Temperature- and pH-responsive hydrogel loaded with chlorogenic acid liposomes, preparation method and application thereof

Abstract

The present invention provides a temperature and pH responsive hydrogel encapsulating chlorogenic acid liposomes, a preparation method and an application thereof, and relates to the field of biotechnology. The temperature and pH responsive hydrogel encapsulating chlorogenic acid liposomes contains chlorogenic acid liposomes encapsulating, modified gelatin grafted with phenylboronic acid, and oxidized hyaluronic acid grafted with dopamine, wherein the concentration of the modified gelatin grafted with phenylboronic acid is 3-10 wt%, the concentration of the oxidized hyaluronic acid grafted with dopamine is 3-10 wt%, and the concentration of chlorogenic acid is 1-20 μM. The temperature and pH responsive hydrogel alone or in combination with blue light can effectively promote wound healing.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a temperature- and pH-responsive hydrogel loaded with chlorogenic acid liposomes, a preparation method thereof, and an application thereof. Background Art

[0002] At present, there are a variety of drugs used clinically to promote wound healing, mainly including antibiotics, growth factors, and traditional Chinese medicine preparations, etc. These drugs have played an important role in promoting wound healing, but at the same time, they also have inevitable defects. Common antibiotic drugs mainly include erythromycin ointment, mupirocin ointment, and fusidic acid cream, etc. However, improper use of antibiotics may lead to an increase in bacterial drug resistance, reduce the efficacy of drugs, and some antibiotics may cause local skin irritation or allergic reactions. Growth factors mainly include gel agents or topical solutions with recombinant human epidermal growth factor as the main drug component. However, growth factor drugs are usually relatively expensive, which limits their wide application, and growth factors may be inactivated due to environmental factors (such as temperature, pH value) during storage and use. There is a rich variety of traditional Chinese medicine topical drugs for promoting wound healing, which mainly play a role through mechanisms such as antibacterial, anti-inflammatory, and promoting tissue regeneration. However, traditional Chinese medicine preparations have the defects of complex components and unclear action mechanisms, and some patients may have allergic reactions to traditional Chinese medicine components. Therefore, developing more diverse products for promoting wound healing helps to better treat and relieve various clinical traumatic injuries.

[0003] In view of this, the present invention is specifically proposed. Summary of the Invention

[0004] The purpose of the present invention is to provide a temperature- and pH-responsive hydrogel loaded with chlorogenic acid liposomes, a preparation method thereof, and an application thereof, so as to improve the defects of existing products for promoting wound healing.

[0005] To solve the above technical problems, the present invention specifically adopts the following technical solutions:

[0006] In the first aspect, a temperature- and pH-responsive hydrogel loaded with chlorogenic acid liposomes is provided. The temperature- and pH-responsive hydrogel contains chlorogenic acid liposomes, modified gelatin grafted with phenylboronic acid, and oxidized hyaluronic acid grafted with dopamine;

[0007] In the temperature- and pH-responsive hydrogel, the concentration of modified gelatin grafted with phenylboronic acid is 3-10 wt%, the concentration of oxidized hyaluronic acid grafted with dopamine is 3-10 wt%, and the concentration of chlorogenic acid is 1-20 μM.

[0008] In a second aspect, a method for preparing the temperature- and pH-responsive hydrogel encapsulating chlorogenic acid liposomes as described in the first aspect is provided, including: mixing the formulated chlorogenic acid liposomes, modified gelatin grafted with phenylboronic acid, and oxidized hyaluronic acid grafted with dopamine to obtain the temperature- and pH-responsive hydrogel encapsulating chlorogenic acid liposomes.

[0009] In a third aspect, the application of the temperature- and pH-responsive hydrogel encapsulating chlorogenic acid liposomes as described in the first aspect, or the preparation method as described in the second aspect in the preparation of a product for promoting wound healing in a subject is provided.

[0010] In a fourth aspect, a dressing for promoting wound healing is provided, and the dressing contains the temperature- and pH-responsive hydrogel encapsulating chlorogenic acid liposomes as described in the first aspect.

[0011] In a fifth aspect, a product for promoting wound healing is provided, and the product contains the temperature- and pH-responsive hydrogel encapsulating chlorogenic acid liposomes as described in the first aspect, or the dressing as described in the fourth aspect; the product further contains a device for providing blue light.

[0012] In a sixth aspect, a method for promoting ATP generation in a subject for non-diagnostic and therapeutic purposes is provided, and the method includes contacting the subject with chlorogenic acid, or the temperature- and pH-responsive hydrogel encapsulating chlorogenic acid liposomes as described in the first aspect, or the dressing as described in the fourth aspect;

[0013] or, the method includes contacting the subject with chlorogenic acid, or the temperature- and pH-responsive hydrogel encapsulating chlorogenic acid liposomes as described in the first aspect, or the dressing as described in the fourth aspect, and then performing blue light irradiation.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention provides a temperature- and pH-responsive hydrogel encapsulating chlorogenic acid. This hydrogel can rapidly release chlorogenic acid in an acidic environment, and this characteristic enables it to accurately respond to the acidic environment of the wound, thereby efficiently releasing the drug and promoting wound healing. At the same time, at the human physiological temperature (37 °C), this hydrogel can continuously release chlorogenic acid, providing lasting drug support for wound healing. The main active ingredient for treatment in the hydrogel provided by the present invention is chlorogenic acid, which has no cytotoxic side effects within the dosage range defined in the present invention, and chlorogenic acid has been widely used in the fields of medicine and food, so it has good safety. It has been experimentally confirmed that the temperature- and pH-responsive hydrogel provided by the present invention can promote endothelial cell migration, and in animal experiments, this hydrogel alone or in combination with blue light can effectively promote wound healing in animal models. Description of the Drawings

[0016] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the accompanying drawings required for the description of the specific embodiments or the prior art. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can also be obtained based on these drawings.

[0017] Figure 1 It is the change curve of the cumulative release amount of chlorogenic acid over time for different groups of hydrogels in Example 1 under the conditions of pH 5.5 (37 °C) and pH 7.4 (37 °C). The percentages in the figure are mass percentages.

[0018] Figure 2 It is the change curve of the cumulative release amount of chlorogenic acid over time for different groups of hydrogels in Example 2 under the conditions of 25 °C (pH 7.4) and 37 °C (pH 7.4). The percentages in the figure are mass percentages.

[0019] Figure 3 It is the proliferation result of CCK-8 detection of cells after co-culturing different groups of hydrogels with human fibroblasts in Example 2.

[0020] Figure 4 It is the photo of each experimental group well plate in Example 3 placed under an optical microscope at 0 h, 24 h, and 72 h.

[0021] Figure 5 It is the migration rate of endothelial cells in each experimental group in Example 3 at 24 h and 72 h.

[0022] Figure 6 It is the ATP concentration of fibroblasts in each experimental group in Example 4.

[0023] Figure 7 It is the wound photos of each experimental group of animals at days 0, 5, and 9 in Example 5.

[0024] Figure 8 It is the wound healing rate of each experimental group at days 5 and 9 in Example 5. Specific Embodiments

[0025] The following will clearly and completely describe the technical solutions of the present invention in combination with the embodiments. Obviously, the described embodiments are some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0026] In this text, unless otherwise specified, all the embodiments and preferred implementation methods mentioned herein can be combined with each other to form new technical solutions. All the technical features and preferred features mentioned herein can be combined with each other to form new technical solutions. The various components or their preferred components involved can be combined with each other to form new technical solutions.

[0027] In this text, unless otherwise specified, "optionally", "optional", "selectable" or "select" means that the subsequent described event or circumstance can but does not have to occur, and this description includes the occasions where the event or circumstance occurs or does not occur.

[0028] In this text, the term "comprising" or "including" means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps.

[0029] In this text, the terms "subject", "patient" or "individual" can be used interchangeably and include human or non-human animals, or cells or tissues derived from human or non-human animals, such as mammals including humans, monkeys, mice, rats, rabbits, donkeys, cows, horses, pigs or dogs.

[0030] Chlorogenic acid (CGA) is a caffeoylquinic acid derivative that widely exists in the plant kingdom and has various biological activities and pharmacological functions, such as antibacterial, antiviral and antioxidant effects. The present invention discovers that chlorogenic acid can effectively promote wound healing. Based on this efficacy, in the first aspect, a temperature- and pH-responsive hydrogel encapsulating chlorogenic acid liposomes is provided. The temperature- and pH-responsive hydrogel contains chlorogenic acid liposome-encapsulating, modified gelatin grafted with phenylboronic acid, and oxidized hyaluronic acid grafted with dopamine.

[0031] In the temperature- and pH-responsive hydrogel, the concentration of the modified gelatin grafted with phenylboronic acid is 3-10 wt%, for example, it can be but is not limited to 3, 4, 5, 6, 7, 8, 9 or 10 wt%, preferably 3-9 wt%, and more preferably 6-9 wt%.

[0032] In the temperature- and pH-responsive hydrogel, the concentration of the oxidized hyaluronic acid grafted with dopamine is 3-10 wt%, for example, it can be but is not limited to 3, 4, 5, 6, 7, 8, 9 or 10 wt%, preferably 3-9 wt%, and more preferably 6-9 wt%.

[0033] In the temperature- and pH-responsive hydrogel, the concentration of chlorogenic acid is 1-20 μM, for example, it can be but is not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 μM, preferably 1-10 μM, and more preferably 5-10 μM.

[0034] In an alternative embodiment, the concentration of the modified gelatin grafted with phenylboronic acid in the temperature- and pH-responsive hydrogel is 6-9 wt%, the concentration of the oxidized hyaluronic acid grafted with dopamine is 6-9 wt%, and the concentration of chlorogenic acid is 1-10 μM.

[0035] In an alternative embodiment, the concentration of the modified gelatin grafted with phenylboronic acid in the temperature- and pH-responsive hydrogel is 6 wt%, the concentration of the oxidized hyaluronic acid grafted with dopamine is 6 wt%, and the concentration of chlorogenic acid is 5 μM.

[0036] In an alternative embodiment, the content of chlorogenic acid in the chlorogenic acid-loaded liposomes is 0.1-60 mg / mL, and can be, for example but not limited to, 0.1, 0.2, 0.5, 0.7, 1, 1.5, 2, 2.5, 3, 5, 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 mg / mL, preferably 1-10 mg / mL, and more preferably 0.7 mg / mL.

[0037] In an alternative embodiment, the liposomes for loading chlorogenic acid in the chlorogenic acid-loaded liposomes include but are not limited to natural phospholipids (such as phosphatidylcholine), synthetic phospholipids (dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC) or dioleoyl phosphatidylcholine (DOPC)), cholesterol, polyethylene glycol, phosphatidylglycerol, phosphatidic acid, sphingomyelin, sterols, saponins and chitosan, or one or more thereof.

[0038] In an alternative embodiment, the liposomes for loading chlorogenic acid in the chlorogenic acid-loaded liposomes include L-α-phosphatidylcholine and cholesterol.

[0039] In an alternative embodiment, the liposomes for loading chlorogenic acid in the chlorogenic acid-loaded liposomes include L-α-phosphatidylcholine and cholesterol, and the mass ratio of the feed of L-α-phosphatidylcholine to cholesterol is 5:1.

[0040] In a second aspect, a method for preparing the temperature- and pH-responsive hydrogel of the chlorogenic acid-loaded liposomes described in the first aspect is provided, and the preparation method includes: mixing the formulated chlorogenic acid-loaded liposomes, the modified gelatin grafted with phenylboronic acid, and the oxidized hyaluronic acid grafted with dopamine to obtain the temperature- and pH-responsive hydrogel of the chlorogenic acid-loaded liposomes.

[0041] In an alternative embodiment, the chlorogenic acid-loaded liposomes and the modified gelatin grafted with phenylboronic acid are first mixed evenly in water, and then mixed with the oxidized hyaluronic acid grafted with dopamine, and after mixing, it is cooled to obtain the temperature- and pH-responsive hydrogel. The cooling is to form the hydrogel at a temperature lower than the temperature of the mixing step.

[0042] In an alternative embodiment, the temperature of any mixing step is independently 40 to 50 °C, for example, but not limited to, 40, 45 or 50 °C.

[0043] In an alternative embodiment, after mixing, it is cooled at 4 °C for 1 to 3 h to obtain the temperature- and pH-responsive hydrogel.

[0044] In an alternative embodiment, it includes preparing chlorogenic acid-loaded liposomes using the thin film hydration ultrasound method.

[0045] In an alternative embodiment, in the preparation of the dopamine-grafted oxidized hyaluronic acid, the molar ratio of the component providing hyaluronic acid to the component providing dopamine is (0.5 to 2):1, for example, but not limited to, 0.5:1, 1:1 or 2:1, and preferably 1:1.

[0046] In an alternative embodiment, the component providing hyaluronic acid includes oxidized hyaluronic acid.

[0047] In an alternative embodiment, the component providing dopamine includes dopamine hydrochloride.

[0048] In an alternative embodiment, oxidized hyaluronic acid and dopamine are linked by an amide bond.

[0049] In an alternative embodiment, the method for preparing the dopamine-grafted oxidized hyaluronic acid includes: providing oxidized hyaluronic acid; activating the carboxyl group of oxidized hyaluronic acid using EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide), and then reacting with dopamine hydrochloride to obtain the dopamine-grafted oxidized hyaluronic acid.

[0050] In an alternative embodiment, in the preparation of the phenylboronic acid-grafted modified gelatin, the mass ratio of phenylboronic acid to gelatin is 1:(1 to 20), for example, but not limited to, 1:1, 1:2, 1:5, 1:10, 1:12, 1:15, 1:18 or 1:20, and preferably 1:5.

[0051] In an alternative embodiment, phenylboronic acid and gelatin are linked by an amide bond.

[0052] In an alternative embodiment, the method for preparing the phenylboronic acid-grafted modified gelatin includes: activating the carboxyl group of gelatin using EDC and NHS, and then reacting with phenylboronic acid to obtain the phenylboronic acid-grafted modified gelatin.

[0053] In a third aspect, there is provided the use of the temperature- and pH-responsive hydrogel loaded with chlorogenic acid liposomes described in the first aspect, or the preparation method described in the second aspect, in the preparation of a product for promoting wound healing in a subject.

[0054] In an alternative embodiment, the wound includes a full-thickness skin defect.

[0055] In a fourth aspect, there is provided a dressing for promoting wound healing, the dressing comprising the temperature- and pH-responsive hydrogel loaded with chlorogenic acid liposomes as described in the first aspect.

[0056] In an alternative embodiment, the dressing for promoting wound healing further comprises a pharmaceutically acceptable excipient, the excipient including, but not limited to, one or more of an antibacterial agent, a humectant, and a lubricant.

[0057] In an alternative embodiment, the dressing for promoting wound healing further comprises a pharmaceutically acceptable substrate, the substrate including, but not limited to, one or more of a non-woven fabric, a gauze, a polyurethane film, and a polyethylene film.

[0058] In a fifth aspect, there is further provided a product for promoting wound healing, the product comprising the temperature- and pH-responsive hydrogel loaded with chlorogenic acid liposomes as described in the first aspect, or the dressing as described in the fourth aspect; the product further comprises a device for providing blue light.

[0059] In an alternative embodiment, the product further comprises a device for providing blue light with a wavelength of 400 - 480 nm.

[0060] In a sixth aspect, there is further provided a method for promoting ATP generation in a subject for non-diagnostic and non-therapeutic purposes, the method comprising contacting the subject with chlorogenic acid, or the temperature- and pH-responsive hydrogel loaded with chlorogenic acid liposomes as described in the first aspect, or the dressing as described in the fourth aspect;

[0061] alternatively, the method comprises contacting the subject with chlorogenic acid, or the temperature- and pH-responsive hydrogel loaded with chlorogenic acid liposomes as described in the first aspect, or the dressing as described in the fourth aspect, and then performing blue light irradiation.

[0062] In an alternative embodiment, the blue light includes blue light with a wavelength of 400 - 480 nm.

[0063] In an alternative embodiment, the subject includes fibroblasts, preferably human fibroblasts.

[0064] The present invention will be further illustrated by specific examples below. However, it should be understood that these examples are only for more detailed illustration and should not be construed as limiting the present invention in any form.

[0065] Example 1

[0066] Preparation of a hydrogel material loaded with chlorogenic acid:

[0067] (1)The liposomes loaded with chlorogenic acid were prepared by the thin-film hydration ultrasonic method. L-α-phosphatidylcholine and cholesterol were mixed at a mass ratio of 5:1 and added to the chloroform solution, and magnetically stirred until completely dissolved. At 40 °C, it was rotary evaporated under vacuum on a rotary evaporator until the chloroform was completely evaporated to form a uniform thin film, and then rotary dried for another 30 min. Diethyl ether was added, and stirred at room temperature until the film was completely dissolved. An aqueous solution of chlorogenic acid was added, with an oil-water ratio of 3.5:1. It was sonicated in an ice bath for 15 min using a ultrasonic cell disruptor, and rotary evaporated under vacuum at 40 °C until the diethyl ether was completely evaporated. It was hydrated for 2 h, and then sonicated in an ice bath for 15 min (3 s on, 3 s off) to obtain liposomes loaded with chlorogenic acid (LIP@CA). The content of chlorogenic acid in the liposomes was 0.1 - 60 mg / mL.

[0068] (2)5 parts by mass of hyaluronic acid (HA) was added to 500 parts by volume of water. After stirring well, 1 - 1.5 parts by mass of sodium periodate was added, and the reaction was carried out in the dark for 6 - 12 h, and then ethylene glycol was added to terminate the reaction. The solution after terminating the oxidation reaction was dialyzed in a neutral environment for 72 h and freeze-dried to obtain oxidized hyaluronic acid (OHA).

[0069] OHA was added to water, 1 - 2 parts by mass of EDC and 1 - 2 parts by mass of NHS were added, and the pH was adjusted to 4.6 - 4.8 with 1 M HCl solution. After stirring at room temperature for 30 min, dopamine hydrochloride was added to the solution, and the pH was adjusted to 4.6 - 4.8 with 1 M HCl solution. Under N 2 protection, the reaction was stirred in the dark for 12 h. Then the solution was transferred to a dialysis bag and dialyzed with deionized water with pH = 4.6 for 2 d, and then dialyzed with deionized water with pH = 5.5 for 1 d, changing the water three times a day. Finally, the dialysis solution was freeze-dried to obtain oxidized hyaluronic acid grafted with dopamine (ODA). The molar ratio of OHA to dopamine hydrochloride used was 1:1.

[0070] (3)10 parts by mass of gelatin (Gel) was added to 100 parts by volume of water. After stirring well at 40 °C, 1 - 2 parts by mass of EDC and 1 - 2 parts by mass of NHS were added, and the pH was adjusted to 5.0 - 6.0 with 1 M HCl solution. After stirring at room temperature for 30 min, phenylboronic acid was added to the solution, and the reaction was carried out for 24 h. Then the solution was transferred to a dialysis bag and dialyzed with deionized water at 40 °C for 3 d, changing the water three times a day. Finally, the dialysis solution was freeze-dried to obtain modified gelatin grafted with phenylboronic acid (GBA). The mass ratio of phenylboronic acid to gelatin used was 1:5.

[0071] (4) Add the chlorogenic acid-loaded liposomes and modified gelatin to water, mix well, and then mix with ODA. Add 1 μL of 0.7 mg / mL chlorogenic acid liposomes to every 200 μL of the hydrogel precursor solution, and then add it to 200 μL of the above hydrogel precursor solution to obtain a 10 μM chlorogenic acid thermosensitive hydrogel. After heating and mixing evenly at 40 - 50 °C, cool it at 4 °C for 1 - 3 h to form a gel, and obtain the thermosensitive hydrogel ODA-GBA-LIP@CA loaded with chlorogenic acid liposomes. According to the above steps, prepare hydrogels with the content of modified gelatin GBA being 3 wt% and the content of ODA being 3 wt%; hydrogels with the content of gelatin GBA being 6 wt% and the content of ODA being 6 wt%; hydrogels with the content of gelatin GBA being 9 wt% and the content of ODA being 9 wt%; among which the content of chlorogenic acid is 10 μM for all.

[0072] (5) pH and temperature-responsive release test of the loaded chlorogenic acid: Put the thermosensitive hydrogel ODA-GBA-LIP@CA loaded with chlorogenic acid liposomes and the hydrogel HA-Gel-LIP@CA (commercial hyaluronic acid-gelatin hydrogel encapsulating chlorogenic acid liposomes (6 wt% HA - 6 wt% Gel), the content of hyaluronic acid is 6 wt%, and the content of gelatin is 6 wt%) into a dialysis bag (cut-off molecular weight = 500 Da), add 5 mL of PBS buffer solutions with different pH values (5.5 and 7.4) (temperature is 37 °C), place the dialysis bag in 20 mL of PBS buffer solution with the corresponding pH value, and the blank hydrogel is the blank control group. Place it in a constant temperature shaker at 25 and 37 °C and shake (r = 150 rpm). Take out 2 mL of PBS containing the released drug (chlorogenic acid) at specific time intervals, then add 2 mL of fresh PBS, and measure the absorbance of chlorogenic acid in the taken-out PBS at 324 nm with a UV spectrophotometer. To detect the pH and temperature-responsive release performance of the ODA-GBA-LIP@CA hydrogel for chlorogenic acid. At the same time, directly use the commercial hyaluronic acid-gelatin (6 wt% HA - 6 wt% Gel) hydrogel encapsulating chlorogenic acid liposomes as a comparison to see whether it can release the drug in response to temperature and pH.

[0073] At 37 °C and under different pH conditions, the curve of the cumulative release amount of chlorogenic acid versus time is as Figure 1 shown. Under different pH conditions, the commercial hyaluronic acid-gelatin (6 wt% HA - 6 wt% Gel) hydrogel encapsulating chlorogenic acid liposomes releases all the chlorogenic acid within the first hour, without pH-responsive drug release. All the hydrogels in the modified groups release chlorogenic acid rapidly in the acidic (pH = 5.5) environment, which is beneficial for the release of the drug chlorogenic acid in response to the acidic environment of the wound. At pH = 7.4 and under different temperature conditions, the curve of the cumulative release amount of chlorogenic acid versus time is as Figure 2As shown, at 37 °C, the commercially available hyaluronic acid - gelatin (6 wt% HA - 6 wt% Gel) hydrogel encapsulated chlorogenic acid liposomes released all the chlorogenic acid within the first hour, without temperature-responsive drug release. In the modified groups, two hydrogels of Example 1 and Example 3 (6 wt% ODA - 6 wt% GBA and 9 wt% ODA - 9 wt% GBA) could continuously respond to release chlorogenic acid at the human physiological temperature of 37 °C. Considering the raw material cost factor, the 6 wt% ODA - 6 wt% GBA hydrogel with the best comprehensive performance was consistently selected to encapsulate chlorogenic acid liposomes in the subsequent experiments.

[0074] Example 2

[0075] Cytotoxicity of the hydrogel material loaded with chlorogenic acid: For better comparison, the final contents of modified gelatin GBA and ODA in the following examples were 6 wt% respectively. The content of chlorogenic acid in the hydrogel was adjusted to obtain five hydrogels with different chlorogenic acid contents. 0.1, 0.5, 1, and 2 μL of 0.7 mg / mL chlorogenic acid liposomes were respectively added to 200 μL of the above hydrogel precursor solution (the final contents of modified gelatin GBA and ODA were 6 wt% respectively) to prepare chlorogenic acid thermosensitive hydrogels with chlorogenic acid contents of 0, 1, 5, 10, and 20 μM (the hydrogel group was the 0 μM chlorogenic acid thermosensitive hydrogel), and a well plate without adding hydrogel was set as a control group for cell culture experiments. The Transwell chamber was used for co-culture with human fibroblasts. After seeding the cells in the well plate, the Transwell chamber was covered, and 200 μL of the hydrogel prepared from the precursor solution was placed in the chamber, and then 800 μL of culture medium was added. All six groups of cells were co-cultured in a constant temperature incubator at 37 °C containing 5 % v / v CO 2 for 1 day, and the CCK-8 kit was used to detect the cell proliferation. The results are as Figure 3 shown. As can be seen from Figure 3 the cell viability statistical chart, when the chlorogenic acid content in the hydrogel reached 10 μM, the cell viability was the best after co-culturing the hydrogel with cells for 1 day. Therefore, the 10 μM chlorogenic acid hydrogel was used in the subsequent experiments.

[0076] Example 3

[0077] The chlorogenic acid hydrogel promotes endothelial cell migration: In this experiment, the Transwell chamber was used, and the indirect contact method of cell scratch experiment was used to evaluate the effect of the chlorogenic acid hydrogel on the migration ability of HUVECs. The specific experimental operation steps are as follows:

[0078] (1) Add 2 mL of HUVECs cell suspension (1×10 5 cells / mL) to each well of a 12-well culture plate and culture for 24 h. At this time, the cells have covered the well plate, and the culture medium is aspirated.

[0079] (2) Use a 200 μL pipette tip to draw a straight line along the ruler, wash it twice with PBS, add 1.5 mL of cell culture medium containing only 0.1% v / v FBS (99.8% v / v DMEM + 0.1% v / v FBS + 0.1% v / v PS), place the well plate under an optical microscope for photographing and observation, and record the scratch area at this time.

[0080] (3) Add Transwell chambers to a 12-well plate, add 100 μL of sterile chlorogenic acid hydrogel to each chamber, and then add 0.5 mL of cell culture medium containing only 0.1% v / v FBS to the chambers. Place the well plate in a cell culture incubator at 37 °C, 95% relative humidity and 5% v / v CO 2 2.

[0081] (4) After culturing the cells for 24 and 48 h, take out the Transwell chambers, place the well plate under an optical microscope for photographing and observation, and record the scratch area at this time. Calculate the cell migration rate based on the size of the scratch area at different times. As Figure 4 can be seen, the chlorogenic acid hydrogel prepared in the example can promote the migration of endothelial cells, and the 10 μM chlorogenic acid hydrogel has the best effect on promoting cell migration ( Figure 5 ).

[0082] Example 4

[0083] Chlorogenic acid combined with blue light promotes ATP production: Use an enhanced ATP detection kit to detect the change of ATP in human fibroblasts after treatment. According to the treatment conditions, it is divided into a control group, a hydrogen peroxide group, a hydrogen peroxide + chlorogenic acid group, and a hydrogen peroxide + chlorogenic acid + fluorescent light energy group. Those without any treatment are used as the control group; the hydrogen peroxide group uses 100 μM of H 2 O 2 to treat fibroblasts for 24 h and then change to normal culture medium for culturing for 24 h; the hydrogen peroxide + chlorogenic acid group uses 100 μM of H 2 O 2 to treat fibroblasts for 24 h, and then use 10 μM of chlorogenic acid to treat the cells for 24 h; the hydrogen peroxide + chlorogenic acid + fluorescent light energy group uses 100 μM of hydrogen peroxide to treat fibroblasts for 24 h, then uses 10 μM of chlorogenic acid to treat the cells for 30 min, and then irradiates with blue light of 400 - 480 nm for 3 min, with a dose of 180 mJ / cm 2 . After irradiating with blue light, use 10 μM of chlorogenic acid to treat the cells for 24 h. After changing the culture medium for the four groups of cells, they are all cultured in a constant temperature incubator at 37 °C containing 5% v / v CO 2 for 24 h, and then use an enhanced ATP detection kit to detect the change of ATP in fibroblasts.

[0084] The results were as follows Figure 6 shown. The ATP content of cells in the hydrogen peroxide group decreased, while the ATP content of cells in the hydrogen peroxide + chlorogenic acid group and the hydrogen peroxide + chlorogenic acid + fluorescent light energy group increased. In particular, the hydrogen peroxide + chlorogenic acid + fluorescent light energy group could significantly increase the production of cellular ATP and had a reparative effect on the damage caused by reactive oxygen species.

[0085] Example 5

[0086] Promotion of wound healing by chlorogenic acid hydrogel combined with mitochondrial fluorescence regeneration technology:

[0087] Male SD rats at 8 weeks of age were selected to establish a full-thickness skin defect animal model. The experimental groups were divided into 5 groups: A: control group (3M Tegaderm dressing); B: blank hydrogel control group (hydrogel group with chlorogenic acid content of 0, hydrogel specification: length × width × height = 30 mm × 30 mm × 1 mm, applied for 24 h); C: hydrogel + chlorogenic acid (final chlorogenic acid content was 10 μM, the hydrogel directly loaded with chlorogenic acid, hydrogel specification: length × width × height = 30 mm × 30 mm × 1 mm, applied for 24 h); D: chlorogenic acid hydrogel group (final chlorogenic acid content was 10 μM, the hydrogel loaded with liposome-encapsulated chlorogenic acid, hydrogel specification: length × width × height = 30 mm × 30 mm × 1 mm, applied for 24 h); E: chlorogenic acid hydrogel + fluorescent light energy group (final chlorogenic acid content was 10 μM, the hydrogel loaded with liposome-encapsulated chlorogenic acid, hydrogel specification: length × width × height = 30 mm × 30 mm × 1 mm, after administration for 24 h, the hydrogel was removed and blue light irradiation was applied for 3 min; the dose was 180 mJ / cm 2 ). The wound area was recorded on days 0, 5, and 9.

[0088] The treatment effects were as follows Figure 7 and Figure 8 shown. It can be seen from Figure 7 and 8 that the treatment effect of the chlorogenic acid hydrogel group (hydrogel loaded with liposome-encapsulated chlorogenic acid) was better than that of the hydrogel + chlorogenic acid group (hydrogel directly loaded with chlorogenic acid), but the chlorogenic acid hydrogel + fluorescent light energy group could effectively promote skin wound healing, and its effect of promoting wound healing was the best.

[0089] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A temperature and pH responsive hydrogel encapsulating chlorogenic acid liposomes, characterized in that: Containing chlorogenic acid-entrapped liposomes, modified gelatin grafted with phenylboronic acid, and oxidized hyaluronic acid grafted with dopamine; In the temperature and pH responsive hydrogel, the concentration of modified gelatin grafted with phenylboronic acid is 6-9 wt %, the concentration of oxidized hyaluronic acid grafted with dopamine is 6-9 wt %, and the concentration of chlorogenic acid is 1-20 μM.

2. The temperature and pH responsive hydrogel according to claim 1, characterized in that The concentration of chlorogenic acid is 1~10μM.

3. The temperature and pH responsive hydrogel according to claim 1 or 2, characterized in that: The content of chlorogenic acid in the chlorogenic acid-loaded liposome is 0.1-60 mg / mL.

4. The method for preparing the temperature and pH responsive hydrogel encapsulating chlorogenic acid liposomes according to any one of claims 1 to 3, characterized in that: The method comprises: mixing a formula amount of chlorogenic acid-encapsulated liposomes, modified gelatin grafted with phenylboronic acid and oxidized hyaluronic acid grafted with dopamine to obtain the temperature and pH responsive hydrogel of the chlorogenic acid-encapsulated liposomes.

5. The preparation method according to claim 4, characterized in that: The method comprises firstly mixing chlorogenic acid-encapsulated liposomes and modified gelatin grafted with phenylboronic acid in water, then mixing with oxidized hyaluronic acid grafted with dopamine, and cooling after mixing to obtain the temperature and pH responsive hydrogel.

6. The preparation method according to claim 4 or 5, characterized in that: The preparation method comprises preparing chlorogenic acid-loaded liposomes using a thin film hydration ultrasound method; and / or, In the preparation of the oxidized hyaluronic acid grafted with dopamine, the molar ratio of the component providing hyaluronic acid to the component providing dopamine is (0.5-2):1; And / or, in the preparation of the modified gelatin grafted with phenylboric acid, the mass ratio of phenylboric acid to gelatin is 1:(1-20).

7. Use of the temperature- and pH-responsive hydrogel encapsulating chlorogenic acid liposomes according to any one of claims 1 to 3, or the temperature- and pH-responsive hydrogel encapsulating chlorogenic acid liposomes obtained by the preparation method according to any one of claims 4 to 6 in the preparation of a product for promoting wound healing in a subject.

8. A dressing for promoting wound healing, characterized in that: A temperature- and pH-responsive hydrogel comprising chlorogenic acid-loaded liposomes as described in any one of claims 1 to 3.

9. A product for promoting wound healing, characterized in that: A temperature and pH responsive hydrogel comprising chlorogenic acid liposomes according to any one of claims 1 to 3, or a dressing according to claim 8; The product also includes a device for providing blue light.

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