Preparation method and application of pH / glucose double-response chronic wound dressing containing frankincense essential oil

Abstract

The invention discloses a preparation method of a pH / glucose double-response chronic wound dressing containing frankincense essential oil, which comprises the following steps: (1) respectively forming Schiff base and boric acid ester bonds with carboxymethyl chitosan and two hyaluronic acids with different molecular weights by using 4-formylphenylboronic acid as a cross-linking agent; preparing adhesive hydrogel which can be used for skin surface wound surface filling and double-dynamic covalent cross-linking, and freeze-drying for later use; (2) preparing the frankincense essential oil into microcapsules by an emulsification and condensation method, and freeze-drying for later use; and (3) mixing the hydrogel with the freeze-dried powder of the frankincense essential oil microcapsules, adding deionized water or PBS, stirring and redissolving to form the pH / glucose double-response hybrid hydrogel dressing coated with the frankincense essential oil microcapsules. The composite dressing prepared by the invention has the effects of resisting inflammation, resisting oxidation, resisting bacteria and promoting healing, is quick and simple to prepare, has stimulation response capability on acid / high-glucose microenvironment of diabetic wound surfaces, and has important practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of tissue repair and biomedical materials, and specifically relates to a preparation method and application of a pH / glucose dual-responsive chronic wound dressing containing frankincense essential oil. Background Art

[0002] Diabetes is a chronic and frequently occurring disease, with an increasing incidence rate worldwide. Due to long-term abnormal glucose metabolism, high blood sugar levels in diabetics lead to certain abnormalities in the microvasculature, nervous system, and immune system. Therefore, once a wound occurs, including surgical procedures required for other diseases, it is very easy to form chronic inflammation that is difficult to heal, manifested by the presence of a large number of inflammatory immune cells, inflammatory cytokines, ROS, and proteases, as well as a local acidic microenvironment caused by repeated bacterial infections, which is difficult to heal for a long time.

[0003] The treatment basis of chronic diabetic foot ulcers includes measures to control blood sugar and infection through drugs, as well as regular debridement, infection treatment and good wound care. Currently, the dressings that directly contact chronic diabetic wound repair include hydrocolloid materials, foam dressings, hydrogel dressings, alginate dressings, hydrophilic cellulose dressings, silver / honey antibacterial dressings, collagen, fibrin, chitosan and hyaluronic acid dressings. However, these dressings have some problems in mechanical properties and drug release properties during basic treatment, and still cannot meet the treatment needs of some patients with refractory chronic diabetic foot ulcers. More and more research and clinical practice are still exploring new methods for the treatment of refractory chronic diabetic foot ulcers.

[0004] Frankincense is a resin exuded from the bark of Boswellia carterii Birdw. and B. bhaw-dajiana Birdw., a plant of the same genus in the olive family. There are about 30 species in the world. It is recorded in ancient medical books such as "Compendium of Materia Medica" and "Compendium of Materia Medica" for its medicinal properties of promoting blood circulation and stretching tendons, clearing the meridians throughout the body, promoting blood circulation and relieving pain, supporting sores and eliminating carbuncles. Modern pharmaceutical research shows that frankincense has anti-inflammatory, antioxidant, antibacterial and immunomodulatory effects. More importantly, frankincense extract 3-acetyl-11-carbonyl-β-boswellic acid and boswellic acids have obvious therapeutic effects on skin ulcers, oral ulcers and gastric ulcers. The mechanism may be related to inhibiting inflammatory factors and alleviating oxidative stress. The chemical components of frankincense include volatile oils, terpenes, alkanes, polysaccharides, etc. Among them, volatile oils, namely frankincense essential oil, are the key components for the efficacy and fragrance of frankincense (Liu Di, et al. Chinese Herbal Medicine, 2020, 51(22):5900-5914). However, the physical and chemical defects of frankincense essential oil, such as poor water solubility, poor stability and high volatility, make it difficult for it to exert its efficacy on the wound surface for a long time, which greatly limits its use occasions and effects. Putting frankincense essential oil into microcapsules can improve these usage defects.

[0005] On the other hand, medical hydrogel is a material form formed by the cross-linked network of hydrophilic polymers swelling with water. Its characteristics are high water content (97% to 99% water content) and biocompatibility. In particular, in 1962, Dr. Winter's animal experiments confirmed that wounds heal better in a moist environment than in an open dry environment, and proposed the moist wound healing theory (Winter G.D. Nature, 1962, 193: 293-294). Under the guidance of the moist wound healing theory, hydrogels, as the most ideal cell scaffolds and drug carriers, have attracted more and more attention in the fields of tissue repair and modern wound dressing research and development, production and application. Adhesive hydrogels can not only adhere firmly to the uneven ulcer wound surface, but also have the function of responding to external physical or chemical signal stimuli, such as pH, glucose, reactive oxygen species (ROS), enzymes, etc. However, when considering replacing or removing adhesive hydrogel dressings, secondary damage may be caused to unhealed wounds. Therefore, a new type of hydrogel that can effectively improve the wound repair of diabetic ulcers, meet the requirements of removability / solubility, and avoid the obstruction of wound repair caused by secondary damage has attracted much attention worldwide.

[0006] As an acidic mucopolysaccharide, hyaluronic acid is widely present in organisms and has physiological functions such as promoting wound healing, lubricating joints, anti-oxidation and anti-aging. Hyaluronic acid of different molecular weights has different functions. On the one hand, high molecular weight hyaluronic acid can protect cell tissues from invasion by bacteria and ultraviolet rays, and on the other hand, it can form cell scaffolds, which is conducive to rapid scar healing of cell tissues. Low molecular weight hyaluronic acid is easily absorbed, and has the effects of promoting blood circulation, improving metabolism, and anti-oxidation. However, existing research on the modification of hyaluronic acid may hinder its original biological activity (Luo Z., et al. Adv. Funct. Mater., 2023, 33, 2306554), and may seriously affect the repair of wounds in organisms. Therefore, it is of great significance to introduce hyaluronic acid into hydrogel dressings without chemical modification. Summary of the invention

[0007] Diabetic patients are very likely to develop chronic inflammatory wounds that are difficult to heal due to long-term abnormal glucose metabolism. In view of this clinical problem, the present invention aims to provide a preparation method of a pH / glucose dual-responsive chronic wound dressing containing frankincense essential oil. As a novel preparation method of a pH / glucose microenvironment dual-responsive frankincense essential oil microcapsule hybrid hydrogel multifunctional dressing, the present invention mainly includes three aspects: first, a dual-responsive medical hydrogel that can be decomposed under pH / glucose stimulation; second, a stabilization method of frankincense essential oil, i.e., a preparation method of frankincense essential oil microcapsules; and third, a preparation method of a pH / glucose dual-responsive chronic wound dressing containing frankincense essential oil and chronic wound application are provided.

[0008] The present invention is achieved by adopting the following technical solutions: A method for preparing a pH / glucose dual-responsive chronic wound dressing containing frankincense essential oil comprises the following steps: (1) Synthesis of dual dynamic covalent cross-linked network adhesive hydrogel: a certain amount of high molecular weight hyaluronic acid and low molecular weight hyaluronic acid (HA) were mixed. H Abbreviated as "H", low molecular weight hyaluronic acid HA L A certain amount of carboxymethyl chitosan (CMCS, referred to as "C") was completely dissolved in PBS to obtain a HA solution; a certain amount of 4-formaldehyde phenylboronic acid (4-FPBA, referred to as "F") was completely dissolved in anhydrous ethanol to obtain a 4-FPBA solution. The HA solution, 4-FPBA solution, and CMCS solution were stirred vigorously with a glass rod in order to form a "CFHh" hydrogel, and the hydrogel freeze-dried powder was obtained after freeze-drying.

[0009] (2) Preparation of frankincense essential oil microcapsules by emulsification coagulation method: At room temperature, a certain amount of chitosan (CS) was added to an acetic acid solution and stirred overnight to obtain a chitosan solution. The mixture was centrifuged at 9000 rpm for 30 minutes, and the supernatant was taken to obtain a chitosan acetic acid solution. A certain amount of Tween-80 was added to the chitosan acetic acid solution as a surfactant, and stirred at a certain temperature for 2 hours to obtain a uniform mixture solution, i.e., the water phase. A certain amount of frankincense essential oil was dissolved in dichloromethane to obtain a dichloromethane solution of frankincense essential oil, i.e., the oil phase. Under the homogenization condition of a high-speed shearing machine at 13000 rpm, the oil phase was gradually dripped into the water phase to obtain an oil-in-water emulsion, and then a certain amount of sodium tripolyphosphate (TPP) solution was added dropwise to the stirred emulsion and stirred for a certain period of time. The microcapsule particles were collected by centrifugation and washed, washed several times with deionized water, and finally ultrasonicated in an ice bath with an ultrasonic generator for 3 to 5 minutes to obtain a uniform suspension, which was freeze-dried to obtain freeze-dried powder of frankincense essential oil microcapsules.

[0010] (3) One-pot stirring and re-dissolving to form a pH / glucose dual-responsive hybrid hydrogel dressing coated with frankincense essential oil microcapsules: After mixing the hydrogel freeze-dried powder and the frankincense essential oil microcapsule freeze-dried powder in a certain proportion, add a certain amount of water or PBS and stir and re-dissolve to obtain the frankincense essential oil microcapsule hybrid hydrogel dressing.

[0011] In the method of the present invention, frankincense essential oil is prepared in the form of microcapsules, and hyaluronic acid that has not been chemically modified is used as a raw material and 4-formaldehyde phenylboronic acid is used as a cross-linking agent to prepare a pH / glucose dual-responsive hydrogel dressing, and then the hydrogel is used to coat the frankincense essential oil microcapsules to prepare a hybrid hydrogel. The frankincense essential oil can be significantly improved in stability under the dual wrapping of the microcapsules and the hydrogel. The hydrogel is gradually decomposed under the stimulation of the diabetic microenvironment and slowly releases the frankincense essential oil to the chronic diabetic wound. At present, the above-mentioned application innovation of frankincense essential oil has not been disclosed, that is, the application of the dressing prepared by the method of the present invention in chronic diabetic skin ulcers and inflammatory skin wounds, and the application of frankincense essential oil in the pH / glucose dual-responsive hybrid hydrogel dressing coated with frankincense essential oil microcapsules prepared by the method of the present invention.

[0012] Preferably, in step (1), the high molecular weight HA and the low molecular weight HA are raw materials that have not undergone any chemical modification, and the biological activity of HA is completely retained, wherein the molecular weight ranges of the high molecular weight HA and the low molecular weight HA are 100,000 to 1.5 million and 2,000 to 5,000, respectively. Further preferably, the molecular weights of the high molecular weight HA and the low molecular weight HA are 800,000 to 1.5 million and 3,000.

[0013] The concentration of the CMCS solution is 5wt%; the concentration of the 4-FPBA solution is 3wt%; the concentration of the HA solution is 8wt%, wherein the concentrations of the high and low molecular weight HA are both 4wt%. When mixed, the volume ratio of the HA solution, 4-FPBA solution, and CMCS solution is 2:0.5:2.

[0014] Further preferably, in step (2), the concentration of the CS solution is 1wt%; the concentration of the TPP solution is 0.4wt%; the concentration of the glacial acetic acid solution is 1wt%; the concentration of the dichloromethane solution of frankincense essential oil is 0.1g / mL. The weight ratio of CS to frankincense essential oil is 1:0.8. The stirring temperature when Tween-80 is added is 45°C.

[0015] Preferably, in step (3), the mass ratio of the hydrogel freeze-dried powder to the frankincense essential oil microcapsule freeze-dried powder is 1:1, that is, the frankincense essential oil and the microcapsule are 7wt% each, and the solid mass fraction in the obtained dressing is 14wt%.

[0016] The preparation method of the present invention is that 4-FPBA is used as a cross-linking agent to form a Schiff base bond responsive to pH and a borate bond responsive to glucose with CMCS and two HAs of different molecular weights, thereby synthesizing a dual dynamic covalent cross-linking network adhesive hydrogel, and at the same time, HAs of different molecular weights that are not chemically modified are introduced into the hydrogel matrix by forming borate bonds; frankincense essential oil microcapsules are prepared by an emulsification coagulation method; and the frankincense essential oil is coated on the hydrogel by a one-pot rapid re-dissolution synthesis method to finally prepare a pH / glucose dual-responsive microcapsule hybrid hydrogel dressing containing frankincense essential oil. As the hybrid hydrogel dressing is slowly decomposed on a chronic diabetic wound, the frankincense essential oil is released, thereby providing a new application of the frankincense essential oil.

[0017] The present invention has the following advantages and beneficial effects: First, the present invention introduces HA of different molecular weights into the dressing without chemical modification, thereby completely retaining its biological activity.

[0018] Second, the dressing prepared by the present invention exhibits good adhesion and plasticity, and has good wound surface conformity and mechanical properties, that is, it can be completely attached to the skin surface and fill the irregular plane of the wound.

[0019] Third, the present invention has the ability of removability / solubility under the pH and glucose microenvironment, has the ability to sustain the release of frankincense essential oil, innovates a way of using essential oil, and provides a new treatment strategy for diabetic ulcer repair.

[0020] The invention has reasonable design and adopts safe and readily available raw materials to prepare the hydrogel. Firstly, the insoluble and volatile frankincense essential oil is made into microcapsules and applied to the field of wound repair, and a new application form of the frankincense essential oil is proposed. Secondly, two high-molecular-weight and low-molecular-weight hyaluronic acids with different biological activities are prepared with chitosan in a form without chemical modification to prepare a hydrogel dressing matrix, so that the composite dressing has the effects of anti-inflammatory, anti-oxidation, antibacterial and promoting healing, and the preparation is fast and simple, and the composite dressing has the stimulation response ability to the acidic / high-sugar microenvironment of the diabetic wound surface, and has important practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 The chemical reaction principle of forming hydrogel based on Schiff base reaction and borate ester bond reaction is shown.

[0024] Figure 2 Representation of the degradation / acid response of the Schiff base bonds of the CFHh hydrogel (A) and the degradation / sugar response of the boronate ester bonds of the CFHh hydrogel (B).

[0025] Figure 3 Schematic diagram showing the animal experiment process.

[0026] Figure 4 Typical photos showing the wound recovery of animal wounds under different conditions and at different times.

[0027] Figure 5 Schematic diagram showing the results of HE staining of animal wound tissues taken on the 14th day.

[0028] Figure 6 Schematic diagram showing the results of Masson staining of animal wound tissues taken on the 14th day. DETAILED DESCRIPTION

[0029] In order to more clearly understand the above-mentioned objectives, features and advantages of the present invention, the scheme of the present invention will be further described below. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0030] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein; it is obvious that the embodiments in the specification are only part of the embodiments of the present invention, rather than all of the embodiments.

[0031] The specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0032] A method for preparing a pH / glucose dual-responsive chronic wound dressing containing frankincense essential oil comprises the following steps: (1) using 4-formaldehyde phenylboronic acid as a cross-linking agent to form a Schiff base and a borate ester bond with carboxymethyl chitosan and two hyaluronic acids of different molecular weights, respectively, to prepare a hydrogel capable of filling skin surface wounds and dual dynamic covalent cross-linking adhesion, and freeze-drying the hydrogel for later use; (2) preparing the frankincense essential oil into microcapsules by emulsification coagulation, and freeze-drying the microcapsules for later use; (3) mixing the hydrogel with the freeze-dried powder of the frankincense essential oil microcapsules in a certain proportion, adding deionized water or PBS to stir and dissolve in one pot to form a pH / glucose dual-responsive hybrid hydrogel dressing coated with the frankincense essential oil microcapsules. Example 1

[0033] Step 1: Preparation of CFHh hydrogel 1) First, 0.1 g of carboxymethyl chitosan (CMCS) powder was completely dissolved in 2 mL of PBS (0.01 M, pH 7.4) solution under room temperature ultrasound and strong stirring to obtain a CMCS solution (concentration of 5 wt%).

[0034] 2) Secondly, under the same conditions, two different molecular weight hyaluronic acids (HA H , HA L ) powder, 0.08 g each, was completely dissolved in 2 mL PBS (0.01 M, pH 7.4) solution at a ratio of 1:1 to obtain HA solution (HA H , HA L The concentration of each was 4wt%, and the total HA concentration was 8wt%).

[0035] 3) Then, 0.015 g of 4-formaldehyde phenylboronic acid (4-FPBA) powder was completely dissolved in 0.5 mL of anhydrous ethanol solution under ultrasound at room temperature to obtain a 4-FPBA solution (concentration of 3 wt%).

[0036] 4) Finally, HA solution, 4-FPBA solution, and CMCS solution were stirred vigorously with a glass rod in the order of solution volume ratio of 2:0.5:2 to form C5F3H4h4 (the numbers after the letters represent the mass percentage of the components in the material) hydrogel (structural formula as shown in FIG. Figure 1 The hydrogel freeze-dried powder was obtained after freeze drying.

[0037] Step 2: Preparation of frankincense essential oil microcapsules 1) Preparation of chitosan solution (concentration of 1 wt%): 0.2 g of chitosan was dissolved in 20 mL of glacial acetic acid solution (concentration of 1 wt%) and stirred overnight to obtain a chitosan solution. The chitosan solution was centrifuged at 9000 rpm for 30 minutes to obtain the supernatant to obtain a chitosan glacial acetic acid solution (concentration of 1 wt%).

[0038] 2) 0.225 g of Tween-80 as a surfactant was added to the chitosan glacial acetic acid solution (20 mL) and stirred at 45 °C for 2 h to obtain a uniform mixture solution as the aqueous phase.

[0039] 3) According to the mass ratio of chitosan to frankincense essential oil of 1:0.8, 0.16 g of frankincense oil was dissolved in 2 mL of dichloromethane to obtain a dichloromethane solution of frankincense essential oil as the oil phase.

[0040] Then, the oil phase was gradually dropped into the mixture solution (20 mL) in step 2) within 5 min of homogenization in a laboratory emulsifier (Laboratory emulsfier, Shanghai Yiwei Electromechanical Equipment Co., Ltd.) at a speed of 13000 rpm to obtain an oil-in-water emulsion.

[0041] 4) Then, sodium tripolyphosphate (TPP) solution (solute mass 0.8 g, concentration 4 wt%, 20 mL) was added dropwise into the stirred oil-in-water emulsion; stirring was continued for 40 min.

[0042] 5) The formed microcapsule particles were collected by high-speed centrifugation (HC-3018, Anhui Zhongke Zhongjia Scientific Instrument Co., Ltd.) at 9000×g for 30 min; then washed three times with deionized water.

[0043] 6) Finally, ultrasonic treatment was performed in an ice bath for 5 min using an ultrasonic generator to obtain a uniform suspension.

[0044] 7) Use a freeze dryer to freeze-dry for 48 hours, and then you can get the frankincense essential oil microcapsule freeze-dried powder.

[0045] Step 3: Mix 50 mg of hydrogel freeze-dried powder and 50 mg of frankincense essential oil microcapsule freeze-dried powder, add them into 600 μL of water, and quickly reconstitute them to obtain a new pH / glucose microenvironment dual-responsive frankincense essential oil microcapsule hybrid hydrogel multifunctional dressing (solid content of 14 wt%). Example 2

[0046] The difference between Example 2 and Example 1 is that 5 mg of the hydrogel freeze-dried powder and 5 mg of the frankincense essential oil microcapsule freeze-dried powder are mixed and added into 1000 μL of water, and the mixture is quickly reconstituted to obtain a pH / glucose dual-responsive chronic wound dressing containing frankincense essential oil (solid content is 1 wt %). Example 3

[0047] The difference between Example 3 and Example 1 is that 10 mg of hydrogel freeze-dried powder and 10 mg of frankincense essential oil microcapsule freeze-dried powder are mixed and added into 1000 μL of water, and then quickly reconstituted to obtain a novel pH / glucose microenvironment dual-responsive frankincense essential oil microcapsule hybrid hydrogel multifunctional dressing (solid content is 2 wt%). Example 4

[0048] The difference between Example 4 and Example 1 is that 125 mg of hydrogel freeze-dried powder and frankincense essential oil microcapsule freeze-dried powder are mixed and added into 1000 μL of water, and then quickly reconstituted to obtain a novel pH / glucose microenvironment dual-responsive frankincense essential oil microcapsule hybrid hydrogel multifunctional dressing (solid content of 20 wt%). The material performance analysis of each embodiment is as follows:

[0049] 1. Degradation / acid response of Schiff base bonds of CFHh hydrogel (simulating the response to the acid environment of ulcer wound microbial metabolism) (1) Take 2 mL of glacial acetic acid solution (pH = 5.5) and add it to the C5F3H4H4 hydrogel. After standing for 30 seconds and 2 minutes, turn the glass bottle upside down to observe whether it liquefies. (2) Take 2 mL of dilute hydrochloric acid solution (pH = 5.5) and add it to the C5F3H4H4 hydrogel. After standing for 30 seconds and 2 minutes, turn the glass bottle upside down to observe whether it liquefies. (3) Take 2 mL of vitamin C solution (pH = 5.5) and add it to the C5F3H4H4 hydrogel. After standing for 30 seconds and 2 minutes, turn the glass bottle upside down to observe whether it liquefies. (4) Take 2 mL of PBS solution (pH = 7.4) and add it to the C5F3H4H4 hydrogel. After standing for 30 seconds and 2 minutes, turn the glass bottle upside down to observe whether it liquefies.

[0050] Analysis results: Figure 2 As can be seen in A, the addition of inorganic acid glacial acetic acid and dilute hydrochloric acid with a pH of 5.5 did have significant acid degradation / response compared to adding only PBS solution. Among them, the fastest degradation rate was organic acid vitamin C with a pH of 5.5, which could completely degrade CFHh hydrogel in about 30 seconds. The reason is that vitamin C solution is not only acidic, but also has a chemical structure similar to sugar, and also has ortho-hydroxyl groups that compete with HA to produce borate ester bonds.

[0051] 2. Degradation of boronate bonds / sugar response of CFHh hydrogels (simulating the response to high sugar concentrations in the microenvironment of diabetic ulcer wounds) (1) When the glucose concentration is 6.1 mmol / L, weigh 0.0110 g of glucose and dissolve it in 10 mL of water. Take 2 mL of glucose solution and add it to the C5F3H4H4 hydrogel. After standing for 10 s and 30 s, turn the glass bottle upside down to observe whether it liquefies. (2) When the glucose concentration is 16.7 mmol / L, weigh 0.0301 g of glucose and dissolve it in 10 mL of water. Take 2 mL of glucose solution and add it to the C5F3H4H4 hydrogel. After standing for 10 s and 30 s, turn the glass bottle upside down to observe whether it liquefies.

[0052] Analysis results: Figure 2 As can be seen in B, adding glucose at two different concentrations, 6.1mmol / L and 16.7mmol / L, did have significant acid degradation / response compared to adding only PBS solution. The fastest degradation rate was in the 16.7mmol / L glucose sample, which could completely degrade the CFHh hydrogel in about 30s.

[0053] 3. Construction of mouse skin defect model and testing of the repair effect of CFHh hydrogel and frankincense essential oil microcapsule hybrid hydrogel on diabetic ulcer wounds: BALB / C mice purchased from Beijing Sibeifu Biotechnology Co., Ltd. were used. Figure 3 A STZ diabetic ulcer wound skin defect model (8 weeks, 21±0.5 g) was prepared, and the in vivo chronic wound healing effects of CFHh hydrogel and frankincense essential oil microcapsule hybrid hydrogel (sample prepared in Example 1) were observed and evaluated.

[0054] (1) Establishment of STZ diabetes model: 1) Preparation of sodium citrate buffer: Add 2.1g citric acid to 100mL sterile water to prepare citric acid mother solution, called A solution; add 2.94g trisodium citrate to 100mL sterile water to prepare sodium citrate mother solution, called B solution; mix solution A and solution B in a ratio of 1:1.32, measure the pH value with a pH test strip, and adjust the solution pH to about 4.0, which is the 0.1mol / L sodium citrate buffer required for preparing STZ.

[0055] 2) Preparation of STZ solution: Dissolve STZ in 0.1 mol / L sodium citrate buffer to prepare a 10 mg / mL STZ solution, and filter and sterilize it with a bacteria filter. Prepare in a dark place and prepare it before use.

[0056] 3) Model establishment: Fast for 12 hours before surgery, and intraperitoneally inject STZ at a dose of 120 mg / kg. The blank control group was given the same dose of sodium citrate buffer. Blood sugar was measured 7 days after injection. If blood sugar was higher than 16.7 mmol / L, the type 1 diabetes model was successfully established and included in the formal experiment.

[0057] (2) Establishment of STZ diabetic ulcer wound skin defect model 1) To create a STZ diabetic ulcer skin defect model, first shave the hair on the back of the mouse with an electric razor, and then use a depilatory cream to remove the hair to fully expose the surgical site. After intraperitoneal injection of avertin anesthetic at a dose of 0.2 mL / 10 g based on the mouse's body weight, an ulcer wound was established on the back of each mouse using a puncher (6 mm in diameter).

[0058] 2) Establish a blank control group (normal mice without diabetes, establish an ulcer wound, and do not add the test sample).

[0059] 3) Establish a negative control group (STZ diabetic mice, establish ulcer wounds, and do not add the test samples).

[0060] 4) Establishment of experimental groups (STZ diabetic mice, establishment of ulcer wounds, and addition of test samples).

[0061] 5) Sample preparation: C5F3H4h4 hydrogel group: 100 mg of C5F3H4h4 hydrogel was reconstituted in a mold by adding 600 μL of sterile water, and then the sample was transferred to the ulcer wound on the back of the mouse and bandaged with medical gauze and pressure-sensitive tape.

[0062] Frankincense essential oil microcapsule hybrid hydrogel group (mass ratio 1:1): 50 mg C5F3H4h4 hydrogel + 50 mg frankincense essential oil microcapsules were reconstituted in the mold with 600 μL sterile water, and then the sample was transferred to the ulcer wound on the back of the mouse and bandaged with medical gauze and pressure-sensitive tape.

[0063] 6) Closely observe the healing process of the ulcer wound and take photos of the ulcer wound on the 1st, 3rd, 7th, 10th and 14th days.

[0064] Analysis results: Figure 4 As shown in the figure, the wound area of ​​all groups decreased to a certain extent over time. Compared with the blank control group and the negative control group, the wound healing speed of the C5F3H4h4 hydrogel group and the frankincense essential oil microcapsule hybrid hydrogel group was faster after the 7th day.

[0065] 4. Pathological sections The wound tissues of mice in the blank control group, negative control group, C5F3H4h4 hydrogel group and frankincense essential oil microcapsule hybrid hydrogel group were taken for pathological sections on the 14th day, and HE staining and Masson staining were performed. The results are as follows Figure 5 , Figure 6 shown.

[0066] from Figure 5 , Figure 6 It can be seen that the amount of new cells and collagen in the frankincense essential oil microcapsule hybrid hydrogel group is greater than that in the C5F3H4h4 hydrogel group and the negative control group, and the wound cell lumen microstructure in the frankincense essential oil microcapsule hybrid hydrogel group is richer than that in the hydrogel group and close to that in the normal group.

[0067] The above is only a specific implementation of the present invention, which enables those skilled in the art to understand or implement the present invention. Although detailed descriptions are given with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions recorded in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments, and they should all be covered by the protection scope of the claims.

Claims

1. A method for preparing a pH / glucose dual-responsive chronic wound dressing containing frankincense essential oil, characterized in that: The steps include: (1) Synthesis of dual dynamic covalent cross-linked network adhesive hydrogel: high molecular weight and low molecular weight hyaluronic acid were mixed and dissolved in PBS to obtain HA solution; carboxymethyl chitosan was completely dissolved in PBS to obtain CMCS solution; 4-formaldehyde phenylboronic acid was completely dissolved in anhydrous ethanol to obtain 4-FPBA solution; The HA solution, 4-FPBA solution and CMCS solution are mixed and stirred in sequence to form CFHh hydrogel, and then freeze-dried to obtain hydrogel freeze-dried powder; (2) Preparation of frankincense essential oil microcapsules: chitosan is added to an acetic acid solution at room temperature and stirred overnight to obtain a chitosan solution, the supernatant is collected after centrifugation to obtain a chitosan acetic acid solution, Tween-80 is added to the chitosan acetic acid solution as a surfactant, and a uniform mixture is obtained after stirring, i.e., an aqueous phase; frankincense essential oil is dissolved in dichloromethane to obtain a dichloromethane solution of frankincense essential oil, i.e., an oil phase; under homogenization conditions, the oil phase is gradually dripped into the aqueous phase to obtain an oil-in-water emulsion, and then a sodium tripolyphosphate solution is dropwise added to the stirred emulsion to stir, and the microcapsule particles are collected by centrifugation and washing, and a uniform suspension is obtained after washing and ultrasonic treatment, and freeze-dried to obtain freeze-dried powder of frankincense essential oil microcapsules; (3) Forming a pH / glucose dual-responsive hybrid hydrogel dressing coated with frankincense essential oil microcapsules: taking the hydrogel freeze-dried powder and the frankincense essential oil microcapsule freeze-dried powder and mixing them, adding them to water or PBS and stirring to dissolve them, thereby obtaining a frankincense essential oil microcapsule hybrid hydrogel dressing.

2. The method for preparing the pH / glucose dual-responsive hybrid hydrogel dressing coated with frankincense essential oil microcapsules according to claim 1, characterized in that: In step (1), the molecular weight ranges of high molecular weight HA and low molecular weight HA are 100,000 to 1.5 million and 2,000 to 5,000, respectively.

3. The method for preparing the pH / glucose dual-responsive hybrid hydrogel dressing coated with frankincense essential oil microcapsules according to claim 2, characterized in that: In step (1), the molecular weight ranges of high molecular weight HA and low molecular weight HA are 800,000 to 1.5 million and 3,000, respectively.

4. The method for preparing the pH / glucose dual-responsive hybrid hydrogel dressing coated with frankincense essential oil microcapsules according to claim 3, characterized in that: In step (1), the concentration of the CMCS solution is 5wt%; the concentration of the 4-FPBA solution is 3wt%; the concentration of the HA solution is 8wt%, wherein the concentrations of the high molecular weight and low molecular weight HA are both 4wt%; when mixed, the volume ratio of the HA solution, the 4-FPBA solution, and the CMCS solution is 2:0.5:

2.

5. The method for preparing the pH / glucose dual-responsive hybrid hydrogel dressing coated with frankincense essential oil microcapsules according to claim 1, characterized in that: In step (2), the concentration of the CS solution is 0.1 to 1 wt %; the concentration of the glacial acetic acid solution is 0.5 to 5 wt %; the concentration of the TPP solution is 0.1 to 1 wt %; the concentration of the dichloromethane solution of frankincense essential oil is 0.01 to 0.5 g / mL; the weight ratio of CS to frankincense essential oil is 1:0.1 to 0.8; and the stirring temperature after adding Tween-80 is 20 to 50° C.

6. The method for preparing the pH / glucose dual-responsive hybrid hydrogel dressing coated with frankincense essential oil microcapsules according to claim 5, characterized in that: In step (2), the concentration of the CS solution is 1wt%; the concentration of the glacial acetic acid solution is 1wt%; the concentration of the TPP solution is 0.4wt%; the concentration of the dichloromethane solution of frankincense essential oil is 0.1g / mL; the weight ratio of CS to frankincense essential oil is 1:0.8; and the stirring temperature after adding Tween-80 is 45°C.

7. The method for preparing the pH / glucose dual-responsive hybrid hydrogel dressing coated with frankincense essential oil microcapsules according to claim 1, characterized in that: In step (3), the mass ratio of the hydrogel freeze-dried powder to the frankincense essential oil microcapsule freeze-dried powder is 1 to 10:

1.

8. The method for preparing the pH / glucose dual-responsive hybrid hydrogel dressing coated with frankincense essential oil microcapsules according to claim 7, characterized in that: In step (3), the mass ratio of the hydrogel freeze-dried powder to the frankincense essential oil microcapsule freeze-dried powder is 1:1, that is, the frankincense essential oil and the microcapsule are 7wt% each, and the solid mass fraction in the obtained dressing is 14wt%.

9. Use of the dressing prepared by the method according to any one of claims 1 to 8 in treating diabetic chronic skin ulcers and inflammatory skin wounds.

10. Use of frankincense essential oil in pH / glucose dual-responsive hybrid hydrogel dressing coated with frankincense essential oil microcapsules prepared by the method according to any one of claims 1 to 8.

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