Dendrobium nano-vesicle complex as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medicines, in particular to a dendrobium nano-vesicle complex as well as a preparation method and application thereof. According to the preparation method disclosed by the invention, nano-scale vesicles are extracted from hydroponic dendrobium nobile by utilizing an ultrasonic pretreatment and ultracentrifugation method, and then the nano-scale vesicles are mixed with trehalose with a specific concentration, so that the dendrobium nobile nano-vesicle (EX0) complex with high purity and high activity is prepared. Experiments show that the complex can promote proliferation and repair of corneal epithelial cells and improve tear secretion, and has a good curative effect on xerophthalmia. The invention further provides eye drops containing the complex, the stability and effectiveness of the exosome are optimized, and the effect of the exosome in treatment of xerophthalmia and cornea is enhanced.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a dendrobium nanovesicle complex and a preparation method and application thereof. Background Art

[0002] Dry eye is a common ophthalmic disease, the main characteristics of which are insufficient tear secretion or abnormal tear quality, leading to corneal epithelial damage and eye discomfort. The occurrence of the disease may be related to a variety of factors, including environmental factors, age, drug use, hormone levels, etc. Traditional treatments usually include artificial tears and anti-inflammatory drugs. Although these methods can temporarily relieve symptoms, their effects are often limited and may cause side effects. For example, artificial tears cannot permanently solve the problem of insufficient tears, and anti-inflammatory drugs may bring side effects of long-term use. Therefore, a new treatment option is urgently needed to more effectively promote corneal epithelial repair and improve dry eye symptoms.

[0003] Existing treatments for dry eyes mainly include artificial tears, anti-inflammatory drugs, etc. Artificial tears can only provide temporary lubrication and can temporarily relieve dry eyes, but cannot fundamentally improve insufficient tear secretion or abnormal tear quality. Although anti-inflammatory drugs such as steroid drugs can relieve inflammation, they usually have limited effects on improving tear secretion. Long-term use of artificial tears or anti-inflammatory drugs may bring side effects, such as drug dependence, eye irritation or other systemic adverse reactions, which not only brings inconvenience to patients, but also fails to achieve long-term therapeutic effects. Although other wetting agents and eye drops can provide a certain sense of comfort, their effects are usually limited and require frequent use. The common disadvantage of these methods is that they cannot fundamentally solve the problem of insufficient tears or abnormal tear quality.

[0004] Plant intranalvesicles refer to membrane-enclosed vesicles inside plant cells, which participate in a variety of biological processes within the cells. They play an important role in the normal function of plant cells and in maintaining the stability of the intracellular environment. Plant extranalvesicles are small vesicles secreted by a variety of cells, with a diameter of about 30-500 nanometers and composed of a lipid bilayer membrane. They are widely present in body fluids, including blood, tears, urine, etc., and are rich in biological molecules such as nucleic acids, proteins and lipids. Extranalvesicles play an important role in intercellular communication and have an important impact on physiological and pathological processes such as immune regulation, tumor development, and tissue repair.

[0005] In recent years, the research on plant nanovesicles has gradually increased, and it has been found that they have better effects than traditional plant extracts in regulating cellular inflammation. Compared with traditional plant extracts, plant extracellular vesicles have the following advantages: 1) Strong targeting: Plant extracellular vesicles can specifically target specific cells or tissues, improving the precise delivery of bioactive ingredients; 2) High stability: Plant extracellular vesicles are usually more stable than traditional plant extracts and can maintain activity for a longer time in the in vivo and in vitro environment; 3) Biocompatibility: Plant extracellular vesicles have good biocompatibility and reduce potential side effects; 4) Diverse functions: Studies have found that plant extracellular vesicles have multiple biological functions in immunomodulation, anti-inflammatory, and antioxidant, showing a wider application prospect than traditional plant extracts.

[0006] As an emerging biomaterial, plant nanovesicles have received extensive attention in recent years. Studies have shown that plant nanovesicles have shown good effects in improving cell growth, anti-oxidation and anti-inflammation. However, research on how to efficiently extract and utilize plant nanovesicles is still evolving.

[0007] The main challenges are as follows:

[0008] 1. Problems with freeze-dried vesicle membrane structure and aggregation: Ice crystals will form during the freeze-drying process due to the cooling treatment, which will lead to the destruction of the vesicle membrane structure and the loss of contents. At the same time, the vesicles will directly adhere and aggregate during freeze-drying.

[0009] 2. The problem of vesicle water content: How to increase the water content of vesicles is still the main difficulty in the use of nanovesicles.

[0010] Dendrobium officinale is a traditional Chinese medicine with a long history of use, mainly distributed in the Qinling Mountains and the south of the Huaihe River in China. According to the Flora of China and Shennong's Herbal Classic, Dendrobium officinale is believed to have the effects of nourishing yin and clearing heat, benefiting the stomach and promoting fluid production, and can enhance the body's immunity, scavenge free radicals and delay aging. Modern research shows that Dendrobium officinale contains rich active ingredients such as polysaccharides, alkaloids, flavonoids and stilbenoid compounds, which are widely used in cosmetics and health products, mainly in the fields of moisturizing, delaying aging and whitening. Studies have reported that Dendrobium officinale Night Light Pills (made from more than 20 traditional Chinese medicinal components such as Dendrobium officinale, ginseng, yam, Poria cocos, licorice, and Cistanche deserticola) combined with other traditional Chinese medicine formulas (such as Mingmu Dihuang Pills) or some conventional eye drops (such as sodium hyaluronate eye drops or polyvinyl alcohol eye drops) have a certain effect on dry eye. So far, there has been no research report on the use of Dendrobium alone to treat dry eye syndrome, and there has been no research report on the use of Dendrobium nanovesicles to treat dry eye syndrome.

[0011] The main forms of dendrobium products include capsules, granules, tablets, oral liquids, etc. These products are mostly used to enhance immunity, resist fatigue and improve sleep. In recent years, the application of dendrobium extracts in cosmetics has also increased, such as moisturizing, whitening and delaying aging. However, these products usually involve chemical solvent extraction, which may cause ingredient loss or chemical residues, and the utilization rate of their bioactive substances is low. Therefore, it is of great value and significance to provide a new dosage form of dendrobium for the treatment of dry eye. Summary of the invention

[0012] In view of this, the present invention provides a dendrobium nanovesicle complex and its preparation method and application. Experiments show that the complex can promote the proliferation and repair of corneal epithelial cells, improve tear secretion, and has a good therapeutic effect on dry eye. Eye drops containing the complex optimize the stability and efficacy of exosomes and enhance their effect in the treatment of dry eye.

[0013] Application of Dendrobium nanovesicle complexes in the preparation of drugs for treating diseases related to epithelial cell damage;

[0014] The epithelial cell damage includes: corneal epithelial defects and / or wounds and ulcers caused by diabetic foot;

[0015] The diseases associated with epithelial cell damage include dry eye, diabetic ulcer, superficial keratitis, corneal ulcer and / or skin ulcer.

[0016] In the above application, the dendrobium nanovesicle complex is prepared by sequentially subjecting the dendrobium bulb to ultrasonic extraction, homogenization, centrifugation, filtration and ultracentrifugation.

[0017] In the present invention, the dendrobium includes Dendrobium officinale, Dendrobium Huoshanense or Dendrobium chrysantha. In a specific embodiment of the present invention, the dendrobium is hydroponic Dendrobium officinale.

[0018] The results of electron microscopy showed that the dendrobium nanovesicle complex prepared by the present invention contained vesicles outside the dendrobium bulb and vesicles inside the plant tissue.

[0019] The present invention has found that the dendrobium nanovesicle complex can effectively promote the proliferation and repair of corneal epithelial cells, improve tear secretion, and has a good therapeutic effect on dry eye. Compared with the prior art scheme of combining dendrobium night light pills with eye drops and dendrobium night light pills with eye-brightening prescriptions, the present invention has the advantages of easy absorption, good permeability, less raw materials, less side effects, low production cost, and good patient compliance while ensuring good drug efficacy.

[0020] The present invention also provides a method for preparing the dendrobium nanovesicle complex, comprising:

[0021] Step (1): extracting the dendrobium bulb by ultrasonic method, homogenizing, centrifuging and filtering to obtain a supernatant containing dendrobium nanovesicles;

[0022] Step (2): After ultracentrifugation of the supernatant obtained in step (1), the precipitate is collected and a buffer solution containing 60-85 mM trehalose is added, and the mixture is freeze-dried to obtain a nanovesicle complex.

[0023] In step (1) of the present invention, the ultrasonic extraction is performed twice, and the conditions for ultrasonic extraction are: 4° C., ultrasonic extraction is performed once every 10 minutes, and each ultrasonic extraction lasts for 5 minutes.

[0024] In step (1) of the present invention, the homogenization conditions are 4°C and 3000g centrifugation for 30 minutes.

[0025] In step (1) of the present invention, the centrifugation includes: centrifugation at 4°C and 3000g for 30 minutes, and centrifugation of the supernatant at 4°C and 10000g for 60 minutes.

[0026] In step (1) of the present invention, the filtration is 0.22 μm filter membrane filtration.

[0027] In step (2) of the present invention, the number of ultracentrifugation is 2 times, and the conditions of each ultracentrifugation are 4°C, 150000g ultracentrifugation for 120 minutes.

[0028] In step (2) of the present invention, the concentration of trehalose in the trehalose-containing buffer may be 60mM, 65mM, 70mM, 75mM, 80mM, or 85mM; and the buffer A comprises at least one of PBS buffer, Hanks' balanced salt solution, and 0.9% sodium chloride solution.

[0029] In an embodiment of the present invention, the ultracentrifugation is performed twice, the first time is to ultracentrifuge the supernatant obtained in step (1), and the second time is to mix the precipitate of the first ultracentrifugation with a buffer solution not containing trehalose.

[0030] After the second ultracentrifugation, the precipitate is collected and a buffer solution containing 60-85mM trehalose is added, and then a freeze-drying step is performed. The obtained nanovesicle complex is a freeze-dried vesicle complex. It can be stored at room temperature, avoiding the problem that traditional liquid Dendrobium exosomes need to be transported and stored under low temperature conditions in order to avoid exosome denaturation. Experiments show that after the freeze-dried vesicles are rehydrated, they not only maintain the bilayer membrane structure of the exosomes, prevent aggregation, retain the complete structure of the nanovesicles, but also enhance their hydration ability, so that they can be effectively stored and conveniently transported at room temperature.

[0031] In the present invention, the freeze-drying includes pre-freezing, primary drying and secondary drying. Specifically:

[0032] The pre-freezing comprises: lowering the temperature to -193°C to 196°C for 1-2 hours;

[0033] The primary drying temperature includes: raising the temperature to -40°C to -30°C, the pressure is lower than 100 bar, and the holding time is 24-48 hours;

[0034] The secondary drying includes: slowly raising the temperature to -10°C to 0°C, the pressure is lower than 10 bar, and the holding time is 12-24 hours.

[0035] The invention also provides a dendrobium nanovesicle complex prepared by the method.

[0036] The present invention also provides the use of the dendrobium nanovesicle complex in the preparation of a drug for treating diseases related to epithelial cell damage;

[0037] The epithelial cell damage includes: corneal epithelial defects, or wounds and ulcers caused by diabetic foot;

[0038] The diseases associated with epithelial cell damage include dry eye, diabetic ulcer, superficial keratitis, corneal ulcer and / or skin ulcer.

[0039] The treatment includes: promoting the proliferation and repair of corneal epithelial cells, and / or improving tear secretion.

[0040] The present invention also provides an eye drop for treating dry eye, comprising the dendrobium nanovesicle complex of the present invention and a pharmaceutically acceptable auxiliary material.

[0041] Furthermore, the auxiliary material includes at least one of glycerol, ascorbic acid, vitamin E, PBS or sodium chloride.

[0042] In some embodiments of the present invention, the eye drops include the Dendrobium nanovesicle complex of the present invention, glycerol, ascorbic acid, vitamin E, PBS and sodium chloride.

[0043] In some specific embodiments, the eye drops include vesicle components and matrix components; the vesicle components include water and dendrobium nanovesicle complexes; the concentration of the dendrobium nanovesicle complexes is 1.2-4.8×10^7 vesicles / μl;

[0044] The matrix component includes PBS buffer and the following components in percentage by mass and volume:

[0045] 2%-10% w / v glycerol, 0.1%-0.5% w / v ascorbic acid, 0.01%-0.05% w / v vitamin E, 0.9% w / v sodium chloride.

[0046] In some specific embodiments, the matrix component includes PBS buffer and the following components in mass volume percentage:

[0047] Glycerol 2% (w / v), ascorbic acid 0.2% (w / v), vitamin E 0.05% (w / v), sodium chloride 0.9% (w / v).

[0048] The above-mentioned vesicle components and B matrix components can be packaged in two tubes. When in use, the matrix components can be injected into the vesicle components. This design further ensures the high purity and activity of the extracellular vesicles.

[0049] Through the above measures, the present invention not only provides a new dry eye treatment solution, but also solves the problems of effectiveness, stability and storage in the prior art, providing a safer and more effective treatment solution for dry eye patients. Specifically, the present invention has at least one of the following advantages:

[0050] (1) Significant therapeutic effect: The present invention utilizes nanovesicles (including inner vesicles and outer vesicles) derived from hydroponic Dendrobium bulbs, which can promote the proliferation and repair of corneal epithelial cells and improve tear secretion, thereby providing a more effective treatment for dry eye.

[0051] (2) Reduced side effects: Compared with traditional drugs, the Dendrobium exocyst eye drops of the present invention are pure natural products, free of preservatives, will not cause drug dependence or eye irritation, and are suitable for long-term use.

[0052] (3) Improved the activity and stability of Dendrobium nanovesicles

[0053] ① Enhance the utilization rate of active ingredients: Through freeze-drying-rehydration treatment, the present invention can not only effectively preserve the nanovesicles, but also maintain the stability of its active ingredients during the rehydration process, enhance its water content, moisturize the eye surface more lastingly, and improve its protective effect on the corneal epithelium.

[0054] ② Convenient storage and transportation: Freeze-dried vesicles can be stored at room temperature, avoiding the need for traditional liquid Dendrobium exosomes to be transported and stored under low temperature conditions. If this process is not properly stored, it will lead to exosome denaturation. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The electron microscopy and particle size analysis of the rehydrated nanovesicles are shown. Figure 1 A is the particle size result. The particle detection concentration of Dendrobium nanovesicles is 5.7E+7Particles / mL, the dilution factor is 4000, the original concentration is 2.3E+11Particles / mL, and the Median (×50) is 113.6nm; Figure 1 B shows the internal and external vesicles that can be detected in the sample;

[0056] Figure 2 It showed that the Dendrobium nanovesicle complex was absorbed and internalized by corneal cells; Figure 2 A is a hexagonal human corneal epithelial cell cultured under a microscope; Figure 2 B is the green fluorescence-PKH67 labeled Dendrobium nanovesicle complex; Figure 2 C is green fluorescence and can be absorbed and internalized by human corneal epithelial cells;

[0057] Figure 3 The results showed that the dendrobium nanovesicle complex can promote the proliferation of corneal epithelial cells, and the high concentration of dendrobium nanovesicle complex inhibits the proliferation of corneal epithelial cells; A is the protective effect of different concentrations of dendrobium nanovesicle complex on normal corneal epithelial cells, and high concentration shows a negative effect on the proliferation of corneal epithelial cells; B is the confirmation of the concentration of NaCl hyperosmotic model, IC50 = 122.4769nM; C is the result of first using NaCl to create a hyperosmotic corneal epithelial cell model, and then using dendrobium nanovesicles for treatment; D is first using dendrobium nanovesicles for protection, and then using NaCl to create a hyperosmotic corneal epithelial cell model to investigate the protective effect of dendrobium nanovesicle complex on NaCl corneal epithelial cells; HDO-EVs: the abbreviation of hydroponic dendrobium bulb nanovesicle complex;

[0058] Figure 4 The results of fluorescein staining of goblet cells in different treatment groups (A), tear secretion (B) and sodium fluorescein scoring (C) are shown;

[0059] Figure 5 The figures show the staining results of conjunctival goblet cells of animals after treatment with Dendrobium nanovesicle eye drops (A), the statistical results of goblet cell numbers (B), and the changes in corneal stroma thickness (C);

[0060] Figure 6 The electron microscopy and particle size analysis results of the nanovesicles of the control group A after rehydration are shown; A is the particle size result; B is the inner and outer vesicles that can be detected in the sample;

[0061] Figure 7 The electron microscopy and particle size analysis results of the nanovesicles in the control group B after rehydration are shown; A is the particle size result; B is the inner and outer vesicles that can be detected in the sample. DETAILED DESCRIPTION

[0062] The present invention provides a dendrobium nanovesicle complex and a preparation method and application thereof. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0063] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0064] The present invention provides a preparation of a dendrobium nanovesicle complex and an eye drop containing the complex, which specifically includes the following advantages:

[0065] 1. Source of new plant nanovesicles: This invention uses hydroponic Dendrobium

[0066] The nanovesicles were obtained from the bulbs of Dendrobium officinale, which contained vesicles outside the bulbs and vesicles inside the plant tissues. The isolated plant nanovesicle complex was used as a new active ingredient for the treatment of dry eye. Dendrobium nanovesicles have unique biological properties, such as promoting the proliferation and repair of corneal epithelial cells and increasing tear secretion. These properties make it an ideal choice for the treatment of dry eye.

[0067] 2. Optimized freeze-drying-rehydration technology: In order to increase the water content of the dendrobium nanovesicles and ensure their activity, the present invention adopts an innovative freeze-drying-rehydration treatment technology. This technology not only retains the complete structure of the nanovesicles, but also enhances their hydration capacity, so that they can be effectively stored at room temperature and conveniently transported.

[0068] 3. Unique eye drop formula: The present invention has developed a formula for eye drops of Dendrobium bulb nanovesicle complex, including hydroponic Dendrobium nanovesicle complex, ascorbic acid, vitamin E, PBS and sodium chloride. In particular, the eye drops are composed of two tubes, one tube containing an effective concentration of vesicle complex, and the other tube containing a matrix component. When used, the matrix component only needs to be injected into the outer vesicle tube to complete the preparation. This design not only simplifies the use process, but also ensures the high purity and activity of the outer vesicle.

[0069] 4. Preclinical effect verification: After testing on a mouse dry eye model, the eye drops of the present invention have shown significant effects in promoting tear secretion, protecting conjunctival goblet cells and promoting corneal epithelial repair. The product is pure natural, preservative-free, suitable for long-term use, will not cause eye irritation or lacrimal punctum embolism, and its nano-level active substances will not hinder the normal discharge of tears.

[0070] In summary, the present invention not only solves the limitations of existing dry eye treatment methods, but also provides a better solution in the extraction, preservation and application of Dendrobium nanovesicle complexes, significantly improving the therapeutic effect of dry eye.

[0071] The present invention will be further described below in conjunction with embodiments:

[0072] Example 1 Preparation of Dendrobium bulb nanovesicle complex

[0073] 1. Extraction of Nanovesicle Complex from Hydroponic Dendrobium Corm

[0074] (1) Wash and cut 100g of hydroponic Dendrobium stems into 1cm pieces 3 Small pieces of corms are then placed in an ultrasonic machine for ultrasonic treatment to destroy the plant cell wall and enhance the production of exosomes. Ultrasonic conditions: the temperature is set to 4°C, the time is 5min / time, and the interval is 10min. The ultrasonic program is performed again, and this program is performed three times in total.

[0075] (2) homogenizing the sample after ultrasound, adding 100 ml of ultrapure water, homogenizing using a homogenizer, and collecting the homogenate;

[0076] (3) The homogenate was centrifuged at 3000 g for 30 min at 4°C to remove dead cells and the supernatant was collected;

[0077] (4) The supernatant was centrifuged at 10,000 g for 60 min at 4°C to remove cell debris and the supernatant was collected;

[0078] (5) 0.22 μm filter membrane to remove pollutants;

[0079] (6) For further purification, the filtered supernatant was subjected to ultracentrifugation at 150,000 g for 120 min at 4°C in a swing-out rotor to collect the precipitate;

[0080] (7) After the precipitate is dissolved with PBS, repeat the above ultracentrifugation conditions again, collect the precipitate, dissolve it with 1 ml PBS containing 75 mM trehalose, and store it at -80°C for subsequent operations.

[0081] 2. Lyophilization and rehydration of nanovesicle complexes

[0082] (1) The frozen nanovesicle sample is freeze-dried using a freeze dryer to remove moisture.

[0083] (2) Freeze-drying parameters and procedures

[0084] 1) Pre-freezing: quick freezing temperature: between -193°C and 196°C; quick freezing time: 1-2 hours, to ensure that the nanovesicle complex suspension forms small ice crystals during rapid freezing, reducing damage to the bilayer structure, because the vesicle dissolving solution contains trehalose, which can protect the bilayer of the outer vesicle from damage.

[0085] 2) Primary drying stage (main drying): Temperature: set between -40℃ and -30℃; Pressure: below 100bar. Time: 24-48 hours. The key to this stage is to slowly increase the temperature to avoid direct evaporation of water molecules and damage to the membrane structure.

[0086] 3) Secondary drying stage (desorption drying): Temperature: slowly raised to -10℃ to 0℃; Pressure: below 10bar. Time: 12 to 24 hours. The purpose of this stage is to remove the residual bound water and ensure the stability of the final product.

[0087] (3) Rehydration parameters and procedures

[0088] 1) Reconstitution solution: Use PBS solution containing 5% dextran. Reconstitution temperature: room temperature (20-25°C); Reconstitution time: 15-30 minutes, ensuring slow and sufficient reconstitution. Reconstitution method: Slowly add the lyophilized powder to the reconstitution solution and gently stir until completely dissolved.

[0089] (II) Electron microscopic identification and particle size analysis of nanovesicle complexes

[0090] The rehydrated nanovesicles were analyzed by electron microscopy and particle size analysis. Figure 1 .

[0091] The results show that the average particle size of the dendrobium nanovesicle complex prepared in the present invention is 113.6 nm.

[0092] Embodiment 2 preparation of eye drops

[0093] (1) Preparation of tube A: The rehydrated nanovesicle complex of Example 1 was placed into tube A at a concentration of 1.2×10 7 ,2.4×10 7 ,3.6×10 7 ,4.8×10 7 vesicles / μl), the optimal concentration was 3.6×10 7 vesicles / μl, volume 500 μl;

[0094] (2) Preparation of tube B: The excipients of the eye drops (i.e., the matrix component) are prepared in tube B with a volume of 2 ml, wherein the matrix component includes 2 ml of PBS buffer and the following components in the following concentrations: 2% (w / v) glycerol, 0.2% (w / v) ascorbic acid, 0.05% (w / v) vitamin E, and 0.9% (w / v) sodium chloride.

[0095] (3) Mixed use: When using, press tube B into tube A and mix to obtain eye drops.

[0096] Example 3 Effect Evaluation Test

[0097] 1) Proof that the Dendrobium nanovesicle complex can be absorbed and internalized by cells:

[0098] Depend on Figure 2 It can be seen that the hydroponic Dendrobium nanovesicle complex extracted by the present invention can be absorbed and internalized by corneal cells.

[0099] 2) Protective effect of Dendrobium nanovesicle complex on hyperosmotic corneal epithelial cells in vitro:

[0100] Different dilution concentrations of Dendrobium nanovesicle complex (1.2×10 7 ,2.4×10 7 ,3.6×10 7 ,4.8×10 7 , 6×10 7 The corneal cells were pretreated with 130 mM NaCl for 24 h, and then the corneal cells were treated with 130 mM NaCl for 24 h to create a hypertonic cell model. CCK8 was used to detect the protective effect of extracellular vesicles on corneal cells damaged by hypertonicity for 24 h and 48 h. The results are shown in Figure 3 Middle A~D.

[0101] The results showed that the Dendrobium nanovesicle complex was within a certain concentration range (1.2×10 7 ~4.8×10 7 vesicles / μl) can promote the proliferation of corneal epithelial cells, and high concentrations of Dendrobium nanovesicle complexes (6×10 7 The results showed that a certain concentration of Dendrobium nanovesicle complex has a significant cell protection effect.

[0102] 2.1 First, observe the different dilution concentrations of Dendrobium nanovesicle complexes (1.2×10 7 ,2.4×10 7 ,3.6×10 7 ,4.8×10 7 , 6×10 7 vesicles / μl) have adverse effects on the growth of normal corneal epithelial cells. Figure 3 -A shows that different concentrations of Dendrobium nanovesicles acted on corneal epithelial cells for 24 hours, but had no effect on normal cultured corneal epithelial cells. However, after 48 hours, high concentration (6×10 7 vesicles / μl) group had a damaging effect on normal cultured corneal epithelial cells. 7 ~4.8×10 7 Dendrobium nanovesicles at a diluted concentration of vesicles / μl had no side effects on the normal cultured corneal epithelium.

[0103] 2.2 Preparation of hyperosmotic corneal epithelial cell model to simulate the hyperosmotic state of corneal cells when dry eye occurs, and calculation of NaCl concentration by IC50

[0104] 2.3 The hyperosmotic corneal epithelial cell model was prepared using the NaCl concentration explored in 2.2, and then different dilution ratios of Dendrobium nanovesicles were administered to observe their protective effects on cell damage. Figure 3 Figure C shows that when corneal epithelial cells were damaged by hyperosmotic injury, the repair treatment with different dilution ratios of Dendrobium nanocapsules had no significant repair effect compared with the corneal cells in the model group. However, when the corneal epithelial cells were treated with different dilutions of Dendrobium nanocapsules before hyperosmotic modeling, it can be seen that Dendrobium nanocapsules can effectively protect the epithelial cells. The concentration range was (2.4×10 7 ~6×10 7 vesicles / μl) can promote the proliferation of corneal epithelial cells, see Figure 3 Middle D.

[0105] 2) Study on the effect of patented eye drops on mouse dry eye model

[0106] ① Experimental grouping and dosing: C57 male mice (6-8 weeks, weighing about 25 g) were selected for the experiment, with 6 mice in each group. Scopolamine (0.3 ml, 0.25 mg / ml, subcutaneous injection behind the ear) and continuous modeling in a drying oven (humidity below 30%) were used for 14 days. At the same time, Dendrobium nanovesicle complex eye drops (prepared according to the method of Example 2) were given three times a day, 5 ul each time, and an auxiliary material control group was set up.

[0107] After modeling, on the 7th and 14th days, first use 1-2 drops of 0.5% tetracaine eye drops to anesthetize the ocular surface, and wait for about 30 seconds to 1 minute for the anesthetic to take effect; then use sodium fluorescein test paper to stain the corneal epithelial defects. When there is a defect in the corneal epithelium, sodium fluorescein can bind to the protein in the tears and penetrate into the area of ​​the corneal epithelium defect. The normal corneal epithelium is intact and can prevent the entry and attachment of fluorescein, so there is no coloring. After moistening one end of the fluorescein test paper with saline, gently touch the inner side of the lower eyelid conjunctiva to transfer a small amount of sodium fluorescein to the conjunctival sac for about 5-10 seconds to allow the fluorescein to be fully distributed in the eye; after staining, use a slit lamp microscope and adjust the light source to cobalt blue light. Open the mouse eyes and aim the microscope objective at the eyes for observation. Under the irradiation of cobalt blue light, the normal corneal area (see Figure 4 The corneal area shown in the normal mouse eyeball position in A does not emit fluorescence, while the corneal epithelial defect will show bright green fluorescence, which is used to evaluate the degree of corneal damage. Figure 4 The area of ​​corneal epithelial defect was converted into the area of ​​fluorescein staining and graded. Figure 4 C. At the same time, tear secretion test strips were used to count tear secretion, and the results are shown in Figure 4 Middle B. Day 7 and Day 14 are the results of the 7th and 14th days of administration of Dendrobium nanovesicles, respectively.

[0108] After the 14th day of observation, the whole cornea was taken for paraffin section fixation, and the epithelial tissue of the conjunctiva of the upper and lower eyelids was stained with Alcian blue-periodic acid Schiff (PAS) to observe the goblet cells. Goblet cells contain abundant mucin granules. The main component of mucin granules is mucin, which belongs to the polysaccharide substance and can be stained with PAS to appear purple-red, so it can be observed and identified under a microscope. The results are shown in Figure 5 A, and count the number of goblet cells (see Figure 5 HE staining was used to observe the integrity and thickness of each corneal layer and to measure the thickness of the corneal stroma (see Figure 5 Middle C).

[0109] ②Results analysis

[0110] Depend on Figure 4 It can be seen that compared with the model group and the auxiliary material control group, the Dendrobium nanovesicle complex group can significantly promote corneal epithelial repair, improve dry eye symptoms, and promote tear secretion.

[0111] Depend on Figure 5 It can be seen that compared with the model group and the auxiliary material control group, the Dendrobium nanovesicle complex group can significantly promote the number of goblet cells, reduce corneal edema, and protect corneal tissue.

[0112] Example 4 Comparison of the protective effects of different concentrations of trehalose under the same freeze-drying conditions

[0113] 1) The experiment set up a 38mM trehalose group and a group without trehalose

[0114] Control group A: 38 mM trehalose group: The nanovesicle complex of Dendrobium bulb was extracted according to the method of Example 1, then freeze-dried and rehydrated, except that 1 ml PBS containing 38 mM trehalose was used for dissolution in step (7). The rehydrated nanovesicle complex was subjected to electron microscopy and particle size analysis. The results are shown in Figure 6 .

[0115] Control group B: The nanovesicle complex of Dendrobium bulb was extracted according to the method of Example 1, then freeze-dried and rehydrated, except that 1 ml PBS (without trehalose) was used for dissolution in step (7). The rehydrated nanovesicle complex was subjected to electron microscopy and particle size analysis. The results are shown in Figure 7 .

[0116] The results showed that when 38 mM trehalose was contained, the nanovesicles aggregated to form vesicles of uneven sizes, and the double-layer lipid-soluble membrane disk of the vesicles was destroyed. When trehalose was not contained, the nanovesicles were either degraded or aggregated to form large vesicles after freeze-drying and rehydration, and the vesicles were obviously destroyed. The PBS buffer solution of 75 mM trehalose of the present invention can effectively protect the double molecular layer of the outer vesicles during the freeze-drying process to prevent structural damage.

[0117] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of Dendrobium officinale nanovesicle complexes in the preparation of drugs for the treatment of diseases related to epithelial cell damage; The epithelial cell damage includes: Corneal epithelial defects and / or skin wounds and ulcers caused by diabetes; The diseases associated with epithelial cell damage include dry eye, diabetic ulcer, superficial keratitis, corneal ulcer and / or skin ulcer.

2. A method for preparing a dendrobium nanovesicle complex, characterized in that: include: Step (1): extracting the dendrobium bulb by ultrasonic method, homogenizing, centrifuging and filtering to obtain a supernatant containing dendrobium nanovesicles; Step (2): After ultracentrifugation of the supernatant obtained in step (1), the precipitate is collected and added to buffer A containing 60-85 mM trehalose, and lyophilized to obtain a nanovesicle complex.

3. The preparation method according to claim 2, characterized in that: In step (1): The ultrasonic extraction was performed twice under the following conditions: 2-8°C, ultrasonic extraction was performed once every 5-15 minutes, and each ultrasonic extraction lasted for 5-10 minutes; The homogenization conditions are 2-8°C, 2000g-5000g centrifugation for 10-30min; The centrifugation comprises: centrifugation at 2-8°C and 3000g for 10-30min, taking the supernatant and centrifuging at 2-8°C and 8000g-15000g for 40-90min; The filtration is carried out by 0.22 μm filter membrane filtration.

4. The preparation method according to claim 2, characterized in that: In step (2), the ultracentrifugation was performed twice, and the conditions for each ultracentrifugation were: 4°C, 150,000 g, ultracentrifugation for 120 minutes; The buffer A includes at least one of PBS buffer, Hanks balanced salt solution and 0.9% sodium chloride solution.

5. The preparation method according to claim 1, characterized in that: The freeze-drying includes pre-freezing, primary drying and secondary drying; The pre-freezing comprises: lowering the temperature to -193°C to 196°C for 1-2 hours; The primary drying temperature includes: raising the temperature to -40°C to -30°C, the pressure is lower than 100 bar, and the holding time is 24 to 48 hours; The secondary drying includes: slowly raising the temperature to -10°C to 0°C, the pressure is lower than 10 bar, and the holding time is 12-24 hours.

6. The preparation method according to any one of claims 2 to 5, characterized in that: After the freeze-drying, the method further comprises a rehydration step; the solution used for the rehydration is buffer A containing 5% dextran.

7. The Dendrobium nanovesicle complex prepared by the preparation method according to any one of claims 2 to 6.

8. Use of the dendrobium nanovesicle complex prepared by the preparation method of any one of claims 2 to 6 or the dendrobium nanovesicle complex according to claim 9 in the preparation of a drug for treating diseases related to epithelial cell damage; The epithelial cell damage includes: Corneal epithelial defects, or wounds and ulcers caused by diabetic foot; The epithelial cell damage-related diseases include dry eye.

9. An eye drop for treating dry eyes, characterized in that: Includes vesicle components and matrix components; The vesicle component is: the Dendrobium nanovesicle complex prepared by any one of the preparation methods of claims 2 to 6 or the Dendrobium nanovesicle complex described in claim 7; The matrix component includes water and at least one of the following components: Glycerol, ascorbic acid, vitamin E, PBS, sodium chloride.

10. The eye drops according to claim 9, characterized in that: The eye drops include the dendrobium nanovesicle complex, glycerol, ascorbic acid, vitamin E, PBS and sodium chloride; The vesicle components include water and dendrobium nanovesicle complex; the concentration of the dendrobium nanovesicle complex is 1.2-4.8×10^7 vesicles / μl; The matrix components include PBS buffer, glycerol, ascorbic acid, vitamin E and sodium chloride; Preferably, the matrix component includes PBS buffer and the following components in percentage by weight and volume: 2%-10% w / v glycerol, 0.1%-0.5% w / v ascorbic acid, 0.01%-0.05% w / v vitamin E, 0.9% w / v sodium chloride.

Images