation index of vesicle uniformity.

[0052] (1) Optimization of differential centrifugation parameters:

[0053] This step uses centrifugation at 1000×g, 2000×g, and 10000×g for 30 minutes each as controls, and centrifugation at 5000×g, 8000×g, and 12000×g for 30 minutes each as the control in Example 1. The 12000×g centrifugation step can be repeated 2-5 times as the experimental group. Only the differential centrifugation parameters are changed; other steps are the same as in Example 1. The control group is evaluated separately. Figure 3 A) and experimental group ( Figure 1 B) Evaluation of the vesicle homogeneity of the extracted avocado-derived exosome-like nanovesicles, as shown in the following results. Figure 1 B and Figure 3 Figure A shows that the AELNs obtained in the experimental group have a more concentrated particle size distribution, indicating higher purity. This confirms that a higher rotation speed combination can more effectively remove tissue debris and subcellular organelles, thereby enriching nanovesicles with better purity and uniformity.

[0054] (2) Optimization of ultracentrifugation parameters:

[0055] Based on the optimization results of step (1), the parameters for fixed differential centrifugation are 5000×g, 8000×g, and 12000×g, each for 30 minutes, with the 12000×g centrifugation step repeated 2-5 times. The parameters for ultracentrifugation are adjusted, except for the 100,000×g centrifugation for 1.5 hours as in Example 1. Figure 1 In addition to B), centrifuge at 120,000 × g for 2 hours. Figure 3 B) and centrifuged at 150,000 × g for 2 hours ( Figure 3 C) The optimized control group, with other steps the same as in Example 1, evaluated the effects of different ultracentrifugation rates on the vesicle uniformity of avocado-derived exosome-like nanovesicles. The results are as follows: Figure 1 B and Figure 3 B, Figure 3 As shown in C.

[0056] As can be seen from the figure, the vesicles obtained by Example 1 have the most concentrated particle size distribution and the best uniformity, making them more suitable as the preferred conditions for large-scale production.

[0057] In summary, centrifugation at 5000×g, 8000×g, and 12000×g for 30 minutes each was selected as the reaction parameters for differential centrifugation. The 12000×g centrifugation step can be repeated 2 to 5 times until there is no visible precipitate at the bottom of the tube. Centrifugation at 100,000×g for 1.5 hours was selected as the reaction parameter for ultracentrifugation.

[0058] Example 3:

[0059] This embodiment first uses D-galactose (D-gal) to induce dermal fibroblasts (DFs) to construct a dermal fibroblast senescence model, and then examines the effect of AELNs in the cell model. The specific steps are as follows:

[0060] (1) Isolation, culture and identification of DFs:

[0061] The dermal fibroblasts (DFs) used in this step were primary cells that were isolated and cultured independently. Immunofluorescence staining confirmed that they expressed α-SMA and Vimentin, but did not express CK10, consistent with the molecular marker characteristics of DFs. The specific isolation, culture, and identification steps are as follows:

[0062] Pulpy SD rats (purchased from the Experimental Animal Center of Jiangsu University) were euthanized by cervical dislocation and then disinfected by immersion in 75% alcohol for 15–30 min. Skin from the back and abdomen of the pulpy rats was excised and placed in PBS solution containing 1% penicillin-streptomycin. The back skin was peeled off, and the adipose tissue was scraped off with forceps. The cells were cut into small pieces and placed on the bottom of a medium-sized dish. After air-drying, the cells were cultured in α-MEM medium containing 10% fetal bovine serum for 3–7 days until cells emerged from the tissue blocks. The medium was changed every 48 h. After the cells exhibited vortex growth, the tissue blocks were removed, washed with PBS, and passaged using 0.25% trypsin.

[0063] The isolated cells were identified using immunofluorescence, and the results showed that they expressed α-SMA ( Figure 2 B) and Vimentin Figure 2 C), without expressing CK10 ( Figure 2 A), consistent with the characteristics of dermal fibroblasts. The obtained cells were confirmed to be dermal fibroblasts. The identified dermal fibroblasts (DFs) were used for subsequent experiments.

[0064] (2) Construction of a dermal fibroblast senescence model:

[0065] This step first established a normal control group (CTR) and model groups with different concentrations of D-galactose (10 mg / mL, 20 mg / mL, 30 mg / mL) to determine the appropriate conditions for D-galactose (D-gal)-induced senescence of dermal fibroblasts (DFs). The specific steps are as follows:

[0066] After digestion and resuspension of DFs, 4 × 10⁻⁶ ppm were used per 4 Cells were seeded at a density of [number] cells per well in 6-well cell culture plates. After cell attachment, 1 mL of D-galactose solution of the corresponding concentration was added to each model group, and stimulation was performed for 24 h and 48 h, respectively. The results are as follows: Figure 2 As shown.

[0067] Based on the combined results of cell viability and molecular marker detection, stimulation with 20 mg / mL D-galactose solution for 24 hours was determined to be the optimal condition for constructing a dermal fibroblast senescence model. Under these conditions, cell viability can be maintained above 80%. Figure 2 D), and Western blot analysis ( Figure 2 E) Significant changes in the expression of aging-related markers were confirmed, namely, decreased COL1A1 protein expression and increased P21 protein expression, indicating that the cells have entered a typical aging state. Therefore, subsequent model construction all followed the standard procedure: dermal fibroblasts were cultured at 4 × 10⁶ cells per well. 4Inoculate the cells at a density of 100 cells / well in a 6-well plate. After adhesion, add D-galactose solution to a final concentration of 20 mg / mL and treat for 24 hours.

[0068] (3) Evaluation of the effects of AELNs in a cell senescence model:

[0069] This step uses the CCK-8 assay to examine the cytotoxicity of AELNs to dermal fibroblasts (DFs) in order to evaluate their biocompatibility. The specific steps are as follows:

[0070] DFs are arranged in a pattern of 5×10 per hole. 3 Cells were seeded in 96-well plates. After cell adhesion, three groups were set up: a normal control group (CTR), a PBS solvent control group (PBS), and an AELNs treatment group (AELNs). The AELNs group was treated with 1×10⁻⁶ PBS. 10 AELNs were added in groups of 1 particle / mL, and an equal volume of PBS was added to the PBS group.

[0071] After co-incubating the above groups for 24 h, 48 h, and 72 h respectively, the culture medium was discarded, and CCK-8 reagent was added. The mixture was then incubated in a 37 ℃, 5% CO2 incubator in the dark for 2 h. The absorbance was measured at 450 nm, and the results are as follows: Figure 3 As shown in Figure A, there was no significant difference in cell viability between the AELNs group and the control group at any time point, indicating that AELNs had no significant cytotoxicity to DFs and possessed good cell compatibility and biosafety.

[0072] This step also established a D-galactose (D-gal)-induced cell senescence model to evaluate the anti-aging effects of AELNs. DFs were distributed at 4 × 10⁻⁶ cells per well. 4 Individuals were inoculated into 6-well plates and, after adhesion, were divided into three groups: normal control group (CTR), D-galactose model group (D-gal), and AELNs pretreatment group (AELNs). The AELNs group received 1×10⁻⁶ cells / well. 10 Pretreatment with AELNs at a concentration of 1 particle / mL for 24 h, followed by stimulation with D-gal solution at a final concentration of 20 mg / mL for 24 h in both the D-gal group and the AELNs group. After stimulation, the effect of AELNs on the cell senescence model was examined, and the results are as follows: Figure 3 As shown.

[0073] As shown in the figure, compared with the D-gal model group, the cell viability of the AELNs pretreatment group was significantly restored, indicating that AELNs can effectively alleviate the decrease in cell viability caused by D-gal and protect cell proliferation function. Figure 3 B); After AELNs treatment, the proportion of senescent positive cells stained blue decreased significantly, suggesting that cellular senescence was alleviated. Figure 3C); AELNs pretreatment significantly reduced intracellular ROS levels, confirming its ability to alleviate oxidative stress (C). Figure 3 D). In addition, immunofluorescence staining ( Figure 3 E, Figure 3 F, Figure 3 G), Western blot ( Figure 3 H) and quantitative PCR (H) Figure 3 I) Further investigation revealed the role of AELNs at the protein and gene transcription levels. Results showed that compared to the D-gal model group, the protein expression levels of COL1A1, Ki67, TGF-β, and Lamin B1 were significantly upregulated in the AELNs pretreatment group, while the protein expression of γ-H2AX, P21, and BAX was significantly decreased. Quantitative real-time PCR results were consistent with these protein expression trends: AELNs pretreatment significantly downregulated the mRNA expression levels of P16 and the pro-inflammatory cytokine IL-8, while upregulating the mRNA expression of TGF-β.

[0074] Among the above-mentioned indicators, COL1A1 is a major structural protein of the extracellular matrix of dermal cells, reflecting collagen synthesis capacity; Ki67 is a nuclear protein that marks cell proliferation; γ-H2AX is a histone variant that marks DNA double-strand breakage; downregulation of Lamin B1 is one of the important markers of cellular senescence; TGF-β is a key regulator of fibrosis and collagen synthesis; P21 and P16 are both cyclin-dependent kinase inhibitors, and their upregulation can mediate cell cycle arrest and promote cellular senescence; BAX is a pro-apoptotic protein, and its overexpression is associated with apoptosis and senescence; IL-8 is an important pro-inflammatory factor in senescence-related secretory phenotypes.

[0075] In summary, AELNs can maintain collagen synthesis and promote cell proliferation by upregulating the expression of COL1A1, Ki67, TGF-β, and Lamin B1; while downregulating the expression of γ-H2AX, P21, P16, BAX, and IL-8, thereby inhibiting DNA damage, cell cycle arrest, apoptosis pathways, and inflammatory responses. These results indicate that AELNs can effectively delay D-galactose-induced senescence of dermal fibroblasts from multiple targets and at multiple levels.

[0076] Example 4:

[0077] In this embodiment, a mouse skin aging model was constructed using D-galactose induction, and then the effects of AELNs on the mouse skin aging model were investigated. The specific steps are as follows:

[0078] Twenty-four 6-week-old female ICR mice (purchased and housed at the Experimental Animal Center of Jiangsu University, with all animal experiments conducted in accordance with standard guidelines for the care and use of laboratory animals and approved by the Ethics Committee of Jiangsu University) were acclimatized for one week and then randomly divided into four groups of six mice each: normal control group (CTR group), D-galactose model group (D-gal group), D-galactose + AELNs treatment group (D-gal + AELNs group), and D-galactose + PBS treatment group (D-gal + PBS group).

[0079] Next, a skin aging model was constructed. The specific steps were as follows: D-galactose was dissolved in 70% ethanol to prepare a 10 mg / mL solution. 0.5 mL of this solution was applied evenly to the hair-removed area on the back of mice twice daily for 6 weeks. The treatment methods for each group were as follows: The normal control group (CTR group) received an equal volume of 70% ethanol daily in the same manner to eliminate the potential effects of the solvent on the skin; the D-galactose model group (D-gal group) underwent the modeling process as described above without any other interventions; the D-galactose + AELNs treatment group (D-gal + AELNs group) received 1×10 mg / mL of D-galactose solution applied to the hair-removed area on the back using nanoneedles on the first day of each week, starting from the first week of modeling. 10 AELNs were administered to individual particles weekly for 6 weeks; the D-galactose + PBS treatment group (D-gal + PBS group) received an equal volume of PBS in the same manner. On the first day of each week, the CTR group and the D-gal group received no treatment.

[0080] After the experiment, the mice were euthanized, and the skin tissue from their backs was collected for section analysis. The results are as follows: Figure 5 As shown in the figure, compared with the normal control group, the dermal collagen content of mice in the D-gal group was significantly reduced, while the proportion of collagen fibers was significantly increased after AELNs treatment, indicating that AELNs can effectively alleviate D-gal-induced collagen loss. Figure 5 A). Furthermore, compared to the normal control group, the epidermis of mice in the D-gal group was significantly thinner, while the epidermal thickness increased after AELNs treatment, indicating that AELNs can effectively alleviate D-gal-induced epidermal thinning. The following conclusions can be drawn: long-term application of D-gal can lead to epidermal thinning and dermal collagen loss in mice, while the above pathological changes in mice treated with AELNs were significantly improved, with epidermal thickness and collagen content approaching normal levels. Figure 5 B).

[0081] Therefore, this embodiment systematically evaluated the in vivo anti-aging effect of AELNs by constructing a D-galactose-induced mouse skin aging model. Masson staining and HE staining results consistently showed that long-term application of D-galactose led to thinning of the epidermis and reduction of dermal collagen content in mice, successfully simulating the typical pathological characteristics of skin aging. After treatment with AELNs assisted by nanoneedles, the above pathological changes were significantly improved, the proportion of dermal collagen fibers significantly increased, and the epidermal thickness returned to near-normal levels. These results confirm that AELNs can effectively antagonize D-galactose-induced skin aging in vivo and have good in vivo anti-aging efficacy.

[0082] In summary, this invention, through system condition optimization and screening, has yielded a method for preparing avocado-derived exosome-like nanovesicles. This method offers superior extraction efficiency and product uniformity, making it suitable for large-scale preparation. In vitro and in vivo experimental results demonstrate that the prepared avocado-derived exosome-like nanovesicles can effectively combat skin aging, exhibiting excellent anti-aging activity at both the tissue and cellular levels, and possessing significant practical value.

[0083] The embodiments described above are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for preparing avocado-derived exosome-like nanovesicles, characterized in that, The preparation method includes: (1) Cut the washed, peeled and pitted fresh avocado into chunks, add buffer solution and juice, then filter with 100 mesh gauze to obtain avocado juice; (2) The avocado juice was subjected to differential-ultracentrifugation to obtain the supernatant. Then, the obtained supernatant was ultracentrifuged to obtain the precipitate. The precipitate was dissolved in buffer overnight and resuspended to obtain the resuspended solution. (3) The resuspension was filtered in a sterile filter to obtain sterile avocado-derived exosome-like nanovesicles.

2. The preparation method according to claim 1, characterized in that, In step (1), the avocado includes Hass avocado; The buffer solution includes PBS, and the mass-to-volume ratio of avocado to PBS during juicing is 200g:400mL; The juicing process is as follows: pause the blender every 90 seconds, hold for 30 seconds, and repeat twice.

3. The preparation method according to claim 1, characterized in that, In step (2), the differential-ultracentrifugation process includes: S1. Centrifuge the avocado juice at 4°C at 5000×g for 30 minutes, 8000×g for 30 minutes, and 12000×g for 30 minutes in sequence. After each centrifugation, discard the precipitate and collect the supernatant. S2. Repeat step 2 to 5 of centrifugation at 12000×g until there is no visible precipitate at the bottom of the centrifuge tube; S3. Combine all supernatants, dispense into ultracentrifuge tubes, centrifuge at 100,000×g for 1.5 hours at 4°C, and discard the supernatant; S4. After resuspending the precipitate with phosphate buffer, wash it again by centrifugation at 100,000×g for 1.5 hours at 4°C; discard the supernatant, add an appropriate amount of phosphate buffer to the precipitate, and let it stand overnight at 4°C to dissolve.

4. The preparation method according to claim 1, characterized in that, In step (3), the filtration is performed using a sterile filter with a diameter of 0.22 μm.

5. The avocado-derived exosome-like nanovesicles obtained by the method according to any one of claims 1-4, characterized in that, The avocado-derived exosome-like nanovesicles are saucer-like, have a phospholipid bilayer structure, an average particle size of 164.8 nm, and an average zeta potential of -31.6 ± 0.43 mV.

6. The application of the avocado-derived exosome-like nanovesicles as described in claim 5 in the preparation of anti-aging products.

7. The application according to claim 6, characterized in that, The products include medical devices, tissue engineering products, and cosmetics.

8. An anti-aging product, characterized in that, The anti-aging product comprises the avocado-derived exosome-like nanovesicles as described in claim 5.

9. The use of the avocado-derived exosome-like nanovesicles of claim 5 in the preparation of products for the prevention and / or treatment of skin damage.

10. The application according to claim 9, characterized in that, The skin damage was caused by D-galactose; The products include medical devices, tissue engineering products, and cosmetics.

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