Preparation, dressing and application of aloe exosome

Abstract

The invention belongs to the technical field of medical preparations, and particularly relates to preparation, dressing and application of aloe exosome. The preparation method of the aloe exosome comprises the four steps of raw material treatment, enzymolysis, centrifugal net capturing and freeze drying. Extraction, separation and purification are carried out by utilizing enzymolysis and a temperature-control and temperature-speed-control centrifugal method, so that the purity and the stability of the aloe exosome are improved. In addition, the formula of the dressing is improved, the permeability of the aloe exosome is improved, and the dressing is beneficial to exerting a curative effect on local diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical preparations, and particularly relates to the preparation, dressing and application of aloe exosomes. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the understanding of the overall background of the present invention, and is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] With the rapid development of radiophysics technology, radiotherapy technology has become increasingly mature and has become an important part of the treatment of 40% of curable malignant tumor patients. At the same time, radiotherapy can also be used for the treatment of advanced or recurrent malignant tumors to relieve patients' symptoms, extend their survival time and improve their quality of life. Although breakthroughs have been made in the accuracy and standardization of radiotherapy, the complications caused by radiotherapy are still important problems in clinical treatment and nursing. Nearly 85% - 95% of tumor patients have varying degrees of radiation skin injury, which not only increases the risk of infection for patients, seriously affects their quality of life, but also brings huge mental pressure and economic burden to patients, and even affects the treatment and ultimately interrupts the progress of radiotherapy.

[0004] Currently, the methods for preventing and treating radiation skin injury at home and abroad mainly include drug treatment and surgical treatment. Drug treatment mostly uses corticosteroids and statins, but the drugs used at present lack specificity for radiation skin injury and are mostly common drugs for the treatment of other skin diseases. To some extent, it relieves the pain of patients with radiation skin injury and enhances comfort, but it does not fundamentally solve the pathological causes of the injury in such patients. Surgical treatment takes a long time, requires high-precision equipment, and has high requirements for doctors' professional levels. For patients, severe radiation injury until ulcer will greatly increase the surgical risk, and complications such as infectious necrosis, poor healing and septic shock are more dangerous. Therefore, there is an urgent need in the market for a targeted, highly effective, rapid-acting and easy-to-use skin injury repair preparation after radiotherapy.

[0005] Aloe vera is a perennial herbaceous plant of the Liliaceae family. The active ingredients in it have the effects of promoting the healing of wounds and skin burns and scalds, antibacterial and anti-inflammatory properties, and beauty characteristics. Existing literature has shown that aloe vera exosomes exhibit good cell compatibility in human skin cells and can easily enter HaCaT cells through clathrin- and caveolae-mediated endocytosis. Aloe vera exosomes promote the migration of epidermal cells and fibroblasts by upregulating the mRNA expression of Nrf2, HO-1, CAT, and SOD, thereby reducing the generation of intracellular free radicals under stress conditions and playing a role in promoting wound healing. Therefore, aloe vera-derived exosomes have good potential for extended application in the field of topical preparations for the repair of skin damage after radiotherapy. (Zeng Lupeng, Wang Huaying, Shi Wanhua, et al. Wound repair effect based on aloe vera cortical exosome-like vesicles [J]. Acta Universitatis Medicinalis Fujianensis, 2022, 56(6): 489-497.)

[0006] Chinese Patent with Publication No. CN 117264869 A discloses a preparation method and application of aloe vera-derived exosomes, and high-purity aloe vera exosomes are obtained by combining differential centrifugation and high-speed centrifugation. It solves the problems of low bioavailability of aloe vera and insufficient research on the efficacy of aloe vera exosomes in the prior art, provides a treatment means for the treatment of radioactive skin damage, and is beneficial to the further development of the treatment of diseases such as cancer. Summary of the Invention

[0007] Overcoming the deficiencies of the prior art, the present invention provides a preparation method of aloe vera exosomes, and the prepared aloe vera exosomes have high purity and good stability.

[0008] The preparation method of aloe vera exosomes of the present invention includes the following steps:

[0009] (1) Raw material treatment: Wash fresh aloe vera leaves, remove the epidermis and yellow juice, cut the aloe vera gel tissue into pieces, and set aside;

[0010] (2) Enzymolysis: Mix 50 parts by weight of aloe vera gel tissue blocks with an enzymolysis solution containing 0.1-2 parts by weight of sodium salicylate, 5-15 U / ml of cellulase, and 2-8 U / ml of pectinase, and stir at 30°C-40°C for 1-3 hours;

[0011] (3) Centrifugal filtration: Centrifuge the enzymolyzed solution by differential centrifugation, discard the supernatant, add an equal volume of PBS buffer for washing, centrifuge at 0°C-5°C and 10,000-15,000 rpm for 30-45 min, discard the supernatant, and perform tangential flow filtration to collect extracts <200 nm;

[0012] (4) Freeze-drying: Add 0.1-1 part by weight of glycine to the extract, mix well, freeze-dry, and seal to obtain the product.

[0013] Traditional enzymatic hydrolysis processes generally use a weakly acidic environment, believing that a weakly acidic pH is more suitable for enzymatic hydrolysis. During the experiment of adjusting the pH of the enzymatic hydrolysis solution in the present invention, it was found that in the experiment of extracting aloe exosomes, a weakly alkaline environment can more effectively promote the disintegration of the cell walls of aloe tissue cells, and more efficiently release exosomes into the surrounding solution. Subsequently, the inventors used buffer solutions, sodium citrate, sodium tartrate, sodium salicylate, etc. to regulate the enzymatic hydrolysis solution, and found that not all salts of strong bases and weak acids can promote enzymatic hydrolysis. The effect of sodium salicylate is relatively significant, which can improve the purity and yield of aloe exosomes.

[0014] On the one hand, sodium salicylate activates the enzymes used in the enzymatic hydrolysis process. Just like some metal ions (such as magnesium ions for certain enzymes) can act as activators, sodium salicylate also has a similar activation function under appropriate conditions. It can bind to the allosteric site of the enzyme, causing a change in the conformation of the enzyme molecule, making the active center of the enzyme more likely to bind to the substrate, thereby increasing the decomposition efficiency of the enzyme on the components of the aloe tissue cell wall (such as cellulose, pectin, etc.). On the other hand, under the condition of precisely controlling the concentration, sodium salicylate regulates the activity of the enzyme through competitive inhibition, thereby adjusting the speed of enzymatic hydrolysis to a certain extent, helping to optimize the enzymatic hydrolysis process, making the decomposition of aloe tissue more refined and controllable, and facilitating the release of exosomes.

[0015] Further, the differential centrifugation method is as follows: centrifuge at 10°C to 15°C and 1000 - 2000 rpm for 10 - 15 minutes to remove the precipitate, then centrifuge at 5°C to 10°C and 5000 - 8000 rpm for 10 - 15 minutes to remove the precipitate, and then centrifuge at 0°C to 5°C and 10000 - 15000 rpm for 40 - 60 minutes; the freeze-drying procedure is as follows: pre-freeze at normal pressure from -5°C to 5°C for 1 - 3 hours, control the temperature to -30°C to -40°C at normal pressure for 4 - 6 hours, increase the temperature under vacuum decompression to -10°C to 0°C for 2 - 4 hours, lower the temperature under vacuum decompression to -35°C to -45°C for 1 - 3 hours, and then increase the temperature under vacuum decompression to -5°C to 10°C for 12 - 15 hours.

[0016] The present invention also provides an aloe exosome dressing, which includes: 5 - 50 parts by weight of aloe exosomes, 0.2 - 0.8 parts by weight of ethyl maltol, 1 - 5 parts by weight of sodium alginate, 2 - 10 parts by weight of glycerol, 0.5 - 3 parts by weight of sodium hyaluronate, 0.01 - 0.1 parts by weight of preservative, and an appropriate amount of purified water.

[0017] Further, the preservative is selected from at least one of benzalkonium chloride, chlorhexidine, and parabens.

[0018] The present invention also provides the use of the above aloe exosome dressing in the preparation of drugs for repairing skin damage.

[0019] Compared with the prior art, the technical effect of the present invention lies in:

[0020] (1) The present invention improves the extraction process of aloe exosomes. The preliminary enzymatic hydrolysis is carried out with an enzymatic hydrolysis solution containing sodium salicylate, and then controlled temperature differential speed centrifugation is used for separation and purification. The prepared aloe exosomes have high purity and good stability.

[0021] (2) The present invention improves the dressing formula and finds that ethyl maltol may be able to improve lipophilicity, help the active ingredients of the drug penetrate the lipid layer of the skin, thereby improving the drug permeability and being beneficial to the treatment of local diseases. Description of the Drawings

[0022] Figure 1 : Standard curve of the total protein content of aloe excretions.

[0023] Figure 2 : Total protein content of aloe excretions prepared with enzymatic hydrolysis solutions of different pH values.

[0024] Figure 3 : Influence of the dosage of sodium salicylate in the enzymatic hydrolysis solution on the total protein content of aloe excretions.

[0025] Figure 4 : Influence of the dosage of sodium citrate in the enzymatic hydrolysis solution on the total protein content of aloe excretions.

[0026] Figure 5 : Particle size stability of aloe exosomes in Examples 1 to 3 and Comparative Examples 1 to 4.

[0027] Figure 6 : Total protein content of aloe exosomes in Example 1 and those in Group A, Group B, and Group C.

[0028] Figure 7 : VEGF expression levels before and after drug administration in each group of rats.

[0029] Figure 8 : TGF-β expression levels before and after drug administration in each group of rats. Detailed Embodiments

[0030] In order to make the purpose and technical solutions of the present invention clearer, the following further describes the present invention with reference to the embodiments. However, the protection scope of the present invention is not limited to these embodiments, and the embodiments are only used to explain the present invention. Those skilled in the art should understand that any change or equivalent substitution that does not deviate from the concept of the present invention is included in the protection scope of the present invention.

[0031] Example 1 Aloe Exosomes and Preparation Method

[0032] (1) Raw material treatment: Wash fresh aloe leaves, remove the epidermis and yellow juice, cut the aloe gel tissue into pieces, and set aside.

[0033] (2) Enzymolysis: Put 50 g of aloe gel tissue pieces into a mixed solution of 500 ml of cellulase at a concentration of 10 U / ml and pectinase at a concentration of 5 U / ml dissolved with 1.5 g of sodium salicylate, and stir at 35 °C for 2 hours.

[0034] (3) Centrifugation and filtration: Centrifuge the enzymolyzed solution at 12 °C and 15000 rpm for 12 min to remove the precipitate, then centrifuge at 8 °C and 6000 rpm for 12 min to remove the precipitate, then centrifuge at 2 °C and 12000 rpm for 50 min, discard the supernatant, add an equal volume of PBS buffer for washing, centrifuge at 2 °C and 14000 rpm for 40 min, discard the supernatant, and perform tangential flow filtration to collect the extract < 200 nm.

[0035] (4) Freeze-drying: Add 0.5 g of glycine to the extract, mix well, pre-freeze at 0 °C under normal pressure for 2 h, control the temperature to -35 °C under normal pressure for 5 h, increase the temperature to -5 °C under vacuum decompression for 3 h, lower the temperature to -40 °C under vacuum decompression for 2 h, increase the temperature to 2 °C under vacuum decompression for 14 h, and seal to obtain the product.

[0036] Example 2 Aloe exosomes and preparation method

[0037] (1) Raw material treatment: Wash fresh aloe leaves, remove the epidermis and yellow juice, cut the aloe gel tissue into pieces, and set aside.

[0038] (2) Enzymolysis: Put 50 g of aloe gel tissue pieces into a mixed solution of 500 ml of cellulase at a concentration of 5 U / ml and pectinase at a concentration of 2 U / ml dissolved with 0.1 g of sodium salicylate, and stir at 30 °C for 1 hour.

[0039] (3) Centrifugation and filtration: Centrifuge the enzymolyzed solution at 10 °C and 1000 rpm for 10 min to remove the precipitate, then centrifuge at 5 °C and 5000 rpm for 10 min to remove the precipitate, then centrifuge at 0 °C and 10000 rpm for 40 min, discard the supernatant, add an equal volume of PBS buffer for washing, centrifuge at 0 °C and 10000 rpm for 30 min, discard the supernatant, and perform tangential flow filtration to collect the extract < 200 nm.

[0040] (4) Freeze-drying: Add 0.1 g of glycine to the extract, mix well, pre-freeze at -5 °C under normal pressure for 1 h, control the temperature to -40 °C under normal pressure for 4 h, increase the temperature to -10 °C under vacuum decompression for 2 h, lower the temperature to -45 °C under vacuum decompression for 1 h, increase the temperature to -5 °C under vacuum decompression for 12 h, and seal to obtain the product.

[0041] Example 3 Aloe exosomes and preparation method

[0042] (1) Raw material treatment: Wash fresh aloe vera leaves, remove the epidermis and yellow juice, cut the aloe vera gel tissue into pieces, and set aside;

[0043] (2) Enzymolysis: Put 50 g of aloe vera gel tissue pieces into a mixed solution of 500 ml of cellulase with a concentration of 15 U / ml and pectinase with a concentration of 8 U / ml dissolved with 2 g of sodium salicylate, and stir at 40 °C for 3 hours;

[0044] (3) Centrifugal filtration: Centrifuge the enzymolyzed solution at 15 °C and 2000 rpm for 15 min to remove the precipitate, then centrifuge at 10 °C and 8000 rpm for 15 min to remove the precipitate, then centrifuge at 5 °C and 15000 rpm for 60 min, discard the supernatant, add an equal volume of PBS buffer for washing, centrifuge at 5 °C and 15000 rpm for 45 min, discard the supernatant, and perform tangential flow filtration to collect the extract < 200 nm;

[0045] (4) Freeze-drying: Add 1 g of glycine to the extract, mix well, pre-freeze at 5 °C under normal pressure for 3 h, control the temperature to -30 °C under normal pressure for 6 h, raise the temperature to 0 °C under vacuum decompression for 4 h, lower the temperature to -35 °C under vacuum decompression for 3 h, raise the temperature to 10 °C under vacuum decompression for 15 h, and seal to obtain the product.

[0046] Comparative Example 1 Aloe vera exosomes and preparation method

[0047] (1) Raw material treatment: Wash fresh aloe vera leaves, remove the epidermis and yellow juice, cut the aloe vera gel tissue into pieces, and set aside;

[0048] (2) Enzymolysis: Put 50 g of aloe vera gel tissue pieces into a mixed solution of 500 ml of cellulase with a concentration of 10 U / ml and pectinase with a concentration of 5 U / ml, and stir at 35 °C for 2 hours;

[0049] (3) Centrifugal filtration: Centrifuge the enzymolyzed solution at 12 °C and 15000 rpm for 12 min to remove the precipitate, then centrifuge at 8 °C and 6000 rpm for 12 min to remove the precipitate, then centrifuge at 2 °C and 12000 rpm for 50 min, discard the supernatant, add an equal volume of PBS buffer for washing, centrifuge at 2 °C and 14000 rpm for 40 min, discard the supernatant, and perform tangential flow filtration to collect the extract < 200 nm;

[0050] (4) Freeze-drying: Add 0.5 g of glycine to the extract, mix well, pre-freeze at 0 °C under normal pressure for 2 h, control the temperature to -35 °C under normal pressure for 5 h, raise the temperature to -5 °C under vacuum decompression for 3 h, lower the temperature to -40 °C under vacuum decompression for 2 h, raise the temperature to 2 °C under vacuum decompression for 14 h, and seal to obtain the product.

[0051] Comparative Example 2 Aloe vera exosomes and preparation method

[0052] (1) Raw material treatment: Wash fresh aloe leaves, remove the epidermis and yellow juice, cut the aloe gel tissue into pieces, and set aside.

[0053] (2) Enzymolysis: Put 50 g of aloe gel tissue pieces into 500 ml of a mixed solution of cellulase at a concentration of 10 U / ml and pectinase at a concentration of 5 U / ml dissolved with 1.5 g of sodium citrate, and stir at 35 °C for 2 hours.

[0054] (3) Centrifugal filtration: Centrifuge the enzymolyzed solution at 12 °C and 15000 rpm for 12 min to remove the precipitate, centrifuge at 8 °C and 6000 rpm for 12 min to remove the precipitate, centrifuge at 2 °C and 12000 rpm for 50 min, discard the supernatant, add an equal volume of PBS buffer for washing, centrifuge at 2 °C and 14000 rpm for 40 min, discard the supernatant, and perform tangential flow filtration to collect the extract < 200 nm.

[0055] (4) Freeze-drying: Add 0.5 g of glycine to the extract, mix well, pre-freeze at 0 °C under normal pressure for 2 h, control the temperature to -35 °C under normal pressure for 5 h, increase the temperature to -5 °C under vacuum decompression for 3 h, lower the temperature to -40 °C under vacuum decompression for 2 h, raise the temperature to 2 °C under vacuum decompression for 14 h, and seal to obtain the product.

[0056] Comparative Example 3 Aloe exosomes and preparation method

[0057] (1) Raw material treatment: Wash fresh aloe leaves, remove the epidermis and yellow juice, cut the aloe gel tissue into pieces, and set aside.

[0058] (2) Enzymolysis: Put 50 g of aloe gel tissue pieces into 500 ml of a mixed solution of cellulase at a concentration of 10 U / ml and pectinase at a concentration of 5 U / ml dissolved with 1.5 g of sodium salicylate, and stir at 35 °C for 2 hours.

[0059] (3) Centrifugal filtration: Centrifuge the enzymolyzed solution at 12 °C and 15000 rpm for 12 min to remove the precipitate, centrifuge at 8 °C and 6000 rpm for 12 min to remove the precipitate, centrifuge at 2 °C and 12000 rpm for 50 min, discard the supernatant, add an equal volume of PBS buffer for washing, centrifuge at 2 °C and 14000 rpm for 40 min, discard the supernatant, and perform tangential flow filtration to collect the extract < 200 nm.

[0060] (4) Freeze-drying: Pre-freeze the extract at 0 °C under normal pressure for 2 h, control the temperature to -35 °C under normal pressure for 5 h, increase the temperature to -5 °C under vacuum decompression for 3 h, lower the temperature to -40 °C under vacuum decompression for 2 h, raise the temperature to 2 °C under vacuum decompression for 14 h, and seal to obtain the product.

[0061] Comparative Example 4 Aloe Exosomes and Preparation Method

[0062] (1) Raw material treatment: Wash fresh aloe leaves, remove the epidermis and yellow juice, cut the aloe gel tissue into pieces, and set aside;

[0063] (2) Enzymolysis: Put 50 g of aloe gel tissue pieces into a mixed solution of 500 ml of cellulase at a concentration of 10 U / ml and pectinase at a concentration of 5 U / ml dissolved with 1.5 g of sodium salicylate, and stir at 35 °C for 2 hours;

[0064] (3) Centrifugal filtration: Centrifuge the enzymolyzed solution at 12 °C and 15000 rpm for 12 min, remove the precipitate, centrifuge at 8 °C and 6000 rpm for 12 min, remove the precipitate, centrifuge at 2 °C and 12000 rpm for 50 min, discard the supernatant, add an equal volume of PBS buffer for washing, centrifuge at 2 °C and 14000 rpm for 40 min, discard the supernatant, and perform tangential flow filtration to collect extracts <200 nm;

[0065] (4) Freeze-drying: Add 0.5 g of mannitol to the extract, mix well, pre-freeze at normal pressure at 0 °C for 2 h, control the temperature to -35 °C at normal pressure for 5 h, increase the temperature to -5 °C under vacuum decompression for 3 h, lower the temperature to -40 °C under vacuum decompression for 2 h, raise the temperature to 2 °C under vacuum decompression for 14 h, and seal to obtain.

[0066] Purity of Aloe Exosomes

[0067] Use the standard curve method to measure the purity of aloe exosomes, Figure 1 It is the standard curve for measuring the total protein concentration of exosomes by the BCA method, showing a good linear range of the standard curve. By comparing the absorbance value of the aloe exosome sample with the standard curve, the content of the marker protein can be quantitatively obtained.

[0068] Table 1 Total Protein Content of Aloe Exosomes in Examples 1 - 3 and Comparative Examples 1 - 4

[0069] Total protein content of aloe exudates (mg / ml) Example 1 2.452 Example 2 2.163 Example 3 2.371 Comparative Example 1 1.420 Comparative Example 2 0.545 Comparative Example 3 1.622 Comparative Example 4 1.768

[0070] It can be seen that the aloe exosomes extracted by the preparation method of the present invention have high purity.

[0071] As is well known, the optimal pH range of cellulase is generally between 4.5 and 6.5, and the pH of the enzymatic hydrolysis reaction of pectinase is generally between 2.5 and 6.0, such as Figure 2As shown, after adjusting the pH value of the enzymatic hydrolysate with a buffer solution, the total protein content of the prepared aloe exosomes was measured. It was found that in the experiment of extracting aloe exosomes, a weakly alkaline environment could more effectively promote the disintegration of the cell wall of aloe tissue cells and release exosomes into the surrounding solution more efficiently. Subsequently, the inventors regulated the enzymatic hydrolysate by using buffer solutions, sodium citrate, sodium tartrate, sodium salicylate, etc., and found that adding 0.1 - 2 g of sodium salicylate could promote enzymatic hydrolysis and improve the purity and yield of aloe exosomes.

[0072] Aloe exosome stability

[0073] The aloe exosomes of Examples 1 - 3 and Comparative Examples 1 - 4 were stored under the conditions of a temperature of 25 ± 2 °C and a relative humidity of 60% ± 5% for 120 days. Their morphology was observed regularly and their particle size was detected to evaluate their stability during storage. Figure 5 It was shown that the aloe exosomes of Examples 1 - 3 of the present invention could basically maintain a uniform particle size during storage and had high stability. However, for the aloe exosomes of the comparative examples, with the extension of the storage time, the exosomes gradually ruptured, and their average particle size and deviation gradually increased. In particular, the aloe exosomes of Comparative Example 4 and Comparative Example 5 showed poor stability.

[0074] Effect of temperature control in differential centrifugation on the purity of aloe exosomes

[0075] A single - factor experiment was set up to explore the effect of the temperature in differential centrifugation on the purity of the prepared aloe exosomes.

[0076] Group A: Centrifuge at 4 °C, 15000 rpm for 12 min, remove the precipitate, then centrifuge at 4 °C, 6000 rpm for 12 min, remove the precipitate, then centrifuge at 4 °C, 12000 rpm for 50 min, discard the supernatant, add an equal volume of PBS buffer for washing, and then centrifuge at 4 °C, 14000 rpm for 40 min.

[0077] Group B: Centrifuge at 15 °C, 15000 rpm for 12 min, remove the precipitate, then centrifuge at 15 °C, 6000 rpm for 12 min, remove the precipitate, then centrifuge at 15 °C, 12000 rpm for 50 min, discard the supernatant, add an equal volume of PBS buffer for washing, and then centrifuge at 15 °C, 14000 rpm for 40 min.

[0078] Group C: Centrifuge at 2 °C, 15000 rpm for 12 min, remove the precipitate, then centrifuge at 8 °C, 6000 rpm for 12 min, remove the precipitate, then centrifuge at 12 °C, 12000 rpm for 50 min, discard the supernatant, add an equal volume of PBS buffer for washing, and then centrifuge at 15 °C, 14000 rpm for 40 min.

[0079] Other conditions were the same as in Example 1.

[0080] In traditional exosome extraction methods, in order to maintain the stability of exosomes and prevent their degradation or damage during extraction, centrifugation is usually carried out at 4°C. However, experiments have found that combining gradient temperature with gradient centrifugation speed can maximize the advantages of centrifugal separation, reduce the interaction between exosomes and impurities, precisely separate according to the physical differences between exosomes and impurities, and improve the purity( Figure 6 ).

[0081] Example 4 Aloe Exosome Dressing

[0082] Formula:

[0083]

[0084]

[0085] Preparation method:

[0086] (1) Take an appropriate amount of purified water, slowly sprinkle sodium alginate into it and stir evenly to make it fully swell to form a uniform sodium alginate solution.

[0087] (2) Add glycerol, ethyl maltol and sodium hyaluronate to the sodium alginate solution and stir until completely dissolved.

[0088] (3) Dissolve aloe exosomes with a small amount of purified water, then slowly add it to the above solution and stir while adding to ensure that the exosomes are evenly dispersed in the solution.

[0089] (4) Add a preservative, stir evenly, make up purified water to 100 ml, stir evenly, sterilize, and obtain the aloe exosome dressing.

[0090] Example 5 Aloe Exosome Dressing

[0091] Formula:

[0092]

[0093] Preparation method:

[0094] (1) Take an appropriate amount of purified water, slowly sprinkle sodium alginate into it and stir evenly to make it fully swell to form a uniform sodium alginate solution.

[0095] (2) Add glycerol, ethyl maltol and sodium hyaluronate to the sodium alginate solution and stir until completely dissolved.

[0096] (3) Dissolve aloe exosomes with a small amount of purified water, then slowly add it to the above solution and stir while adding to ensure that the exosomes are evenly dispersed in the solution.

[0097] (4) Add preservatives, stir evenly, make up purified water to 100 ml, stir evenly, and sterilize to obtain aloe exosome dressing.

[0098] Example 6 Aloe Exosome Dressing

[0099] Formula:

[0100]

[0101] Preparation method:

[0102] (1) Take an appropriate amount of purified water, slowly sprinkle sodium alginate into it and stir evenly to make it fully swell to form a uniform sodium alginate solution.

[0103] (2) Add glycerol, ethyl maltol and sodium hyaluronate to the sodium alginate solution and stir until completely dissolved.

[0104] (3) Dissolve aloe exosomes with a small amount of purified water, then slowly add it to the above solution while stirring to ensure that the exosomes are evenly dispersed in the solution.

[0105] (4) Add preservatives, stir evenly, make up purified water to 100 ml, stir evenly, and sterilize to obtain aloe exosome dressing.

[0106] Comparative Example 5 Aloe Exosome Dressing

[0107] Formula:

[0108]

[0109]

[0110] Preparation method:

[0111] (1) Take an appropriate amount of purified water, slowly sprinkle sodium alginate into it and stir evenly to make it fully swell to form a uniform sodium alginate solution.

[0112] (2) Add glycerol and sodium hyaluronate to the sodium alginate solution and stir until completely dissolved.

[0113] (3) Dissolve aloe exosomes with a small amount of purified water, then slowly add it to the above solution while stirring to ensure that the exosomes are evenly dispersed in the solution.

[0114] (4) Add preservatives, stir evenly, make up purified water to 100 ml, stir evenly, and sterilize to obtain aloe exosome dressing.

[0115] Comparative Example 6 Aloe Exosome Dressing

[0116] Formula:

[0117]

[0118] Preparation method:

[0119] (1) Take an appropriate amount of purified water, slowly sprinkle sodium alginate into it and stir evenly to make it fully swell, forming a uniform sodium alginate solution.

[0120] (2) Add glycerol, ethyl maltol and sodium hyaluronate to the sodium alginate solution and stir until completely dissolved.

[0121] (3) Dissolve aloe exosomes with a small amount of purified water, then slowly add it to the above solution while stirring to ensure that the exosomes are evenly dispersed in the solution.

[0122] (4) Add a preservative, stir evenly, make up the purified water to 100 ml, stir evenly, and sterilize to obtain the aloe exosome dressing.

[0123] Aloe exosome dressing of Comparative Example 7

[0124] Formula:

[0125]

[0126] Preparation method:

[0127] (1) Take an appropriate amount of purified water, slowly sprinkle sodium alginate into it and stir evenly to make it fully swell, forming a uniform sodium alginate solution.

[0128] (2) Add glycerol, propylene glycol and sodium hyaluronate to the sodium alginate solution and stir until completely dissolved.

[0129] (3) Dissolve aloe exosomes with a small amount of purified water, then slowly add it to the above solution while stirring to ensure that the exosomes are evenly dispersed in the solution.

[0130] (4) Add a preservative, stir evenly, make up the purified water to 100 ml, stir evenly, and sterilize to obtain the aloe exosome dressing.

[0131] Transdermal permeability test of aloe exosome dressing

[0132] SD rats were selected as experimental animals, with SPF grade, weighing 180 - 220 g, and the license number was SYXK(Shandong)20180008. They were adaptively fed for one week before the experiment. When preparing the excised rat skin, first depilate it with 8% sodium sulfide solution, wash it and then feed it for 24 h. After decapitation, take the abdominal skin, remove the subcutaneous fat, wash it with physiological saline, and blot the water with filter paper for standby. For the transdermal experiment, with the aid of a transdermal instrument, fix the rat abdominal skin between the supply pool and the receiving pool, with the stratum corneum facing the supply pool. Apply the aloe exosome dressings of Examples 4 - 6, Comparative Examples 5 - 7 and the blank dressing (referring to the blank dressing without adding aloe exosomes) with the same area to the supply pool respectively. The receiving solution is 20% ethanol - pH7.4 phosphate buffer solution. Sampling is carried out at 2 min and 15 min to measure the content of aloe exosomes (using protein as a marker), and calculate the cumulative permeation amount of each example according to the formula.

[0133]

[0134] Where V is the total volume of the receiving solution, Cn is the drug concentration (μg / mL) measured at the nth sampling point, C is the drug concentration (μg / mL) measured at the ith sampling point, V is the sampling volume, and A is the diffusion and permeation area (cm 2 ).

[0135] Table 2 Cumulative permeation amount

[0136]

[0137] Table 2 shows that a certain amount of ethyl maltol is added to the aloe exosome dressing of the present invention in the formulation, which can promote the skin penetration ability of aloe exosomes, increase the local drug concentration in the skin, and is beneficial to the drug in treating local diseases to exert its efficacy. The dressings prepared from Examples 2 and 3 according to the dressing formula of Example 4 were also subjected to percutaneous penetration experiments, and the cumulative permeation amounts at 15 min were 1.67 ± 0.15 and 1.70 ± 0.14 μg / cm 2 .

[0138] The effect of the aloe exosome dressing of the present invention in repairing radiotherapy - induced skin damage

[0139] SD rats, SPF grade, weighing 180 - 220 g, with the experimental animal license number: SYXK(Shandong)20180008. Before the experiment, they were adaptively fed under standard conditions for 1 week. Several rats were anesthetized, and the shaved area on the back was irradiated with high-energy electron beams emitted by a 6 MeV linear accelerator at a dose rate of 500 cGy / min. The non-irradiated parts were covered with lead plates; the irradiation field area was 2 cm × 2 cm, and the irradiation dose was 45 Gy to form a rat radiation dermatitis model. It was divided into a model group, Example 4 group, Example 5 group, Example 6 group, Comparative Example 5 group, Comparative Example 6 group, and Comparative Example 7 group, with 10 rats in each group. 10 rats in the normal group were only shaved and not given any treatment, and they were continued to be raised. Each group of rats was given an equal dose of the corresponding aloe exosome dressing, and the rats in the model group and the blank group were dressed with normal saline for 7 consecutive days.

[0140] VEGF and TGF-β level expressions

[0141] Figure 7 It shows the VEGF expression levels in each group of rats before and after administration. It indicates that the vascular endothelial cells in the rat skin tissue are damaged after modeling. Due to vascular damage, the body usually increases the VEGF level in order to repair and reconstruct blood vessels. After administration, the VEGF levels in the rats of Example 4 - 6 groups increased significantly, and there was a significant difference compared with the model group, P < 0.01.

[0142] TGF-β is a multifunctional cytokine that can regulate cell proliferation, differentiation, and the synthesis of the extracellular matrix. It can stimulate the proliferation of fibroblasts and promote the migration of fibroblasts to the damaged skin area, which is a key step in initiating skin tissue repair. Figure 8 It shows the TGF-β expression levels in each group of rats before and after administration. After administration, there was a significant difference in the TGF-β levels between the rats of Example 4 - 6 groups and the model group, P < 0.01.

Claims

1. A preparation method of aloe exosomes, characterized in that, The preparation method of the aloe exosomes comprises the following steps: (1) Raw material treatment: Wash fresh aloe leaves, remove the epidermis and yellow juice, cut the aloe gel tissue into pieces, and set aside; (2) Enzymolysis: Mix 50 parts by weight of aloe gel tissue pieces with an enzymolysis solution containing 0.1 - 2 parts by weight of sodium salicylate, 5 - 15 U / ml of cellulase, and 2 - 8 U / ml of pectinase, and stir at 30°C - 40°C for 1 - 3 hours; (3) Centrifugal filtration: Centrifuge the enzymolyzed solution by differential centrifugation, discard the supernatant, add an equal volume of PBS buffer for washing, centrifuge at 0°C - 5°C, 10000 - 15000 rpm for 30 - 45 min, discard the supernatant, perform tangential flow filtration, and collect the extract <200 nm; (4) Freeze-drying: Add glycine to the extract, mix well, perform freeze-drying, and seal to obtain the product.

2. The preparation method of aloe exosomes according to claim 1, wherein, The enzymolysis solution is a mixed solution of 1.5 parts by weight of sodium salicylate, 10 U / ml of cellulase, and 5 U / ml of pectinase.

3. The preparation method of aloe exosomes according to claim 1, characterized in that, The dosage of glycine is 0.1 - 1 part by weight; preferably, the dosage of glycine is 0.5 part by weight.

4. The preparation method of aloe exosomes according to claim 1, wherein, The differential centrifugation method is as follows: Centrifuge at 10°C - 15°C, 1000 - 2000 rpm for 10 - 15 min, remove the precipitate, centrifuge at 5°C - 10°C, 5000 - 8000 rpm for 10 - 15 min, remove the precipitate, and centrifuge at 0°C - 5°C, 10000 - 15000 rpm for 40 - 60 min; Preferably, the differential centrifugation method is as follows: Centrifuge at 12°C, 15000 rpm for 12 min, remove the precipitate, centrifuge at 8°C, 6000 rpm for 12 min, remove the precipitate, centrifuge at 2°C, 12000 rpm for 50 min, discard the supernatant, add an equal volume of PBS buffer for washing, and centrifuge at 2°C, 14000 rpm for 40 min.

5. The preparation method of aloe exosomes according to claim 1, characterized in that, The freeze-drying procedure is as follows: Pre-freeze at normal pressure of -5°C - 5°C for 1 - 3 h, control the temperature to -30°C - -40°C at normal pressure for 4 - 6 h, increase the temperature to -10°C - 0°C under vacuum decompression for 2 - 4 h, reduce the temperature to -35°C - -45°C under vacuum decompression for 1 - 3 h, and increase the temperature to -5°C - 10°C under vacuum decompression for 12 - 15 h; Preferably, the freeze-drying procedure is as follows: Pre-freeze at normal pressure of 0°C for 2 h, control the temperature to -35°C at normal pressure for 5 h, increase the temperature to -5°C under vacuum decompression for 3 h, reduce the temperature to -40°C under vacuum decompression for 2 h, and increase the temperature to 2°C under vacuum decompression for 14 h.

6. An aloe exosome dressing, characterized in that, The aloe exosome dressing comprises: 5 - 50 parts by weight of aloe exosomes, 0.2 - 0.8 parts by weight of ethyl maltol, 1 - 5 parts by weight of sodium alginate, 2 - 10 parts by weight of glycerol, 0.5 - 3 parts by weight of sodium hyaluronate, 0.01 - 0.1 parts by weight of preservative, and an appropriate amount of purified water.

7. The dressing according to claim 6, wherein The aloe exosome dressing comprises: 25 parts by weight of aloe exosomes, 0.5 parts by weight of ethyl maltol, 3 parts by weight of sodium alginate, 8 parts by weight of glycerol, 2 parts by weight of sodium hyaluronate, 0.02 parts by weight of preservative, and an appropriate amount of purified water.

8. The dressing according to claim 6 or 7, characterized in that, The preservative is selected from at least one of benzalkonium chloride, chlorhexidine, and parabens.

9. Use of the dressing according to claim 6 in the preparation of a medicament for repairing skin damage.

10. The use according to claim 9, characterized in that, The skin damage is skin damage after radiotherapy.

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