Hydrogel containing plant exosome as well as preparation method and application thereof

Abstract

The invention relates to the technical field of hydrogel spray preparations, in particular to hydrogel containing plant exosomes as well as a preparation method and application of the hydrogel. The invention provides hydrogel containing plant exosomes, the hydrogel comprises plant exosome freeze-dried powder and a hydrogel base solution, and the plant exosome freeze-dried powder comprises houttuynia cordata exosome powder, aloe exosome powder and honeysuckle exosome powder in a mass ratio of (1-2): (2-4): (1-2). The plant exosome compound has excellent effects of resisting inflammation, inhibiting bacteria and promoting epidermal cell proliferation; the plant exosome and the temperature-sensitive hydrogel are subjected to complementary synergism in the medication mode and the treatment effect, the temperature-sensitive hydrogel containing the plant exosome can effectively increase the diabetic foot ulcer wound repair speed, and the hydrogel can slowly release the exosome and treatment factors contained in the exosome to achieve the treatment effect. The selected biodegradable hydrogel is quick and long in curative effect, and the influence of waste generated after treatment on the environment is not increased.

Description

Technical Field

[0001] The present application belongs to the technical field of hydrogel spray preparations, and in particular relates to a hydrogel containing plant exosomes and a preparation method and application thereof. Background Art

[0002] Diabetic foot is one of the common complications of diabetic patients. It is caused by ischemic diseases of the lower limbs caused by diabetes. Patients not only have high blood sugar in their bodies, but also have vascular occlusion and blood supply disorders caused by vascular lesions in the lower limbs, which prevent the foot tissue from getting enough nutrients. Minor trauma can cause microbial invasion and infection, and it is easy to spread and form difficult-to-heal ulcers. At present, there are limited clinical treatments for diabetic foot, mostly anti-infective drugs and surgical operations, but long-term use of antibiotics is prone to drug resistance, and amputation seriously affects the quality of life of patients. Therefore, it is of great significance to study a new method that can effectively repair diabetic foot.

[0003] Organic reagents are often used as raw materials for the preparation of thermosensitive hydrogels. Therefore, such gels are prone to biological toxicity and other inflammatory reactions, which reduces the biocompatibility of such hydrogels and limits the application of thermosensitive smart hydrogels involving organic reagents. Choosing a thermosensitive hydrogel that is biodegradable and can respond quickly to gelation can solve these problems well.

[0004] Studies have shown that plant exosomes have anti-inflammatory, antiviral, anti-fibrotic, and anti-tumor effects, and participate in the defense response to pathogen invasion. Therefore, exosomes from medicinal plants may interact with mammalian systems as new bioactive ingredients. Summary of the invention

[0005] The purpose of the present application is to provide a hydrogel containing plant exosomes and a preparation method and application thereof, aiming to solve the problem that there is no plant-based hydrogel spray that can effectively repair diabetic foot in the prior art.

[0006] In order to achieve the above application purpose, the technical solution adopted in this application is as follows:

[0007] In a first aspect, the present application provides a hydrogel containing plant exosomes, wherein the hydrogel comprises plant exosome freeze-dried powder and a hydrogel base liquid, wherein the plant exosome freeze-dried powder comprises Houttuynia cordata exosome powder, aloe exosome powder and honeysuckle exosome powder in a mass ratio of 1-2:2-4:1-2.

[0008] In some embodiments, the hydrogel base liquid includes chitosan, hydroxymethyl chitosan and sodium glycerophosphate in a volume ratio of 4-5:2-3:4-5.

[0009] In some embodiments, the volume of the plant exosome freeze-dried powder accounts for 1 / 7 to 2 / 7 of the total volume of the hydrogel.

[0010] In some embodiments, the hydrogel containing plant exosomes is administered by spraying in the form of a spray.

[0011] In a second aspect, the present application provides a method for preparing a hydrogel containing plant exosomes, comprising the following steps:

[0012] Providing fresh plant samples for exosome preparation to obtain plant exosome freeze-dried powder;

[0013] Mixing the hydrogel base components to obtain a hydrogel base liquid;

[0014] The plant exosome freeze-dried powder and the hydrogel base liquid are mixed, and treated in a water bath at 36-37° C. to obtain a hydrogel containing plant exosomes.

[0015] In some embodiments, the method for preparing the plant exosome freeze-dried powder comprises the following steps:

[0016] Provide fresh plant samples for pre-treatment, grind and collect the mixture;

[0017] The mixture is centrifuged multiple times by differential centrifugation, the supernatant is collected and filtered, and then ultra-high-speed centrifugation is performed to collect the precipitate;

[0018] The precipitate is resuspended to obtain an exosome solution, and then freeze-dried to obtain plant exosome freeze-dried powder.

[0019] In some embodiments, the step of subjecting the mixture to multiple centrifugation treatments using differential centrifugation includes: centrifuging the mixture at 4°C at a speed of 1000-1200g for 10-12 minutes, at a speed of 3000-3200g for 20-22 minutes, and at a speed of 10000-11000g for 30-32 minutes.

[0020] In some embodiments, the ultra-high speed centrifugation treatment is performed at a rotation speed of 100,000 to 120,000 for 70 to 75 minutes.

[0021] In some embodiments, the protein concentration of the exosome solution is 100-300 μg / ml.

[0022] In some embodiments, the method for preparing the hydrogel base liquid comprises the following steps:

[0023] Dissolving chitosan, hydroxymethyl chitosan and sodium glycerophosphate in water respectively to prepare a chitosan solution, a hydroxymethyl chitosan solution and a sodium glycerophosphate solution, wherein the mass percentage of the solute in the chitosan solution is 5% to 8%, the mass percentage of the solute in the hydroxymethyl chitosan solution is 2.5% to 3%, and the mass percentage of the solute in the sodium glycerophosphate solution is 50% to 60%;

[0024] Then mix according to the proportion to obtain a hydrogel base liquid.

[0025] In a third aspect, the present application provides a hydrogel containing plant exosomes or a hydrogel containing plant exosomes prepared by the above-mentioned method for preparing a hydrogel containing plant exosomes in the treatment and repair of diabetic foot ulcers.

[0026] The first aspect of the present application provides a hydrogel containing plant exosomes, which includes plant exosomes and a hydrogel base liquid. The plant exosomes provided are preferably a composite component of exosomes of Houttuynia cordata, aloe vera and honeysuckle. The plant exosome complex has excellent effects of anti-inflammatory, antibacterial and promoting epidermal cell proliferation; the plant exosomes and the thermosensitive hydrogel complement each other in terms of medication mode and therapeutic effect. The thermosensitive hydrogel containing plant exosomes can effectively increase the repair speed of diabetic foot ulcer wounds, and because the hydrogel can achieve the sustained release of exosomes and the therapeutic factors contained therein to achieve the therapeutic effect, the selected biodegradable hydrogel has a fast and long-lasting therapeutic effect and does not increase the environmental impact caused by the waste generated after treatment.

[0027] The second aspect of the present application provides a method for preparing a hydrogel containing plant exosomes. The method first prepares plant exosome freeze-dried powder and hydrogel base liquid, respectively, and then optimizes the mass ratio to ensure that the temperature of the hydrogel phase change does not exceed 37°C. The temperature stimulus response condition of the thermosensitive hydrogel is used to allow the product to be sprayed and administered before use. When the spray liquid contacts the human skin, a hydrogel can be formed within 3-5 minutes. A better and more convenient way of administration can better solve the problems of incomplete administration and low drug utilization in patients with diabetic foot. The preparation method is simple and quick, which is conducive to the rapid preparation of products and convenient administration of the obtained products.

[0028] The third aspect of the present application provides a hydrogel containing plant exosomes, or a hydrogel containing plant exosomes prepared by the above-mentioned method for preparing a hydrogel containing plant exosomes, for use in the treatment and repair of diabetic foot ulcers. Since the plant exosomes in the provided hydrogel containing plant exosomes have anti-inflammatory, antibacterial, and epidermal cell proliferation-promoting effects, the hydrogel can achieve the effect of sustained release of exosomes and therapeutic factors contained therein to achieve therapeutic effects. Therefore, the hydrogel containing plant exosomes is directly sprayed on the diabetic foot ulcer, and a gel film can be formed after 3-5 minutes to better cover the ulcer. The method of use is simple, and the storage conditions are not harsh, which is conducive to its wide application in the treatment and repair of diabetic foot ulcers. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0030] Figure 1 This is the exosome dry powder preparation sample provided in Example 1 of the present application.

[0031] Figure 2 This is the gel solidification condition provided in Example 2 of the present application.

[0032] Figure 3 It is the thermosensitive hydrogel containing plant exosomes provided in Example 3 of the present application.

[0033] Figure 4 This is an analysis diagram of the plant exosome mixture promoting cell proliferation provided in Example 4 of the present application.

[0034] Figure 5 This is a diagram of the plant exosome mixture promoting cell migration provided in Example 5 of the present application.

[0035] Figure 6 This is an analysis diagram of the effect of the plant exosome mixture on the expression of inflammatory factors provided in Example 7 of the present application.

[0036] Figure 7 This is a diagram showing the effect of the plant exosome mixture hydrogel provided in Example 8 of the present application on the repair of skin damage in diabetic rats. DETAILED DESCRIPTION

[0037] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0038] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0039] In this application, "at least one" means one or more, and "plurality" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.

[0040] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0041] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "an" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0042] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass described in the embodiment description of the present application can be a mass unit known in the chemical industry such as μg, mg, g, kg, etc.

[0043] The terms "first" and "second" are used only for descriptive purposes to distinguish objects such as substances from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Thus, features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0044] An embodiment of the present application provides a hydrogel containing plant exosomes, wherein the hydrogel includes plant exosome freeze-dried powder and a hydrogel base liquid, wherein the plant exosome freeze-dried powder includes Houttuynia cordata exosome powder, aloe exosome powder and honeysuckle exosome powder in a mass ratio of 1-2:2-4:1-2.

[0045] A first aspect of an embodiment of the present application provides a hydrogel containing plant exosomes, which includes plant exosomes and a hydrogel base liquid. The provided plant exosomes are preferably a composite component of exosomes of Houttuynia cordata, aloe vera and honeysuckle. The plant exosome complex has excellent effects of anti-inflammatory, antibacterial and promoting epidermal cell proliferation; the plant exosomes and the thermosensitive hydrogel complement each other in terms of medication mode and therapeutic effect. The thermosensitive hydrogel containing plant exosomes can effectively increase the repair speed of diabetic foot ulcer wounds, and because the hydrogel can achieve the sustained release of exosomes and the therapeutic factors contained therein to achieve the therapeutic effect, the selected biodegradable hydrogel has a fast and long-lasting therapeutic effect, and does not increase the environmental impact caused by the waste generated after treatment.

[0046] In some embodiments, the hydrogel base liquid includes chitosan, hydroxymethyl chitosan and sodium glycerophosphate in a volume ratio of 4 to 5: 2 to 3: 4 to 5. Chitosan / carboxymethyl chitosan / sodium glycerophosphate (CS / CMCS / GPS) thermosensitive hydrogel is a composite hydrogel material composed of chitosan (CS), carboxymethyl chitosan (CMCS) and sodium glycerophosphate (GPS), which has temperature-sensitive properties. Chitosan (CS) is a natural polymer derived from the deacetylation product of crustacean shells and has good biocompatibility and biodegradability. Carboxymethyl chitosan (CMCS) is a chemically modified product of chitosan. The water solubility of chitosan is improved by introducing carboxymethyl groups and giving it more functional properties. Sodium glycerophosphate (GPS) is a thermosensitive material that can cause the sol-gel transition of hydrogel within a certain temperature range. It can interact with chitosan and carboxymethyl chitosan in aqueous solution to form a stable network structure.

[0047] In some embodiments, the volume of the plant exosome freeze-dried powder accounts for 1 / 7 to 2 / 7 of the total volume of the hydrogel.

[0048] In some embodiments, the hydrogel containing plant exosomes is administered by spraying in the form of a spray. The hydrogel utilizes the temperature stimulus response condition of the thermosensitive hydrogel, so that the product can be administered by spraying in the form of a spray before use. When the spray liquid contacts the human skin, the hydrogel can be formed within 3-5 minutes. This is a better and more convenient way of administration, which can better solve the problems of incomplete administration and low drug utilization in patients with diabetic foot.

[0049] The second aspect of the embodiment of the present application provides a method for preparing a hydrogel containing plant exosomes, comprising the following steps:

[0050] S01. Providing fresh plant samples for exosome preparation to obtain plant exosome freeze-dried powder;

[0051] S02. The hydrogel base components are mixed to obtain a hydrogel base liquid;

[0052] S03. The plant exosome freeze-dried powder and the hydrogel base liquid are mixed, and treated in a water bath at 36-37° C. to obtain a hydrogel containing plant exosomes.

[0053] The preparation method of the hydrogel containing plant exosomes provided in the second aspect of the embodiment of the present application, the preparation method first prepares the plant exosome freeze-dried powder and the hydrogel base liquid respectively, and then optimizes the mass ratio to ensure that the temperature of the hydrogel phase change does not exceed 37 ° C, and utilizes the temperature stimulus response condition of the thermosensitive hydrogel so that the product can be sprayed in the form of spray before use. When the spray liquid contacts the human skin, it can form a hydrogel within 3-5 minutes. The better and more convenient way of administration can better solve the problems of incomplete administration and low drug utilization in patients with diabetic foot. The preparation method is simple and fast, which is conducive to the rapid preparation of products and the obtained products are convenient for administration.

[0054] In step S01, a fresh plant sample is provided for exosome preparation to obtain plant exosome freeze-dried powder.

[0055] In some embodiments, the method for preparing the plant exosome freeze-dried powder comprises the following steps:

[0056] G01. Provide fresh plant samples for pre-treatment, grind and collect the mixture;

[0057] G02. The mixture is centrifuged multiple times by differential centrifugation, the supernatant is collected and filtered, and then ultracentrifuged to collect the precipitate;

[0058] G03. Resuspend the precipitate to obtain an exosome solution, and then freeze-dry it to obtain plant exosome freeze-dried powder.

[0059] In some embodiments, the step of subjecting the mixture to multiple centrifugation treatments using differential centrifugation includes: centrifuging the mixture at 4°C at a speed of 1000-1200g for 10-12 minutes, at a speed of 3000-3200g for 20-22 minutes, and at a speed of 10000-11000g for 30-32 minutes.

[0060] In some embodiments, the ultra-high speed centrifugation treatment is performed at a speed of 100,000 to 120,000 g for 70 to 75 minutes.

[0061] In some embodiments, the protein concentration of the exosome solution is 100-300 μg / ml.

[0062] In some specific embodiments, the method for preparing plant exosomes specifically comprises the following steps:

[0063] After obtaining fresh Houttuynia cordata, Aloe vera or Honeysuckle samples, wash them several times and cut them into pieces;

[0064] Use a high-speed blender to grind Houttuynia cordata and add an appropriate amount of ice-water mixture to avoid excessive temperature during the process;

[0065] The mixed liquid was centrifuged at 1000 g for 10 min, 3000 g for 20 min, and 10000 g for 30 min at 4 °C by differential centrifugation, and the supernatant was taken each time to remove impurities and large particles.

[0066] After filtering out impurities in the liquid supernatant in the previous step using a 0.45 μm filter, use an ultra-high-speed centrifuge to centrifuge at 100,000 g for 70 min. After the centrifugation is completed, the precipitate is resuspended in PBS solution and stored at -80°C for later use;

[0067] The resuspended exosomes were tested for total protein using the Bio-Tech BCA protein quantitative detection kit, preferably at a concentration of 100-300 μg / ml;

[0068] The extracted exosomes were finally vacuum filtered and freeze-dried for use.

[0069] In step S02, the various hydrogel basic components are mixed to obtain a hydrogel base liquid.

[0070] In some embodiments, the method for preparing the hydrogel base liquid comprises the following steps:

[0071] Dissolving chitosan, hydroxymethyl chitosan and sodium glycerophosphate in corresponding solvents respectively to prepare chitosan solution, hydroxymethyl chitosan solution and sodium glycerophosphate solution, wherein the mass percentage of solute in the chitosan solution is 5% to 8%, the mass percentage of solute in the hydroxymethyl chitosan solution is 2.5% to 3%, and the mass percentage of solute in the sodium glycerophosphate solution is 50% to 60%;

[0072] Then mix according to the proportion to obtain a hydrogel base liquid.

[0073] The present application prefers the above-mentioned thermosensitive hydrogel for subsequent experiments. The preferred configuration scheme is that when the above-mentioned chitosan (CS), carboxymethyl chitosan (CMCS) and sodium glycerophosphate (GPS) components are mixed together in proportion, the preferred mixing ratio of the present invention is: preferably chitosan: hydroxymethyl chitosan: sodium glycerophosphate are mixed in a volume ratio of 4:2:4. This hydrogel is a flowing liquid at room temperature, but when the temperature rises to near human body temperature, it will become a semi-solid gel. Its characteristics can be used for spray treatment. The prepared hydrogel is finally freeze-dried after vacuum filtration for use.

[0074] In step S03, the plant exosome freeze-dried powder and the hydrogel base liquid are mixed and treated in a water bath at 36-37° C. to obtain a hydrogel containing plant exosomes.

[0075] In some specific embodiments, the prepared plant exosome dry powder contains three kinds of plant exosomes, namely, houttuynia cordata, aloe vera and honeysuckle, and the mixing ratio is 1:2:1 by mass, and is pre-mixed into a dry powder preparation. After being mixed with the thermosensitive hydrogel dry powder with deionized water for 3-5 minutes, it can be used for spraying administration.

[0076] A third aspect of the embodiments of the present application provides the use of a hydrogel containing plant exosomes or a hydrogel containing plant exosomes prepared by the above-mentioned method for preparing a hydrogel containing plant exosomes in the treatment and repair of diabetic foot ulcers.

[0077] The use of the plant exosome-containing hydrogel provided in the third aspect of the embodiment of the present application or the plant exosome-containing hydrogel prepared by the above-mentioned method for preparing the plant exosome-containing hydrogel in the treatment and repair of diabetic foot ulcers. Since the plant exosomes in the provided hydrogel have anti-inflammatory, antibacterial and epidermal cell proliferation-promoting effects, the hydrogel can achieve the effect of sustained release of exosomes and therapeutic factors contained therein to achieve the therapeutic effect. Therefore, the plant exosome-containing hydrogel is directly sprayed on the diabetic foot ulcer, and a gel film can be formed after 3-5 minutes to better cover the ulcer. The method of use is simple, the storage conditions are not harsh, and it is conducive to its wide application in the treatment and repair of diabetic foot ulcers.

[0078] The following describes the invention in conjunction with specific embodiments.

[0079] Example 1

[0080] Preparation of plant exosomes

[0081] The present embodiment selects fresh herb houttuynia 20g, aloe 40g, honeysuckle 20g respectively, and the above materials are repeatedly cleaned and cut into pieces. Utilize high-speed agitator (homogenizer) to grind herb houttuynia, aloe, honeysuckle respectively, and add appropriate 4 ℃ phosphate buffer solution 3-5ml in the process to avoid too high stirring temperature. The liquid is obtained through 1000g 10min, 3000g20min, 10000g 30min multiple centrifugation, and the supernatant is taken each time to remove impurities and large particles. After the impurities in the liquid supernatant are filtered out by a 0.45μm filter, an ultracentrifuge is used to carry out 100000g 70min centrifugation, and after centrifugation, the precipitation is resuspended with PBS solution and stored in -80 ℃ for standby use. The resuspended plant exosomes of Houttuynia cordata, aloe vera, honeysuckle and other plants were tested for the total amount of exosome protein according to the instructions of the Bio-Tech BCA protein quantitative detection kit. The concentrations of plant exosomes of Houttuynia cordata, aloe vera, honeysuckle and other plants were adjusted to 150μg / ml, 300μg / ml and 150μg / ml respectively according to the requirements of subsequent experiments. Then, the mixed ratio of the extracted plant exosomes of Houttuynia cordata, aloe vera and honeysuckle was 1:2:1 by mass, freeze-dried in a vacuum dryer, and placed at -20℃ for use. As follows Figure 1 As shown, the prepared exosome dry powder preparation.

[0082] Example 2

[0083] Preparation of thermosensitive hydrogel

[0084] Weigh 5g of chitosan and dissolve it in 100mL of 0.1mol / L hydrochloric acid solution to prepare a 5% chitosan solution. Weigh 2.5g of hydroxymethyl chitosan and dissolve it in 100mL of deionized water to prepare a 2.5% hydroxymethyl chitosan solution. Weigh 50g of sodium glycerol phosphate and dissolve it in 100mL of deionized water to prepare a 50% sodium glycerol phosphate solution. Preferably, mix chitosan: hydroxymethyl chitosan: sodium glycerol phosphate in a volume ratio of 4:2:4. At room temperature and below, the mixed liquid is a flowing liquid. When the temperature rises to 36°C, close to human body temperature, a semi-solid gel can be formed. The prepared hydrogel is finally freeze-dried after vacuum filtration for use. Place the prepared hydrogel upright in a 37°C constant temperature water bath, such as Figure 2 As shown, the test tube is tilted every 30 seconds to observe whether the liquid level follows the tilt of the test tube and determine the gel solidification time.

[0085] Example 3

[0086] Preparation of thermosensitive hydrogel spray containing plant exosomes

[0087] Select 1 mg of the plant exosome dry powder preparation prepared in Example 1, select 1 mg of the chitosan / hydroxymethyl chitosan / sodium glycerophosphate mixed freeze-dried powder preparation prepared in Example 2, add 5 mL of deionized water, and mix well. The prepared hydrogel is a flowing liquid when placed in a room temperature environment. The prepared hydrogel is placed upright in a 37°C constant temperature water bath for 3 minutes to form a hydrogel, as follows Figure 3 As shown, A is the state at room temperature, and B is the state after being taken out of a 37°C water bath.

[0088] Example 4

[0089] Plant exosome mixture promotes cell proliferation experiment

[0090] In this experiment, mouse fibroblasts 3T3 were selected and inoculated into 96 wells at 5000 cells / well. The plant exosomes prepared in Example 1 were dissolved in phosphate buffer. A control group and different concentration experimental groups were set up. The experimental groups were added to the cell culture wells at mixed concentrations of 50μg / ml, 150μg / ml and 300μg / ml. The relative proliferation rate of the cells was calculated according to the instructions of the Biyuntian CCK8 kit at 0 hour before, 24 hours and 48 hours after the addition of exosomes. The results are as follows Figure 4 As shown, compared with the control group, the number of cells in the experimental group increased more and was correlated with the added dose, indicating that the prepared plant exosome mixture has the effect of promoting cell proliferation.

[0091] Example 5

[0092] Hydrogel containing plant exosome mixture promotes cell migration experiment

[0093] In this experiment, human laryngeal cancer epithelial cells Hep2 in the exponential growth phase were selected and cultured at a rate of 1×10 6 Cells / well were inoculated on a 6-well plate. After overnight, a 20μl pipette tip was used to scratch perpendicular to the auxiliary line drawn on the back of the well plate the day before. Two scratches were made in total, with a scratch interval of 1cm. After the scratching was completed, the plate was washed 3 times with sterile PBS. The following were set up separately: Group 1: ordinary gel without exosomes, as the control group. Group 2: gel containing plant exosomes, as the experimental group, wherein the exosome-containing gel preparation scheme used in Group 2 referred to Examples 1-3, and the final concentrations of exosomes contained in Houttuynia cordata, Aloe vera and Honeysuckle were 150μg / ml, 300μg / ml and 150μg / ml, respectively. Before adding the gel, the cells were photographed with an inverted microscope as the 0-hour data. The gel was added according to the grouping and placed in a 37°C cell culture incubator for culture. After 48 hours, the cell culture plate was taken out, photographed and the cell migration was observed. The results are as follows Figure 5As shown, compared with the control well of group 1, the gel of group 2 with added plant exosomes can increase the migration rate of cells after incubation with cells for 48 hours, and has the effect of promoting cell migration.

[0094] Example 6

[0095] Hydrogel containing plant exosome mixture inhibits bacterial growth

[0096] This experiment set up group 1: blank control group, 0.5ml of phosphate buffer was added to the bacterial solution; group 2: blank gel group, 0.5ml of blank gel without plant exosomes was added to the bacterial solution; group 3: exosome gel group, 0.5ml of gel containing plant exosome complex was added to the bacterial solution. The initial bacterial solution contained 10000CPU of Escherichia coli. After the above grouping treatment, the samples were cultured in a bacterial incubator for 18 hours. After taking out, 100μL of the mixed liquid was evenly coated on the agar solid culture plate using the dilution coating plate method, and the colony count on the solid culture medium plate was calculated after being placed back in the bacterial incubator for 24 hours, and the corresponding bacterial growth inhibition rate was calculated. The results are shown in Table 1 below. Compared with the control group, the gel containing exosomes had an inhibition rate of 91.51% on bacteria, which had a good effect in inhibiting the growth of Escherichia coli.

[0097] Table 1

[0098]

[0099] Example 7

[0100] Effects of plant exosome mixture on inflammatory factors

[0101] In this experiment, mouse fibroblasts 3T3 were selected and the number of cells was 5x10 5 Cells / well were inoculated into 6 wells, and the plant exosomes prepared in Example 1 were dissolved in phosphate buffer, and the following were set up: Group 1, control group, adding equal volume of PBS buffer, and setting up different concentration experimental groups, Group 2: 100 μg / ml exosomes, Group 3: 300 μg / ml exosomes, Group 4: 600 μg / ml exosomes. Place in a cell culture incubator overnight for 24 hours, collect cell precipitates to extract RNA, reverse transcribe into cDNA, and detect the transcriptional expression level of inflammatory factors.

[0102] The results are as follows Figure 6 As shown, compared with the control group in group 1, the transcriptional expression levels of pro-inflammatory factors TNF-α, IL6 and IL-1β in groups 2, 3 and 4 with added plant exosomes were reduced, and the transcriptional expression level of the inhibitory inflammatory factor IL10 was increased. This indicates that the addition of a mixture of plant exosomes has the effect of inhibiting inflammatory response.

[0103] Example 8

[0104] Experimental study on repairing skin damage in diabetic rats by hydrogel containing plant exosome mixture

[0105] The present invention selected 15 SD male rats, weighing 220-240g, and raised them for one week before modeling. Streptozotocin solution was injected intraperitoneally once a day for 7 consecutive days. Blood sugar was tested 7 days after administration. If the blood sugar was ≥16.67mmol / L in 3 random blood tests on different days, it indicated that the modeling was successful. Ten rats with successful modeling were selected, and after anesthesia, a skin injury model with a diameter of 2cm was prepared on the back skin with a puncher. The rats were randomly divided into 2 groups according to their body weight, with 5 rats in each group. Group 1 was treated with blank hydrogel, which was replaced every 3 days; Group 2 was treated with hydrogel containing plant exosomes, which was replaced every 3 days for 15 days. The wound healing was observed. The results are as follows Figure 7 As shown, compared with Group 1, the wound area of ​​rats containing the plant exosome mixture was gradually reduced and healed faster.

[0106] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A hydrogel containing plant exosomes, characterized in that: The hydrogel comprises plant exosome freeze-dried powder and a hydrogel base liquid, wherein the plant exosome freeze-dried powder comprises houttuynia cordata exosome powder, aloe exosome powder and honeysuckle exosome powder in a mass ratio of 1-2:2-4:1-2.

2. The plant exosome-containing hydrogel according to claim 1, characterized in that: The hydrogel base liquid comprises chitosan, hydroxymethyl chitosan and sodium glycerophosphate in a volume ratio of 4-5:2-3:4-5; and / or, The volume of the plant exosome freeze-dried powder accounts for 1 / 7 to 2 / 7 of the total volume of the hydrogel.

3. The plant exosome-containing hydrogel according to claim 1, characterized in that: The hydrogel containing plant exosomes is administered by spraying in the form of a spray.

4. A method for preparing a hydrogel containing plant exosomes, characterized in that: The steps include: Providing fresh plant samples for exosome preparation to obtain plant exosome freeze-dried powder; Mixing the hydrogel base components to obtain a hydrogel base liquid; The plant exosome freeze-dried powder and the hydrogel base liquid are mixed, and treated in a water bath at 36-37° C. to obtain a hydrogel containing plant exosomes.

5. The plant exosome-containing hydrogel according to claim 4, characterized in that: The method for preparing the plant exosome freeze-dried powder comprises the following steps: Provide fresh plant samples for pre-treatment, grind and collect the mixture; The mixture is centrifuged multiple times by differential centrifugation, the supernatant is collected and filtered, and then ultra-high-speed centrifugation is performed to collect the precipitate; The precipitate is resuspended to obtain an exosome solution, and then freeze-dried to obtain plant exosome freeze-dried powder.

6. The plant exosome-containing hydrogel according to claim 5, characterized in that: The step of subjecting the mixture to multiple centrifugation treatments by differential centrifugation comprises: centrifuging the mixture at 4°C at a speed of 1000-1200g for 10-12 minutes, at a speed of 3000-3200g for 20-22 minutes, and at a speed of 10000-11000g for 30-32 minutes.

7. The plant exosome-containing hydrogel according to claim 5, characterized in that: The ultra-high speed centrifugation treatment is performed at a rotation speed of 100,000 to 120,000 for 70 to 75 minutes.

8. The plant exosome-containing hydrogel according to claim 5, characterized in that: The protein concentration of the exosome solution is 100-300 μg / ml.

9. The plant exosome-containing hydrogel according to claim 4, characterized in that: The method for preparing the hydrogel base liquid comprises the following steps: Dissolving chitosan, hydroxymethyl chitosan and sodium glycerophosphate in water respectively to prepare a chitosan solution, a hydroxymethyl chitosan solution and a sodium glycerophosphate solution, wherein the mass percentage of the solute in the chitosan solution is 5% to 8%, the mass percentage of the solute in the hydroxymethyl chitosan solution is 2.5% to 3%, and the mass percentage of the solute in the sodium glycerophosphate solution is 50% to 60%; Then mix according to the proportion to obtain a hydrogel base liquid.

10. Use of the plant exosome-containing hydrogel according to any one of claims 1 to 3 or the plant exosome-containing hydrogel prepared by the preparation method of the plant exosome-containing hydrogel according to any one of claims 4 to 9 in the treatment and repair of diabetic foot ulcers.

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