Temperature and pH responsive hydrogel entrapped with chlorogenic acid liposome as well as preparation method and application of temperature and pH responsive hydrogel

Abstract

The invention provides temperature and pH responsive hydrogel entrapped with chlorogenic acid lipidosome as well as a preparation method and application of the temperature and pH responsive hydrogel, and relates to the technical field of biology. The temperature and pH responsive hydrogel entrapping chlorogenic acid lipidosome contains chlorogenic acid lipidosome, phenylboronic acid grafted modified gelatin and dopamine grafted oxidized hyaluronic acid, the concentration of the phenylboronic acid grafted modified gelatin is 3-10 wt%, the concentration of the dopamine grafted oxidized hyaluronic acid is 3-10 wt%, and the concentration of chlorogenic acid is 1-20 [mu] M. The temperature and pH responsive hydrogel can effectively promote wound healing independently or in combination with blue light.

Description

Technical Field

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

[0002] At present, there are many kinds of drugs used in clinical practice to promote wound healing, mainly including antibiotics, growth factors and traditional Chinese medicine preparations. These drugs play an important role in promoting wound healing, but they also have inevitable defects. Common antibiotics mainly include erythromycin ointment, mupirocin ointment and fusidic acid cream, but improper use of antibiotics may lead to increased bacterial resistance, reduce drug efficacy, and some antibiotics may cause local skin irritation or allergic reactions. Growth factors mainly include gels or topical solutions with recombinant human epidermal growth factor as the main drug ingredient, but growth factor drugs are usually expensive, which limits their wide application, and growth factors may be inactivated by environmental factors (such as temperature and pH) during storage and use. There are a wide variety of topical drugs for promoting wound healing in traditional Chinese medicine, which mainly work through antibacterial, anti-inflammatory, and tissue regeneration mechanisms. However, traditional Chinese medicine preparations have complex ingredients and unclear mechanisms of action, and some patients may have allergic reactions to traditional Chinese medicine ingredients. Therefore, the development of more diverse products to promote wound healing will help to better treat and relieve various clinical traumas.

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

[0004] The object of the present invention is to provide a temperature and pH responsive hydrogel encapsulating chlorogenic acid liposomes and a preparation method and application thereof, so as to improve the defects of the prior art products for promoting wound healing.

[0005] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions: In a first aspect, a temperature- and pH-responsive hydrogel encapsulating chlorogenic acid liposomes is provided, wherein the temperature- and pH-responsive hydrogel contains chlorogenic acid-encapsulating 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 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.

[0006] In the second aspect, a method for preparing the temperature- and pH-responsive hydrogel encapsulating chlorogenic acid liposomes as described in the first aspect is provided, comprising: mixing a formulated amount of chlorogenic acid-encapsulating 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.

[0007] In a third aspect, provided is the use of the temperature and pH responsive hydrogel encapsulating chlorogenic acid liposomes described in the first aspect, or the preparation method described in the second aspect in preparing a product for promoting wound healing in a subject.

[0008] In a fourth aspect, a dressing for promoting wound healing is provided, the dressing comprising the temperature and pH responsive hydrogel encapsulating chlorogenic acid liposomes as described in the first aspect.

[0009] In a fifth aspect, a product for promoting wound healing is provided, the product comprising 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 comprises a device for providing blue light.

[0010] In a sixth aspect, a method for promoting ATP production in a subject for non-diagnostic and non-therapeutic purposes is provided, the method comprising 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; Alternatively, the method comprises contacting the subject with chlorogenic acid, or the temperature and pH responsive hydrogel encapsulating chlorogenic acid liposomes described in the first aspect, or the dressing described in the fourth aspect, and then subjecting the subject to blue light irradiation.

[0011] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a temperature and pH responsive hydrogel encapsulating chlorogenic acid. The hydrogel can quickly release chlorogenic acid in an acidic environment, and this property enables it to accurately respond to the acidic environment of the wound, thereby efficiently releasing drugs and promoting wound healing. At the same time, at the physiological temperature of the human body (37°C), the hydrogel can continuously release chlorogenic acid to provide lasting drug support for wound healing. The active therapeutic ingredient in the hydrogel provided by the present invention is mainly chlorogenic acid, which has no cytotoxic side effects within the dosage range specified by the present invention, and chlorogenic acid has been widely used in the fields of medicine and food, so it has good safety. Experiments have confirmed that the temperature and pH responsive hydrogel provided by the present invention can promote endothelial cell migration. In animal experiments, the hydrogel alone or in combination with blue light can effectively promote wound healing in animal models. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0013] Figure 1 The curves of the cumulative release of chlorogenic acid from different groups of hydrogels in Example 1 at pH 5.5 (37°C) and pH 7.4 (37°C) respectively over time, where the percentages are mass percentages; Figure 2 The curves of the cumulative release of chlorogenic acid from different groups of hydrogels in Example 2 at 25°C (pH 7.4) and 37°C (pH 7.4) over time are shown in the figure. The percentages in the figure are mass percentages; Figure 3 The results of cell proliferation detected by CCK-8 after co-culture of each group of hydrogels with human fibroblasts in Example 2; Figure 4 These are photos of the well plates of each experimental group in Example 3 placed under an optical microscope at 0 h, 24 h and 72 h respectively; Figure 5 is the migration rate of endothelial cells in each experimental group at 24h and 72h in Example 3; Figure 6 is the ATP concentration of fibroblasts in each experimental group in Example 4; Figure 7 These are the wound photos of the animals in each experimental group in Example 5 on days 0, 5 and 9; Figure 8 The wound healing rates of each experimental group in Example 5 on the 5th and 9th days. DETAILED DESCRIPTION

[0014] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0015] In this document, unless otherwise specified, all embodiments and preferred implementation methods mentioned herein can be combined with each other to form a new technical solution. All technical features and preferred features mentioned herein can be combined with each other to form a new technical solution. The components involved or their preferred components can be combined with each other to form a new technical solution.

[0016] As used herein, unless otherwise stated, "optionally", "optional", "optional" or "optional" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur.

[0017] Herein, the terms "comprise" or "comprising" are intended to include stated elements, integers or steps, but not to exclude any other elements, integers or steps.

[0018] As used herein, the terms "subject," "patient," or "individual" are used interchangeably and include humans or non-human animals, or cells or tissues derived from humans or non-human animals, such as humans, monkeys, mice, rats, rabbits, donkeys, cows, horses, pigs, or dogs.

[0019] Chlorogenic acid (CGA) is a caffeoylquinic acid derivative, which is widely present in the plant kingdom and has multiple biological activities and pharmacological functions, including antibacterial, antiviral and antioxidant effects. The present invention finds that chlorogenic acid can effectively promote wound healing. Based on this effect, 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 liposomes encapsulating chlorogenic acid, modified gelatin grafted with phenylboronic acid, and oxidized hyaluronic acid grafted with dopamine.

[0020] The concentration of the modified gelatin grafted with phenylboronic acid in the temperature and pH responsive hydrogel is 3-10 wt %, for example, but not limited to 3, 4, 5, 6, 7, 8, 9 or 10 wt %, preferably 3-9 wt %, and more preferably 6-9 wt %.

[0021] The concentration of the oxidized hyaluronic acid grafted with dopamine in the temperature and pH responsive hydrogel is 3-10 wt %, for example, but not limited to 3, 4, 5, 6, 7, 8, 9 or 10 wt %, preferably 3-9 wt %, and more preferably 6-9 wt %.

[0022] The concentration of chlorogenic acid in the temperature and pH responsive hydrogel is 1 to 20 μM, for example, it can be but 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 to 10 μM, and more preferably 5 to 10 μM.

[0023] In an optional embodiment, the concentration of modified gelatin grafted with phenylboronic acid in the temperature and pH responsive hydrogel is 6-9 wt %, the concentration of oxidized hyaluronic acid grafted with dopamine is 6-9 wt %, and the concentration of chlorogenic acid is 1-10 μM.

[0024] In an optional embodiment, the concentration of modified gelatin grafted with phenylboronic acid in the temperature and pH responsive hydrogel is 6 wt %, the concentration of oxidized hyaluronic acid grafted with dopamine is 6 wt %, and the concentration of chlorogenic acid is 5 μM.

[0025] In an optional embodiment, the content of chlorogenic acid in the chlorogenic acid-loaded liposomes is 0.1-60 mg / mL, for example, it can be 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.

[0026] In an optional embodiment, the liposomes used to encapsulate chlorogenic acid in the chlorogenic acid-encapsulated liposomes include but are not limited to one or more of natural phospholipids (such as phosphatidylcholine), synthetic phospholipids (dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC) or dioleoylphosphatidylcholine (DOPC)), cholesterol, polyethylene glycol, phosphatidylglycerol, phosphatidic acid, sphingomyelin, sterols, saponin and chitosan.

[0027] In an optional embodiment, the liposomes used to encapsulate chlorogenic acid in the chlorogenic acid-encapsulated liposomes include L-α-phosphatidylcholine and cholesterol.

[0028] In an optional embodiment, the liposomes used to encapsulate chlorogenic acid in the chlorogenic acid-encapsulated liposomes include L-α-phosphatidylcholine and cholesterol, and the feed mass ratio of L-α-phosphatidylcholine to cholesterol is 5:1.

[0029] In the second aspect, a method for preparing the temperature- and pH-responsive hydrogel encapsulating chlorogenic acid liposomes as described in the first aspect is provided, the preparation method comprising: mixing a formulated amount of chlorogenic acid-encapsulating 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.

[0030] In an optional embodiment, the chlorogenic acid-encapsulated liposomes and the modified gelatin grafted with phenylboronic acid are first mixed uniformly in water, and then mixed with the oxidized hyaluronic acid grafted with dopamine, and then cooled after mixing to obtain the temperature and pH responsive hydrogel. The cooling is to form the hydrogel at a temperature lower than the mixing step temperature.

[0031] In an optional embodiment, the temperature of any mixing step is independently 40-50°C, for example, but not limited to 40, 45 or 50°C.

[0032] In an optional embodiment, the mixture is cooled at 4° C. for 1 to 3 hours after mixing to obtain the temperature and pH responsive hydrogel.

[0033] In an optional embodiment, the chlorogenic acid-loaded liposomes are prepared using a thin film hydration ultrasound method.

[0034] In an optional embodiment, 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, for example, it can be but not limited to 0.5:1, 1:1 or 2:1, preferably 1:1.

[0035] In an alternative embodiment, the component providing hyaluronic acid comprises oxidized hyaluronic acid.

[0036] In an alternative embodiment, the dopamine-providing component comprises dopamine hydrochloride.

[0037] In an alternative embodiment, the oxidized hyaluronic acid and dopamine are linked via an amide bond.

[0038] In an optional embodiment, the preparation method of the oxidized hyaluronic acid grafted with dopamine includes: providing oxidized hyaluronic acid; using EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) to activate the carboxyl group of the oxidized hyaluronic acid, and then reacting with dopamine hydrochloride to obtain the oxidized hyaluronic acid grafted with dopamine.

[0039] In an optional embodiment, in the preparation of the modified gelatin grafted with phenylboric acid, the mass ratio of phenylboric acid to gelatin is 1:(1~20), for example, it can be but not limited to 1:1, 1:2, 1:5, 1:10, 1:12, 1:15, 1:18 or 1:20, preferably 1:5.

[0040] In an alternative embodiment, phenylboronic acid and gelatin are linked via an amide bond.

[0041] In an optional embodiment, the preparation method of the modified gelatin grafted with phenylboronic acid comprises: using EDC and NHS to activate the carboxyl group of gelatin, and then reacting with phenylboronic acid to obtain the modified gelatin grafted with phenylboronic acid.

[0042] In a third aspect, provided is the use of the temperature and pH responsive hydrogel encapsulating chlorogenic acid liposomes described in the first aspect, or the preparation method described in the second aspect in preparing a product for promoting wound healing in a subject.

[0043] In an alternative embodiment, the wound comprises a full-thickness skin defect.

[0044] In a fourth aspect, a dressing for promoting wound healing is provided, the dressing comprising the temperature and pH responsive hydrogel encapsulating chlorogenic acid liposomes as described in the first aspect.

[0045] In an optional embodiment, the dressing for promoting wound healing further comprises pharmaceutically acceptable excipients, including but not limited to one or more of antibacterial agents, moisturizers and lubricants.

[0046] In an optional embodiment, the dressing for promoting wound healing further comprises a pharmaceutically acceptable substrate, and the substrate includes but is not limited to one or more of non-woven fabrics, gauze, polyurethane films and polyethylene films.

[0047] In the fifth aspect, a product for promoting wound healing is also provided, wherein the product comprises 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 also comprises a device for providing blue light.

[0048] In an optional embodiment, the product further comprises a device for providing blue light of 400-480 nm.

[0049] In a sixth aspect, a method for promoting ATP generation in a subject for non-diagnostic and non-therapeutic purposes is also provided, the method comprising 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; Alternatively, the method comprises contacting the subject with chlorogenic acid, or the temperature and pH responsive hydrogel encapsulating chlorogenic acid liposomes described in the first aspect, or the dressing described in the fourth aspect, and then subjecting the subject to blue light irradiation.

[0050] In an optional implementation, the blue light includes blue light of 400-480 nm.

[0051] In an alternative embodiment, the subject comprises fibroblasts, preferably human fibroblasts.

[0052] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0053] Example 1 Preparation of hydrogel material loaded with chlorogenic acid: (1) The chlorogenic acid-encapsulated liposomes were prepared by thin film hydration ultrasound method. L-α-phosphatidylcholine and cholesterol were mixed in a mass ratio of 5:1 and added to chloroform solution. The mixture was stirred magnetically until the solution was uniformly dissolved. The mixture was evaporated in a rotary evaporator at 40°C until the chloroform was completely evaporated to form a uniform film. The film was then dried by rotation for 30 min. Ether was added and stirred at room temperature until the film was completely dissolved. The chlorogenic acid aqueous solution was added with an oil-water ratio of 3.5:1. The mixture was ultrasonically treated in an ice bath with an ultrasonic cell crusher for 15 min. The mixture was vacuum-evaporated at 40°C until the ether was completely evaporated. The mixture was hydrated for 2 h. The mixture was ultrasonically treated in an ice bath with an ultrasonic cell crusher for 15 min (3 s on, 3 s off) to obtain chlorogenic acid-encapsulated liposomes (LIP@CA). The content of chlorogenic acid in the liposomes was 0.1~60 mg / mL.

[0054] (2) Add 5 parts by mass of hyaluronic acid (HA) to 500 parts by volume of water, stir thoroughly, add 1-1.5 parts by mass of sodium periodate, react in the dark for 6-12 hours, and then add ethylene glycol to terminate the reaction. The solution after the oxidation reaction is terminated is dialyzed under a neutral environment for 72 hours and freeze-dried to obtain oxidized hyaluronic acid (OHA).

[0055] 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 1M HCl solution. After stirring at room temperature for 30 minutes, dopamine hydrochloride was added to the solution, and the pH was adjusted to 4.6-4.8 with 1M HCl solution. The reaction was stirred for 12 hours in the dark under N2 protection, and then the solution was transferred to a dialysis bag and dialyzed with deionized water at pH = 4.6 for 2 days, and then dialyzed with deionized water at pH = 5.5 for 1 day, and the water was changed three times a day. Finally, the dialysate was freeze-dried to obtain oxidized hyaluronic acid (ODA) grafted with dopamine. The molar ratio of OHA and dopamine hydrochloride used was 1:1.

[0056] (3) Add 10 parts by mass of gelatin (Gel) to 100 parts by volume of water, stir thoroughly at 40°C, add 1-2 parts by mass of EDC and 1-2 parts by mass of NHS, adjust the pH to 5.0-6.0 with 1M HCl solution, stir at room temperature for 30 minutes, add phenylboric acid to the solution, react for 24 hours, transfer the solution to a dialysis bag, dialyze with deionized water at 40°C for 3 days, and change the water three times a day. Finally, freeze-dry the dialysate to obtain modified gelatin grafted with phenylboronic acid (GBA). The mass ratio of phenylboric acid to gelatin used is 1:5.

[0057] (4) Add chlorogenic acid liposomes and modified gelatin into water, mix well, and then mix with ODA. Add 1 μL of 0.7 mg / mL chlorogenic acid liposomes to 200 μL of the above hydrogel precursor solution to obtain a 10 μM chlorogenic acid thermosensitive hydrogel. Heat at 40-50 °C to mix well, and then cool at 4 °C for 1-3 hours to form a gel, to obtain a thermosensitive hydrogel ODA-GBA-LIP@CA containing chlorogenic acid liposomes. According to the above steps, hydrogels with a modified gelatin GBA content of 3 wt% and an ODA content of 3 wt% were prepared; hydrogels with a gelatin GBA content of 6 wt% and an ODA content of 6 wt%; and hydrogels with a gelatin GBA content of 9 wt% and an ODA content of 9 wt% were prepared, and the chlorogenic acid content was 10 μM in each case.

[0058] (5) pH and temperature responsive release test of chlorogenic acid: The thermosensitive hydrogel ODA-GBA-LIP@CA and hydrogel HA-Gel-LIP@CA (commercial hyaluronic acid-gelatin hydrogel encapsulating chlorogenic acid liposomes (6wt%HA-6wt%Gel), hyaluronic acid content of 6wt%, gelatin content of 6wt%) were placed in a dialysis bag (molecular weight cutoff = 500 Da), and 5 mL of PBS buffer solution (temperature 37°C) with different pH (5.5 and 7.4) was added. The dialysis bag was placed in 20 mL of PBS buffer solution with the corresponding pH. The blank hydrogel was used as the blank control group. It was placed in a constant temperature shaker at 25 and 37°C and shaken (r = 150 rpm). At specific time intervals, 2 mL of PBS containing the released drug (chlorogenic acid) was taken out, and 2 mL of fresh PBS was added. The absorbance of chlorogenic acid in the taken out PBS at 324 nm was measured using a UV spectrophotometer. The pH and temperature response release performance of ODA-GBA-LIP@CA hydrogel was tested. At the same time, commercial hyaluronic acid-gelatin (6wt%HA-6wt%Gel) hydrogel was directly used to encapsulate chlorogenic acid liposomes as a comparison to see whether it can release drugs in response to temperature and pH.

[0059] At 37°C and different pH conditions, the cumulative release of chlorogenic acid changes with time. Figure 1 As shown in the figure, under different pH conditions, commercial hyaluronic acid-gelatin (6wt%HA-6wt%Gel) hydrogel encapsulated chlorogenic acid liposomes released all chlorogenic acid in the first hour, without pH-responsive drug release. All modified hydrogels released chlorogenic acid rapidly in acidic conditions (pH=5.5), which is beneficial for the release of chlorogenic acid in response to the acidic environment of the wound. At pH 7.4 and different temperatures, the cumulative release of chlorogenic acid changes with time as shown in the figure. Figure 2As shown in the figure, at 37°C, the commercial hyaluronic acid-gelatin (6wt%HA-6wt%Gel) hydrogel encapsulated chlorogenic acid liposomes released all chlorogenic acid in the first hour, without temperature-responsive drug release. In the modified group, the hydrogels of Example 1 and Example 3 (6wt%ODA-6wt%GBA and 9wt%ODA-9wt%GBA) can continuously respond to release chlorogenic acid at the human physiological temperature of 37°C. Combined with the cost of raw materials, the 6wt%ODA-6wt%GBA hydrogel encapsulated chlorogenic acid liposomes with the best comprehensive performance were selected in the subsequent experiments.

[0060] Example 2 Cytotoxicity of chlorogenic acid-loaded hydrogel materials: 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 added to 200 μL of the above hydrogel precursor solution (the final contents of modified gelatin GBA and ODA were 6 wt %, respectively), and 0, 1, 5, 10, and 20 μM chlorogenic acid thermosensitive hydrogels were prepared (the hydrogel group was 0 μM chlorogenic acid thermosensitive hydrogel). A group of well plates without hydrogels were set up as the control group for cell culture experiments. Transwell chambers were used to co-culture with human fibroblasts. After the cells were planted in the well plates, the Transwell chambers were covered, and 200 μL of the hydrogel prepared by the precursor solution was placed in the chamber, and 800 μL of culture medium was added. The six groups of cells were co-cultured in a constant temperature incubator at 37°C containing 5% v / v CO2 for 1 day, and the cell proliferation was detected using the CCK-8 kit. The results are as follows Figure 3 As shown. Figure 3 From the cell viability statistics chart, we can see that when the chlorogenic acid content in the hydrogel reaches 10 μM, the cell viability is best after the hydrogel and cells are co-cultured for 1 day, so 10 μM chlorogenic acid hydrogel is used in subsequent experiments.

[0061] Example 3 Chlorogenic acid hydrogel promotes endothelial cell migration: This experiment uses Transwell chambers and indirect contact cell scratch assays to evaluate the effect of chlorogenic acid hydrogel on HUVECs migration. The specific experimental steps are as follows: (1) Add 2 mL of HUVECs cell suspension (1×10 5 cells / mL) for 24 h, at which time the cells had filled the well plate and the culture medium was aspirated.

[0062] (2) Use a 200 μL pipette tip to draw a straight line along the ruler, wash 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, take a picture and observe, and record the scratch area at this time.

[0063] (3) Add Transwell chambers to a 12-well plate, add 100 μL of sterile chlorogenic acid hydrogel to each chamber, then add 0.5 mL of cell culture medium containing only 0.1% v / v FBS to the chamber, and place the well plate in a cell culture incubator at 37 °C, 95% relative humidity, and 5% v / v CO2.

[0064] (4) After 24 and 48 h of cell culture, remove the Transwell chamber, place the well plate under an optical microscope, take a picture and observe, and record the scratch area at this time. Calculate the cell migration rate based on the size of the scratch area at different times. Figure 4 It can be seen that 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 ).

[0065] Example 4 Combination of chlorogenic acid and blue light promotes ATP generation: An enhanced ATP detection kit was used to detect changes in ATP in human fibroblasts after treatment. According to the treatment conditions, the cells were 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. The control group was not treated with any treatment; the hydrogen peroxide group treated fibroblasts with 100 μM H2O2 for 24 hours and then changed to ordinary culture medium for 24 hours; the hydrogen peroxide + chlorogenic acid group treated fibroblasts with 100 μM H2O2 for 24 hours, and then treated cells with 10 μM chlorogenic acid for 24 hours; the hydrogen peroxide + chlorogenic acid + fluorescent light energy group treated fibroblasts with 100 μM hydrogen peroxide for 24 hours, then treated cells with 10 μM chlorogenic acid for 30 minutes, and then irradiated with 400-480 nm blue light for 3 minutes, with a dose of 180 mJ / cm 2 After irradiation with blue light, the cells were treated with 10 μM chlorogenic acid for 24 h. After the culture medium was replaced, the four groups of cells were cultured in a constant temperature incubator at 37°C containing 5% v / v CO2 for 24 h. The enhanced ATP detection kit was used to detect changes in ATP in fibroblasts.

[0066] The results are as follows Figure 6As shown, the cellular ATP content in the hydrogen peroxide group decreased, while the cellular ATP content 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 cellular ATP production and have a repairing effect on the damage caused by reactive oxygen species.

[0067] Example 5 Chlorogenic acid hydrogel combined with mitochondrial fluorescence regeneration technology promotes wound healing: Eight-week-old male SD rats were selected to construct a full-thickness skin defect animal model. The experimental group was divided into 5 groups: A: control group (3M Tegaderm dressing); B: blank hydrogel group (hydrogel group with 0 chlorogenic acid, hydrogel specifications: length × width × height = 30mm × 30mm × 1mm, applied for 24h); C: hydrogel + chlorogenic acid (chlorogenic acid final content was 10μM, hydrogel directly loaded with chlorogenic acid, hydrogel specifications: length × width × height = 30mm × 30mm × 1mm, applied for 24h); D: chlorogenic acid hydrogel group (chlorogenic acid The final content was 10 μM, the hydrogel was loaded with chlorogenic acid encapsulated by liposomes, the hydrogel specifications were: length × width × height = 30 mm × 30 mm × 1 mm, and it was applied for 24 hours); E: chlorogenic acid hydrogel + fluorescent light energy group (the final content of chlorogenic acid was 10 μM, the hydrogel was loaded with chlorogenic acid encapsulated by liposomes, the hydrogel specifications were: length × width × height = 30 mm × 30 mm × 1 mm, 24 hours after administration, the hydrogel was removed and blue light irradiation was applied for 3 minutes; the dose was 180 mJ / cm 2 ). Wound area was recorded on days 0, 5, and 9.

[0068] Its therapeutic effects are Figure 7 and Figure 8 As shown. Figure 7 and 8 It can be seen that the therapeutic effect of the chlorogenic acid hydrogel group (chlorogenic acid encapsulated in hydrogel-loaded liposomes) is better than that of the hydrogel + chlorogenic acid group (hydrogel directly loaded with chlorogenic acid), but the chlorogenic acid hydrogel + fluorescent light energy group can effectively promote skin wound healing, and its effect in promoting wound healing is the best.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, 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 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.

2. The temperature and pH responsive hydrogel according to claim 1, characterized in that 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 1-10 μM.

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 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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