Asiatic acid elastic self-adaptive liposome as well as preparation method and application thereof

Abstract

The invention discloses an asiatic acid elastic self-adaptive liposome as well as a preparation method and application thereof. The betaine and the lipidosome are utilized to synergistically improve the membrane stability, permeability and binding capacity of the lipidosome, the asiatic acid lipidosome synthesized by the asiatic acid instead of cholesterol is used as a delivery carrier of the exosome to improve the skin microenvironment and increase the effective utilization rate, and the Tween 80 is utilized to reduce the membrane surface tension of the lipidosome. Meanwhile, sodium deoxycholate is adopted to enhance the electrostatic interaction between the liposome and the skin, so that the liposome can generate reversible shape change when exposed to external stimulation, and the transdermal efficiency and the stability of the liposome are improved again while certain self-adaption is achieved. Finally, a lipid nano-carrier capable of realizing exosome non-invasive efficient transdermal delivery, remodeling a skin microenvironment and treating skin related diseases is constructed. A new thought is provided for transdermal delivery and clinical application of the exosome.

Description

Technical Field

[0001] The present invention belongs to the field of transdermal delivery of exosomes, and particularly relates to asiatic acid elastic adaptive liposomes, a preparation method thereof and applications thereof. Background Art

[0002] Since exosomes contain DNA, microRNA, proteins and other bioactive compounds, and at the same time have the characteristics of diverse sources, high production cost-effectiveness, simple separation, low immunogenicity, high biocompatibility, low cytotoxicity, etc., they are often applied to the fields of drug delivery, tissue repair and regeneration. Due to its structural characteristics, single exosomes are often administered intravenously or directly by local injection for invasive treatment. Conventional treatment methods cannot achieve good biosafety, tissue targeting, utilization efficiency and stability, resulting in certain limitations in the use of exosome therapy. There is an urgent need for new delivery strategies. Non-invasive transdermal delivery is an extremely attractive method and has made great contributions to medical clinical applications. It avoids the first-pass effect, has the advantages of non-invasive drug delivery, high efficiency, convenience and painlessness, and has been hailed as one of the most promising non-invasive drug delivery routes. It can bring compliance and convenience to patients and has broad application prospects. For example, Malakar et al. developed liposome gels containing insulin by the reverse evaporation method for the treatment of insulin-dependent diabetes and achieved good results (Saudi pharmaceutical journal, 20(2012)355-363.); Kurakula et al. loaded lipid nanoparticles and avanafil into a hydrogel film for transdermal treatment of erectile dysfunction, greatly improving the solubility and bioavailability of the drug (Journal of liposome research, 26(2016)288-296); these two works demonstrated the unique advantages of liposomes in transdermal treatment and the potential for future treatment of skin-related diseases. The carrier for realizing exosome transdermal delivery should have the following points: (1) high encapsulation efficiency; (2) stability during the transdermal process; (3) high transdermal penetration ability; (4) high bioactivity; (5) having a penetration retention effect; Therefore, how to construct a new type of material that can effectively penetrate the skin, achieve high-efficiency penetration and high utilization rate of exosomes in the skin, reshape the skin microenvironment and normal skin immune defense by designing the structure of the material, utilizing transdermal delivery and combining the advantages of exosome therapy to achieve non-invasive and highly efficient treatment of skin diseases has become an important scientific problem that urgently needs to be solved at present.

[0003] In recent years, researchers have proposed various methods to improve the effective utilization rate of exosomes, mainly through local injection or intravenous administration to treat skin-related diseases. However, there are problems such as high pain levels, which are not conducive to the effective release of exosomes, and a relatively long recovery period. Although microneedles loaded with exosomes can change the skin barrier function, solid, drug-coated, or hollow microneedles cannot be completely absorbed by the skin after creating micropores or delivering drugs to skin wounds. At the same time, the wound clot or scar tissue gradually formed during the wound healing process will become a physical barrier, hindering the penetration of exosomes into the deep wound surface, thereby reducing their efficacy. At present, most invasive treatment methods will cause skin damage and affect the normal physiological functions of the skin. Therefore, developing a non-invasive carrier for the transdermal delivery of exosomes has unique advantages.

[0004] As a drug carrier, liposomes have the advantages of reducing the dissolution rate, promoting effective absorption, improving bioavailability, high biocompatibility, low toxicity, and high encapsulation. They are widely used in the field of transdermal delivery. They can encapsulate hydrophilic or lipophilic drugs with poor water solubility and are widely used in skin, pulmonary, intravenous, and oral drug administration. Liposomes as carriers for exosome delivery have been widely studied. For example, the mExos@DSPE-Hyd-PMPC lipid nanoparticles developed by Xiao et al. successfully loaded milk exosomes for oral peptide delivery and applied it to the treatment of type 2 diabetes, verifying the drug delivery ability of milk exosome-liposome hybrid vesicles to evaluate their possible uses in exosome therapy (ACS nano 18.32(2024):21091-21111). Xuan et al. developed novel hybrid lipid-fused exosome nanoparticles using a simple freeze-thaw method. Through this method, hybrid lipid exosome nanoparticles were successfully prepared, demonstrating their excellent tumor cell drug delivery ability and providing an innovative method for enhancing drug delivery and regulating the tumor microenvironment (International Journal of Molecular Sciences 25.7(2024):3645). Multiple studies at home and abroad have proven the effectiveness of liposome nanocarriers in delivering exosomes to treat diseases. However, due to the poor stability of conventional liposomes and the lack of certain controlled release properties, it is difficult to achieve effective transdermal delivery of exosomes. Most treatment methods still use oral administration, injection, or microneedles. The difficulty of achieving non-invasive transdermal treatment with liposome exosomes still needs to be overcome.

[0005] In view of this, the present invention is specifically proposed. Summary of the Invention

[0006] In order to overcome the drawbacks and deficiencies of the prior art, the object of the present invention is to provide a asiatic acid elastic self-adaptive liposome and its preparation method and application. The asiatic acid elastic self-adaptive liposome is a multifunctional liposome with high transdermal efficiency, remodeling of the skin microenvironment and improvement of skin problems, and can achieve transdermal delivery of exosomes.

[0007] The present invention aims at how to achieve non-invasive transdermal delivery of exosomes, efficient penetration and retention effect, remodeling of the skin microenvironment and improvement of skin-related diseases. By using the penetration enhancer betaine and liposomes to synergistically improve the membrane stability, permeability and binding ability of liposomes, using asiatic acid to replace cholesterol to synthesize asiatic acid liposomes as the delivery carrier of exosomes to improve its skin microenvironment while increasing its effective utilization rate, using Tween 80 to reduce the membrane surface tension of liposomes, thereby enhancing its penetration effect on the skin barrier. At the same time, the anionic edge activator sodium deoxycholate is used to enhance the electrostatic interaction between liposomes and the skin, so that it can undergo reversible shape changes when exposed to external stimuli, have a certain degree of self-adaptability, and at the same time improve its transdermal efficiency and stability again. Finally, a lipid nanocarrier that can achieve non-invasive and efficient transdermal delivery of exosomes, remodeling of the skin microenvironment and treatment of skin-related diseases is constructed.

[0008] The object of the present invention is achieved by the following technical solutions:

[0009] One of the technical solutions of the present invention: A preparation method of an asiatic acid elastic self-adaptive liposome, comprising the following steps: dissolving phospholipids, asiatic acid and an anionic edge activator (sodium deoxycholate) in an organic solvent to obtain solution A, dissolving a neutral ionic edge activator (Tween 80) in enzyme-free water to obtain solution B, then slowly adding solution B to solution A, mixing the two evenly, evaporating the organic solvent by the thin film hydration method to remove the solvent to obtain solid C, hydrating solid C with a betaine solution, and obtaining a suspension of asiatic acid elastic self-adaptive liposomes with uniform particle size through ultrasonic and microfluidic high-pressure processes.

[0010] Preferably, the phospholipid includes soy lecithin, and the mass ratio of the phospholipid, asiatic acid, and anionic edge activator (sodium deoxycholate) is (1.5 - 2.5):(0.5 - 1.5):0.5 - 1; the organic solvent includes a mixed solution composed of ethanol and chloroform in a volume ratio of 1:(0.8 - 1); the dosage of the organic solvent is calculated based on adding 0.5 - 0.8 mg of soy lecithin (preferably 0.56 mg) per 1 mL; the mass ratio of the neutral ionic edge activator (Tween 80) to the asiatic acid phospholipid mixture is 1:(4 - 5), where the asiatic acid phospholipid mixture is a mixture of phospholipid, asiatic acid, and anionic edge activator; the mass-to-volume ratio of Tween 80 to enzyme-free water is (3 - 7) mg:2 mL; the slow addition rate is 4 - 5 mL / min; the mass-to-volume ratio of solid C to the betaine solution is (20 - 30) mg:20 mL; the concentration of the betaine solution is 0.52 - 1.25 mg / mL (preferably 1 mg / mL); the thin film hydration method is to use a high-shear dispersion emulsifier [with a power of 200 - 300 W (preferably 300 W), a rotation speed of 6000 - 8000 rpm (preferably 8000 rpm), and each time for 5 min] to mix the two evenly and then perform rotary evaporation. The set temperature is 40 - 60 °C (preferably 60 °C), the rotation speed is 80 - 100 rpm (preferably 100 rpm), the vacuum degree of the evaporation flask is 100 kPa, and rotary evaporation is performed for 1 - 1.5 h; the conditions for ultrasonic treatment are ultrasonic treatment (180 W, 5 s / 5 s) for 30 - 40 min (preferably 30 min); the conditions for the microfluidic high-pressure process are extrusion 3 - 5 times under a pressure of 500 - 800 bar (preferably extrusion 4 times under a pressure of 500 bar).

[0011] Preferably, the preparation method of the betaine solution includes the following steps:

[0012] Dissolve betaine in the enzyme-free water in a round-bottom flask under heating conditions at 50 - 70 °C (preferably 60 °C), and at the same time stir the reaction magnetically at 80 - 120 rpm (preferably 100 rpm), and perform ultrasonic treatment on the obtained solution.

[0013] The dosage of the betaine is calculated based on adding 0.52 - 1.25 mg (preferably 1 mg) of betaine per 1 mL of enzyme-free water; the time for the magnetic stirring reaction is 15 - 45 min (preferably 30 min), and the conditions for the ultrasonic treatment are ultrasonic treatment 5 times, each time lasting 1 - 5 min (preferably 3 min), and the interval between each ultrasonic treatment is 1 - 3 min (preferably 3 min).

[0014] The second technical solution of the present invention: An asiatic acid elastic self-adaptive liposome prepared according to the above preparation method.

[0015] The asiatic acid elastic adaptive liposome can be used as an application for preparing a transdermal transfection delivery carrier of exosomes.

[0016] Technical solution three of the present invention: A complex of asiatic acid elastic adaptive liposome loaded with exosomes, comprising exosomes and the above-mentioned asiatic acid elastic adaptive liposome; further, the mass ratio of the asiatic acid elastic adaptive liposome to exosomes is (80-100):1.

[0017] The preparation method of the complex of asiatic acid elastic adaptive liposome loaded with exosomes comprises the following steps: The complex solution comprises the above-mentioned asiatic acid elastic adaptive liposome suspension and exosomes; specifically, a certain amount of exosomes is added to the asiatic acid elastic adaptive liposome suspension, incubated, then ultrasonicated and finally extruded and homogenized to obtain a stable asiatic acid elastic adaptive liposome-exosome, that is, the complex of asiatic acid elastic adaptive liposome loaded with exosomes.

[0018] Preferably, the exosomes are at least one of broccoli exosomes, bitter gourd exosomes, grape exosomes, apple exosomes, lemon exosomes and ginger exosomes; more preferably ginger exosomes;

[0019] Preferably, the exosomes are obtained by extraction using density gradient ultracentrifugation.

[0020] Preferably, the incubation time is 2-3 h (preferably 3 h).

[0021] Preferably, the incubation temperature is 25-40 °C; further 37 °C.

[0022] Preferably, the ultrasonic time is 10-15 min (preferably 15 min).

[0023] Preferably, the conditions for extrusion and homogenization are to sequentially pass through polycarbonate membranes with pore sizes of 600 nm, 400 nm and 200 nm, and manually push it forward and backward 30-40 times (preferably 40 times).

[0024] Technical solution four of the present invention: The application of the above-mentioned complex of asiatic acid elastic adaptive liposome loaded with exosomes in the preparation of products for enhancing the stability of exosomes;

[0025] Technical solution five of the present invention: The application of the above-mentioned complex of asiatic acid elastic adaptive liposome loaded with exosomes in the preparation of products for enhancing the anti-inflammatory effect, improving the skin microenvironment, promoting angiogenesis, promoting cell proliferation or promoting cell repair of exosomes;

[0026] Sixth technical solution of the present invention: Application of the asiatic acid elastic adaptive liposome complex loaded with exosomes in the preparation of products for improving the transdermal efficiency of exosomes;

[0027] Seventh technical solution of the present invention: Application of the asiatic acid elastic adaptive liposome complex loaded with exosomes in the preparation of products for improving non-invasive delivery of exosomes for treating alopecia;

[0028] Eighth technical solution of the present invention: Application of the asiatic acid elastic adaptive liposome complex loaded with exosomes in the preparation of products for treating skin diseases through non-invasive delivery of exosomes.

[0029] The present invention has the following advantages and effects compared with the prior art:

[0030] (1) Compared with traditional exosome carriers, the present invention uses materials such as betaine, asiatic acid, sodium deoxycholate, phospholipids, and Tween 80 to prepare asiatic acid elastic adaptive liposomes, with safe and simple components; using the penetration enhancer betaine and liposomes to synergistically improve the membrane stability, permeability, binding ability of liposomes, using asiatic acid to replace cholesterol to synthesize asiatic acid elastic adaptive liposomes as the delivery carrier of exosomes to improve its skin microenvironment and increase its effective utilization rate at the same time, using Tween 80 to reduce the membrane surface tension of liposomes, thereby enhancing its penetration effect on the skin barrier, and at the same time using the anionic edge activator sodium deoxycholate to enhance the electrostatic interaction between liposomes and the skin, enabling it to undergo reversible shape changes when exposed to external stimuli, having a certain degree of self-adaptation and at the same time enhancing its transdermal efficiency and stability again. Finally, a lipid nanocarrier that can achieve non-invasive and efficient transdermal delivery of exosomes to reshape the skin microenvironment and treat skin-related diseases is constructed.

[0031] (2) The asiatic acid elastic adaptive liposomes of the present invention are usually smaller, more controllable in volume than traditional liposomes, have a longer circulation time in vivo and a high encapsulation efficiency; they have high stability in vitro and can effectively improve cell inflammation and promote angiogenesis.

[0032] (3) Compared with traditional exosome treatment methods, asiatic acid elastic adaptive liposomes improve the effective utilization rate of exosomes in the skin without affecting their biological activity and at the same time have a high transdermal efficiency, realizing the efficient utilization of exosomes.

[0033] (4) Compared with traditional exosome carriers, the carrier of the present invention has high stability in vitro and effectively improves the effective utilization rate of exosomes. At the same time, this carrier has low cytotoxicity and high angiogenesis ability in in vitro cell experiments, and at the same time has a certain anti-inflammatory effect and can improve the skin microenvironment.

[0034] (5) The carrier of the present invention can efficiently penetrate the skin with exosomes and has certain self - adaptability, effectively promoting its effective utilization in the skin and remodeling the skin microenvironment (improving the microenvironment for hair follicle growth). For example, carrying ginger exosomes can achieve a better therapeutic effect on skin diseases, providing new ideas for the transdermal delivery and clinical application of exosomes, and is expected to fully utilize the advantages of non - invasive delivery of exosomes for treating skin diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0036] Figure 1 Particle size and potential diagrams of different material groups.

[0037] Figure 2 Results diagrams of the deformation index and polydispersity index (PDI) of different material groups.

[0038] Figure 3 Western blot diagrams of ginger exosomes and different material groups loaded with ginger exosomes; among them, "GDNV" represents the ginger exosome group, "LIP@GDNV" represents the cholesterol liposome - ginger exosome group, "AL@GDNV" represents the asiatic acid liposome - ginger exosome group, and "BAL@GDNV" represents the asiatic acid elastic self - adaptive liposome - ginger exosome group.

[0039] Figure 4 Quantification of the fluorescence intensity of GDNV, LIP@GDNV, AL@GDNV, and BAL@GDNV permeating through the skin at different time intervals of 1 h, 3 h, 5 h, 7 h, 12 h, and 24 h.

[0040] Figure 5 Distribution of GDNV, LIP@GDNV, AL@GDNV, and BAL@GDNV in the skin after 24 - hour in vitro permeation in a Franz diffusion cell, scale bar = 100 μm.

[0041] Figure 6 Cytotoxicity diagrams of human umbilical vein endothelial cells in different material treatment groups.

[0042] Figure 7 Proliferation of human dermal papilla cells in different material treatment groups after DHT treatment.

[0043] Figure 8This is the expression graph of VEGF content in human immortalized keratinocytes treated with different materials after irradiation.

[0044] Figure 9 This is the expression graph of TNF-α content in human immortalized keratinocytes treated with different materials after irradiation.

[0045] Note: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. DETAILED DESCRIPTION

[0046] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0047] In addition, for the numerical range in the present invention, it is understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0048] The words “include,” “including,” “have,” “contain,” etc. used in the present invention are open-ended terms, meaning including but not limited to.

[0049] In the examples, the purity of the betaine used is ACS, ≥98%; the asiatic acid used is provided by Aladdin Chemical Co., Ltd.; the sodium deoxycholate, soybean lecithin, chloroform and ethanol used are all purchased from Sigma-Aldrich;

[0050] In the examples, room temperature refers to 10 to 35°C.

[0051] The description will not be repeated below.

[0052] Example 1

[0053] A preparation method of asiatic acid elastic adaptive liposomes comprises the following steps:

[0054] Soybean lecithin, asiatic acid, and sodium deoxycholate were dissolved in an organic solvent and heated with stirring until completely dissolved to obtain an asiatic acid phospholipid mixture solution. The mass ratio of soybean lecithin, asiatic acid, and sodium deoxycholate was 1.5:1:1. The organic solvent was a mixed solution composed of absolute ethanol and chloroform in a volume ratio of 1:1. The amount of the organic solvent added was 0.56 mg of soybean lecithin per 1 mL. Tween 80 was dissolved in enzyme-free water, and the mass-volume ratio of Tween 80 to enzyme-free water was 2 mg / mL. The mass ratio of Tween 80 to the asiatic acid phospholipid mixture was 1:4. It was slowly added (5 mL / min) to the above asiatic acid phospholipid mixture solution. After mixing the two evenly using a high-shear dispersion emulsifier (power 300 W, rotation speed 8000 rpm, each time for 5 min), rotary evaporation was carried out. The set temperature was 60 °C, the rotation speed was 100 rpm, and the vacuum degree of the evaporation flask was 100 kPa. Rotary evaporation was carried out for 1 - 1.5 h until the organic solvent was completely evaporated to form a thin layer of lipid. This lipid was ultrasonically hydrated using a betaine solution (preparation steps of the betaine solution: dissolve betaine in enzyme-free water in a round-bottom flask under heating at 60 °C, and at the same time stir magnetically at 100 rpm for 30 min. The obtained solution was ultrasonically treated 5 times, each time lasting 3 min, and the interval between each ultrasonic treatment was 3 min). The concentration of the betaine solution was 1 mg / mL. The betaine solution was slowly added dropwise to the formed lipid under heating conditions (and then stir magnetically). The mass-volume ratio of the lipid to the betaine solution was 1 mg:1 mL. The formed pre-emulsion was ultrasonically treated (180 W, 5 s / 5 s, 30 min) using an ultrasonic laboratory homogenizer UP100H. Then the sample was cooled at room temperature after being extruded by microfluidic high pressure (extruded 4 times under a pressure of 500 bar) to obtain a suspension of asiatic acid elastic adaptive liposomes modified with anionic edge activator sodium deoxycholate and penetration enhancer betaine with uniform particle size, denoted as BAL.

[0055] Referring to the preparation steps of the asiatic acid elastic adaptive liposomes in this example, only replace the same mass of asiatic acid in this example with cholesterol Cho, denoted as LIP.

[0056] Referring to the preparation steps of the asiatic acid elastic adaptive liposomes in this example, without adding Tween 80, betaine, and sodium deoxycholate, denoted as AL.

[0057] Example 2

[0058] Preparation of a complex of asiatic acid elastic adaptive liposomes loaded with exosomes, including the following steps:

[0059] At room temperature, an asiatic acid elastic self - adapting liposome suspension BAL with uniform particle size was obtained by ultrasonic and micro - jet high - pressure processes. In a 37°C water bath, ginger exosomes were mixed with the asiatic acid elastic self - adapting liposome suspension BAL at a mass - to - volume ratio of 0.01 mg:1 mL (where the mass ratio of ginger exosomes to asiatic acid elastic self - adapting liposomes is 1:100). After mixing evenly, it was placed in a 37°C shaker and incubated for 3 h, then ultrasonicated (for 15 min) and finally extruded and homogenized to obtain stable asiatic acid elastic self - adapting liposome - ginger exosome. The conditions for micro - jet high - pressure were extrusion 3 - 5 times at a pressure of 500 bar; the conditions for extrusion and homogenization were successively passing through polycarbonate membranes with pore sizes of 600 nm, 400 nm, and 200 nm, and manually pushing it forward and backward 40 times to obtain a uniformly mixed and stable asiatic acid elastic self - adapting liposome - ginger exosome suspension, denoted as BAL@GDNV, and stored at 4°C.

[0060] Referring to the preparation steps of an asiatic acid elastic self - adapting liposome complex loaded with exosomes in this example, BAL in this example was replaced with LIP or AL in Example 1 in equal volume, denoted as LIP@GDNV and AL@GDNV.

[0061] Among them, ginger exosomes were obtained by density gradient ultra - centrifugation of ginger; then, 1 mg of ginger exosomes was dissolved in 100 mL of PBS buffer (pH 7.4) to obtain a ginger exosome solution with a concentration of 0.01 mg / mL, denoted as GDNV.

[0062] Example 3

[0063] Disperse 100 μL of LIP, AL, BAL obtained in Example 1, GDNV, LIP@GDNV, AL@GDNV, and BAL@GDNV obtained in Example 2 into 1 mL of pure water respectively. After ultrasonic dispersion, the zeta potential and particle size were measured using a Malvern laser particle size analyzer. Each group was set with 3 replicates, and the results are as Figure 1 shown. It can be seen that the hydrated particle sizes of asiatic acid elastic self - adapting liposome - ginger exosome BAL@GDNV and LIP, AL, BAL, GDNV, AL@GDNV are all below 300 nm and are of uniform size; the addition of sodium deoxycholate, Tween 80, and betaine increased the absolute value of the zeta potential of BAL@GDNV, which fully reflects that liposome anionic edge activators and neutral ionic edge activators can maintain the electric double - layer condition of liposomes, keeping them at a relatively high zeta potential and thus maintaining their stability. At the same time, the polydispersity index (PDI) of different materials within one week was measured as Figure 2The stability of different materials was judged, and it was found that the PDI of BAL@GDNV was the most stable. At the same time, 1 mL of LIP@GDNV, AL@GDNV, and BAL@GDNV were respectively added to a liposome extruder ( Lipids, USA). The liposomes were passed through a 50-nm polycarbonate membrane at a pressure of 1 MPa. After 5 min, the extrusion volume was recorded, and the particle sizes of each formulation before and after extrusion were measured using a laser particle size analyzer. The deformation index was calculated according to the formula. As Figure 2 shown, the deformation index of the asiatic acid liposome-ginger exosome AL@GDNV without adding any edge activators and penetration enhancers was higher than that of the cholesterol liposome-ginger exosome LIP@GDNV with edge activators and betaine added, indirectly reflecting that the elasticity and adaptability of the liposome-ginger exosome AL@GDNV (0.309) synthesized with asiatic acid replacing cholesterol were higher than those of the cholesterol liposome-ginger exosome LIP@GDNV (0.103) with sodium deoxycholate, Tween 80, and betaine added; the deformation index of the liposome-ginger exosome BAL@GDNV with sodium deoxycholate, Tween 80, and betaine added reached 0.597, which was significantly higher than that of other material treatment groups, laying a foundation for the subsequent transdermal application of BAL@GDNV.

[0064]

[0065] Example 4

[0066] The expression of Alix and β-actin in different material groups of GDNV, LIP@GDNV, AL@GDNV, and BAL@GDNV obtained in Example 2 was verified by Western Blotting experiments. Using β-actin as an internal reference for protein expression, after incubation, it was detected using a chemiluminescent imaging system ( Figure 3) The following are the detailed steps. Take 50 μL of GDNV, LIP@GDNV, AL@GDNV, and BAL@GDNV material samples. Add 500 μL of RIPA lysis buffer and 5 μL of 1% protease inhibitor. Incubate on ice for 30 minutes and gently invert to mix evenly. Centrifuge the samples at 12,000 rpm for 15 minutes at 4°C, collect the supernatant, and avoid interfering substances. Use the BCA method to measure the protein concentration of different samples. It is recorded that the protein solution concentrations of GDNV, LIP@GDNV, AL@GDNV, and BAL@GDNV are between 0.2 and 0.4. Prepare a 10% - 12% polyacrylamide gel according to the separation molecular weight required. After casting the gel, use the electrophoresis tank to prepare the electrophoresis system. Add loading buffer (containing SDS and reducing agent) to each sample, then heat to 95°C for 5 minutes to denature the protein. Load the sample into the gel and add protein with molecular weight markers. Electrophorese at 80V until the dye front reaches the bottom of the gel. Then soak the PVDF membrane (or nylon membrane) in the transfer buffer for 10 minutes to ensure the membrane is wet. Stack the gel and the membrane in the transfer device and use the transfer settings (100V, 1h). Prepare 2.5 g of BSA dissolved in 50 mL of 1*TBST for the 5% blocking solution. Remove the transferred membrane, place it protein-side up in a box, and add the 5% blocking solution to block for 2 h (at room temperature). Then dilute the primary antibody against Alix with 4 mL of diluent (at a ratio of 1:500). Immerse the membrane in the diluted primary antibody solution and incubate overnight on a shaker at 4°C. Place the primary antibody at room temperature and then aspirate and recycle it. Wash three times with 1*TBST, changing every 10 minutes to remove unbound primary antibody. Dilute the HRP-labeled secondary antibody (usually at a ratio of 1:2000), immerse the membrane in the secondary antibody solution, and incubate on a shaker at room temperature for 2 hours. Wash the membrane 3 times with TBST, 10 minutes each time. Place the washed membrane in TBST. Prepare the developer according to the instructions and keep it in the dark throughout the process. Use the ECL chemiluminescence reagent, apply it to the membrane according to the instructions, and incubate for 5 minutes. Use the chemiluminescence imaging system to detect and obtain Figure 3 , proving effective binding to exosomes.

[0067] Example 5

[0068] Use a permeation area of 1.767 cm 2In vitro simulated skin penetration experiments were carried out on GDNV, LIP@GDNV, AL@GDNV, and BAL@GDNV labeled with the specific dye PKH-26 using a Franz diffusion cell. The receiving chamber was filled with phosphate buffer solution (pH = 7.4) and continuously stirred at a speed of 200 rpm. The temperature inside the diffusion cell was kept constant at 37 °C through a circulating water bath, and mouse skin of appropriate size (the mouse skin was prepared according to Example 7 of "CN115590774A - Hyaluronic Acid Liposome Assembly and Its Preparation Method and Application") was clamped between the supply chamber and the receiving chamber. The dermis of the mouse skin was contacted with the receptor solution of phosphate buffer solution (pH 7.4), and 2 mL of freshly prepared PKH-26 labeled GDNV, LIP@GDNV, AL@GDNV, and BAL@GDNV sample solutions (the final concentration of ginger exosomes was 0.2% w / v) were respectively given to the stratum corneum facing the supply chamber. Then, 1 mL of the receptor solution was taken out through the sampling port at different time intervals (1 h, 3 h, 5 h, 7 h, 12 h, 24 h) and replaced with an equal volume of phosphate buffer solution (pH = 7.4). The content of ginger exosomes in the receptor solution was analyzed by measuring the fluorescence intensity at 551 nm using a multifunctional microplate reader (Synergy H1). As Figure 4 , the asiatic acid liposome-ginger exosome AL@GDNV without adding any edge activators and penetration enhancers has a higher transdermal efficiency than the cholesterol liposome-ginger exosome LIP@GDNV added with edge activators and betaine, indirectly reflecting that the asiatic acid substituted cholesterol synthesized liposome-ginger exosome AL@GDNV has higher transdermal performance and self-adaptive ability than the cholesterol liposome-ginger exosome LIP@GDNV added with sodium deoxycholate, Tween 80, and betaine; at the same time, the results fully demonstrate that the asiatic acid elastic self-adaptive liposome-ginger exosome BAL@GDNV has the highest transdermal efficiency, laying a foundation for the asiatic acid elastic self-adaptive liposome-ginger exosome to improve the skin microenvironment and treat skin-related diseases in the future.

[0069] Example 6

[0070] After 24 h of in vitro penetration using a Franz diffusion cell in the experiment of Example 5, the excess sample solution on the skin surface was removed, the skin was taken out from the Franz diffusion cell, and the skin surface was washed with physiological saline to ensure no sample residue. The skin tissue was dried with filter paper and cut into pieces with an area of about 1 cm 2The thin slices were embedded with a cryosection embedding agent (OTC compound), fixed with liquid nitrogen, and then the frozen skin tissue was vertically cut into skin sections with a thickness of about 10 μm at -20 °C using a cryostat. The skin tissue sections were adhered to glass slides and the fluorescence distribution in the skin was observed using a high-end inverted fluorescence microscope (AxioCam MRc) (a filter with an emission wavelength of 567 nm and an excitation wavelength of 551 nm was selected). The results are as Figure 5 shown. The ginger exosomes hybridized with asiatic acid elastic adaptive liposomes successfully penetrated the stratum corneum and reached the epidermis and dermis positions.

[0071] Example 7

[0072] Grouping: blank control group (Healthy), experimental groups: LIP, AL, BAL, GDNV, LIP@GDNV, AL@GDNV, BAL@GDNVG groups.

[0073] Human umbilical vein endothelial cells (HUVECs cells) were seeded in 96-well plates at a density of 5×10 3 cells per well. After incubation in an environment of 37 °C and 5% CO2 for 24 h, the culture medium was removed and an equal volume of culture medium was added (wherein, the culture medium contained LIP, AL, BAL, GDNV, LIP@GDNV, AL@GDNV or BAL@GDNVG in a certain volume, and the final concentration of ginger exosomes in the culture medium containing GDNV, LIP@GDNV, AL@GDNV or BAL@GDNVG was 0.02% w / v). The blank control group (Healthy) was only cultured in the culture medium, and the experimental groups were cultured in the culture medium containing different materials. Each group had 5 parallel wells; after 24 h, CCK-8 indicator was added to the experimental wells and the blank control wells. After incubation in the dark for 2 hours, the optical density value of each well at 450 nm was measured using an enzyme-linked immunosorbent assay reader, and the cell proliferation rate after the action of different materials was calculated. Each group had 3 parallel controls. The cytotoxicity results are as Figure 6 shown. The results showed that the BAL@GDNV complex had the lowest toxicity, with obvious advantages compared with the BAL group, indicating that the ginger exosomes hybridized with asiatic acid elastic adaptive liposomes had a certain cell proliferation effect and good biosafety. At the same time, the cell proliferation effect of AL@GDNV was significantly higher than that of LIP@GDNV, indicating that the asiatic acid liposome-ginger exosomes had a certain self-adaptability and stronger cell regulation ability than the cholesterol liposome exosomes added with edge activators and betaine.

[0074] Example 8

[0075] Grouping: blank control group (Healthy), DHT group (Control), experimental groups: LIP, AL, BAL, GDNV, LIP@GDNV, AL@GDNV, BAL@GDNVG groups, and DHT treatment was performed simultaneously.

[0076] Human dermal papilla cells (HDPCs) were cultured in DMEM containing 10% fetal bovine serum and 1% antibiotics. The HDPCs were seeded into 96-well culture plates at a density of 6×10 3 cells per well. After incubation in an environment of 37°C and 5% CO2 for 24 h, the medium was removed and an equal volume of medium was added (wherein the medium contained the same concentration of DHT (dihydrotestosterone), and an equal volume of LIP, AL, BAL, GDNV, LIP@GDNV, AL@GDNV or BAL@GDNVG; and the final concentration of ginger exosomes in the medium containing the same concentration of DHT and an equal volume of GDNV, LIP@GDNV, AL@GDNV or BAL@GDNVG was 0.02%). The blank control group (Healthy) was only cultured in the medium, the DHT group (Control) was cultured in the medium containing DHT (final concentration of 0.02%), and the experimental groups were cultured in the medium containing DHT (final concentration of 0.02% w / v) and different materials. Each group was set with 5 parallel wells. After 24 h, cell viability was measured by the CCK8 assay. After removing the materials, CCK-8 reagent was added to each well. After culturing for 2 h, the absorbance at a wavelength of 450 nm was recorded using a multifunctional microplate reader, and the cell proliferation rate after the action of different materials was calculated. Each group was set with 3 parallel controls. As Figure 7 shown, the results showed that the cell proliferation effects of AL and AL@GDNV were significantly higher than those of LIP and LIP@GDNV, indicating that asiatic acid liposome-ginger exosomes AL@GDNV had a certain self-adaptability and stronger cell repair ability than cholesterol liposome-ginger exosomes LIP@GDNV added with marginal activators and betaine. It had the ability to regulate cell differentiation, maintain cell morphological stability, and restore cell viability under external stimulation. At the same time, the BAL@GDNV complex had the lowest toxicity, with obvious advantages compared with the DHT group, the blank control group and other material groups, indicating that ginger exosomes hybridized with asiatic acid elastic self-adaptive liposomes had the strongest ability to promote cell proliferation and improve the hair follicle growth microenvironment.

[0077] Example 9

[0078] Grouping: blank control group (Healthy), irradiation group (Control), experimental groups: LIP, AL, BAL, GDNV, LIP@GDNV, AL@GDNV, BAL@GDNVG groups, and irradiation was performed first.

[0079] Human immortalized keratinocytes (HaCat cells) were seeded at 5×10 4 cells per well in a 24-well plate. After incubation in an environment of 37 °C and 5% CO2 for 24 h, the medium was removed, and the cells were covered with a thin layer of PBS. Then, they were irradiated using an ULTRA-VITALUX lamp at a distance of 10 cm from the cells, with an energy of 100 mJ / cm 2 . After irradiation, an equal volume of medium was added (wherein the medium contained an equal volume of LIP, AL, BAL, GDNV, LIP@GDNV, AL@GDNV, or BAL@GDNV, and the final concentration of ginger exosomes in the medium containing GDNV, LIP@GDNV, AL@GDNV, or BAL@GDNV was 0.02% w / v). The blank control group (Healthy) was cultured only in the medium, the irradiation group (Control) was cultured in the medium after irradiation, and the experimental groups were cultured in the medium containing different materials after irradiation. Five parallel wells were set in each group. After incubation for 24 h, the sample solution was removed, total RNA was extracted from the cells, reverse transcription was performed using a relevant kit according to the operation instructions, and finally, the expression levels of vascular endothelial growth factor (VEGF) and tumor necrosis factor α (TNF-α) in the cells were measured using a real-time fluorescence quantitative PCR instrument. The results are as Figure 8 and Figure 9 shown. The cell repair effects of AL and AL@GDNV were significantly higher than those of LIP and LIP@GDNV, indicating that asiatic acid liposome-ginger exosomes AL@GDNV have a certain cell regulation ability and stronger cell repair ability than cholesterol liposome-ginger exosomes LIP@GDNV added with marginal activator and betaine. It can effectively regulate signal transduction between cells, activate or inhibit the expression of specific cytokines (such as VEGF, TNF-α, etc.), reduce inflammatory responses, and further promote cell repair. It helps exosomes to be more efficiently taken up by cells and delivered to the damaged site, thereby enhancing the repair effect. At the same time, compared with other material groups, the asiatic acid elastic adaptive liposome-ginger exosomes BAL@GDNV added with marginal activator and betaine had the best repair effect, indicating that BAL@GDNV has obvious advantages in promoting cell repair, inhibiting inflammatory responses, and optimizing drug delivery. It can not only effectively improve the skin microenvironment and regulate the expression of inflammation-related factors but also promote angiogenesis; prevent the aggravation of cell damage and support the natural repair process of cells.

[0080] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for preparing asiatic acid elastic adaptive liposomes, characterized in that: The following steps are involved: The phospholipid, Centella asiatica and anionic edge activator are dissolved in an organic solvent to obtain a solution A, the neutral ionic edge activator is dissolved in enzyme-free water to obtain a solution B, then the solution B is slowly added to the solution A, the two are mixed evenly, the organic solvent is evaporated by a thin film hydration method, the solvent is removed to obtain a solid C, the solid C is hydrated with a betaine solution, and an asiatica acid elastic adaptive liposome suspension with uniform particle size is obtained by an ultrasonic and microfluidization high-pressure process; The anionic edge activator is sodium deoxycholate; the neutral ionic edge activator is Tween 80.

2. The preparation method according to claim 1, characterized in that: The phospholipid comprises soybean lecithin, and the mass ratio of the phospholipid, Centella asiatica and anionic edge activator is (1.5-2.5): (0.5-1.5): 0.5-1; the organic solvent comprises a mixed solution of ethanol and chloroform in a volume ratio of 1: (0.8-1); the amount of the organic solvent is calculated by adding 0.5-0.8 mg of soybean lecithin per 1 mL; the mass ratio of the neutral ion edge activator to the Centella asiatica phospholipid mixture is 1: (4-5), wherein the Centella asiatica phospholipid mixture is a phospholipid , a mixture of Centella asiatica and anionic edge activator; the mass volume ratio of Tween 80 to enzyme-free water is (3-7) mg:2 mL; the speed of slow addition is 4-5 mL / min; the mass volume ratio of solid C to betaine solution is (20-30) mg:20 mL; the concentration of betaine solution is 0.52-1.25 mg / mL; the ultrasonic time is 30-40 min; the conditions of the microfluidic high-pressure process are extrusion at a pressure of 500-800 bar for 3-5 times.

3. An asiatic acid elastic adaptive liposome, characterized in that: Prepared according to the preparation method according to any one of claims 1 to 2.

4. Use of the asiatic acid elastic adaptive liposomes according to claim 3 as a carrier for preparing exosome transdermal transfection delivery.

5. An Asiatic acid elastic adaptive liposome complex loaded with exosomes, characterized in that: It comprises exosomes and the asiatic acid elastic adaptive liposomes as described in claim 3.

6. The Asiatic acid elastic adaptive liposome complex loaded with exosomes according to claim 5, characterized in that: The mass ratio of the asiatic acid elastic adaptive liposomes to the exosomes is (80-100):

1.

7. The method for preparing the exosome-loaded Centella asiatica elastic adaptive liposome complex according to any one of claims 5 to 6, characterized in that: The method comprises the following steps: the complex solution comprises the asiatic acid elastic adaptive liposome suspension and exosomes according to any one of claims 1 to 2; specifically, a certain amount of exosomes are added to the asiatic acid elastic adaptive liposome suspension, incubated, ultrasonicated, and then extruded and homogenized to finally obtain stable asiatic acid elastic adaptive liposome-exosomes, that is, asiatic acid elastic adaptive liposome complex loaded with exosomes.

8. The preparation method according to claim 7, characterized in that: The exosomes are at least one of broccoli exosomes, bitter melon exosomes, grape exosomes, apple exosomes, lemon exosomes and ginger exosomes; The exosomes are extracted by density gradient ultracentrifugation.

9. The preparation method according to claim 7 or 8, characterized in that: The incubation time is 2 to 3 hours; The incubation temperature is 25-40°C; The ultrasonic time is 10 to 15 minutes; The extrusion homogenization condition is to pass through 600nm, 400nm, and 200nm polycarbonate membranes in sequence, and manually push them forward and backward 30 to 40 times.

10. The use of the exosome-loaded Centella asiatica elastic adaptive liposome complex according to claim 5 or 6, characterized in that: Apply one or more combinations of the following: (1) Application in the preparation of products that improve the stability of exosomes; (2) Application of exosomes in the preparation of products that enhance the anti-inflammatory effects, improve the skin microenvironment, promote angiogenesis, promote cell proliferation or promote cell repair; (3) Application in the preparation of products that improve the transdermal efficiency of exosomes; (4) Application in the preparation of products for enhancing the non-invasive delivery of exosomes for the treatment of hair loss; (5) Application in the preparation of products for treating skin diseases via non-invasive delivery of exosomes.

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