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

Abstract

This invention discloses a method for preparing a pH / glucose dual-responsive chronic wound dressing containing frankincense essential oil, comprising: (1) using 4-formaldehyde phenylboronic acid as a crosslinking agent to form Schiff base and borate ester bonds with carboxymethyl chitosan and two different molecular weight hyaluronic acids, respectively, to prepare a hydrogel capable of filling skin surface wounds and performing dual dynamic covalent crosslinking adhesion, and freeze-drying for later use; (2) preparing frankincense essential oil into microcapsules by emulsification coagulation method, and freeze-drying for later use; (3) mixing the hydrogel with the freeze-dried powder of frankincense essential oil microcapsules, adding deionized water or PBS and stirring to reconstitute to form a pH / glucose dual-responsive hybrid hydrogel dressing coated with frankincense essential oil microcapsules. The composite dressing prepared by this invention has anti-inflammatory, antioxidant, antibacterial, and healing-promoting effects, is quick and easy to prepare, and has the ability to respond to the acidic / high-glucose microenvironment of diabetic wounds, and has important practical application value.

Description

Technical Field

[0001] This invention belongs to the field of tissue repair and biomedical materials technology, specifically a method for preparing and applying a pH / glucose dual-responsive chronic wound dressing containing frankincense essential oil. Background Technology

[0002] Diabetes is a chronic and prevalent disease, with its incidence rate increasing year by year worldwide. Due to long-term abnormal glucose metabolism, diabetic patients experience high blood sugar levels, leading to abnormalities in their microvessels, nervous system, and immune system. Therefore, wounds, including those requiring surgery for other diseases, are highly susceptible to developing into chronic, difficult-to-heal inflammations. This is characterized by the presence of large amounts of inflammatory immune cells, inflammatory cytokines, reactive oxygen species (ROS), and proteases, as well as a local acidic microenvironment caused by repeated bacterial infections, resulting in persistent and difficult-to-heal conditions.

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

[0004] Frankincense is the resin exuded from the bark of the Boswellia carterii Birdw. and related species B. bhaw-dajiana Birdw., belonging to the Burseraceae family. There are approximately 30 species worldwide. Its medicinal properties, including promoting blood circulation, relieving pain, treating sores and carbuncles, and alleviating boils, are recorded in ancient medical texts such as the *Compendium of Materia Medica* and *Commentary on the Materia Medica*. Modern pharmaceutical research shows that frankincense possesses anti-inflammatory, antioxidant, antibacterial, and immunomodulatory effects. More importantly, frankincense extracts, such as 3-acetyl-11-carbonyl-β-boswellic acid and boswellic acid derivatives, have significant 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, and polysaccharides. Among them, volatile oil, namely frankincense essential oil, is the key component for frankincense to exert its medicinal effects and aroma (Liu Di, et al. Chinese Herbal Medicines, 2020, 51(22):5900-5914). However, the poor water solubility, poor stability, and volatility of frankincense essential oil make it difficult to exert its medicinal effects on the surface of wounds for a long time, which greatly limits its application and efficacy. Making frankincense essential oil into microcapsules can improve these defects.

[0005] On the other hand, medical hydrogels are a material form formed by the swelling of a cross-linked network of hydrophilic polymers in water. Their characteristics include high water content (97%–99%) and biocompatibility. In particular, Dr. Winter's animal experiments in 1962 demonstrated that wounds heal better in a moist environment than in an open, dry environment, leading to the theory of moist wound healing (Winter G.D. Nature, 1962, 193:293-294). Guided by this theory, hydrogels, as ideal cell scaffolds and drug carriers, have attracted increasing attention in tissue repair and the research, development, production, and application of modern wound dressings. Adhesive hydrogels not only adhere firmly to uneven ulcer surfaces but also respond to external physical or chemical stimuli, such as pH, glucose, reactive oxygen species (ROS), and enzymes. However, replacing or removing adhesive hydrogel dressings may cause secondary damage to unhealed wounds. Therefore, a novel hydrogel that can effectively improve the repair of diabetic ulcers while also being removable / soluble and avoiding secondary damage that could hinder wound repair is attracting worldwide attention.

[0006] Hyaluronic acid, an acidic mucopolysaccharide, is widely found in organisms and possesses physiological functions such as promoting wound healing, lubricating joints, and anti-oxidation and anti-aging. Different molecular weights of hyaluronic acid exhibit different functions. High molecular weight hyaluronic acid protects cells and tissues from bacterial and ultraviolet radiation and forms a cellular scaffold, facilitating rapid scar healing. Low molecular weight hyaluronic acid is easily absorbed and promotes blood circulation, improves metabolism, and has antioxidant effects. However, current research on the modification of hyaluronic acid may hinder its original biological activity (Luo Z., et al. Adv. Funct. Mater., 2023, 33, 2306554), potentially affecting wound repair in severe cases. Therefore, the introduction of unmodified hyaluronic acid into hydrogel dressings is of great significance. Summary of the Invention

[0007] Diabetic patients are prone to developing chronic inflammatory wounds that are difficult to heal due to long-term abnormal glucose metabolism. To address this clinical problem, this invention aims to provide a method for preparing a pH / glucose dual-responsive chronic wound dressing containing frankincense essential oil. This method, as a novel multifunctional dressing for a pH / glucose microenvironment dual-responsive frankincense essential oil microcapsule hybrid hydrogel, mainly includes three aspects: First, a dual-responsive medical hydrogel that can decompose under pH / glucose stimulation; second, a method for stabilizing frankincense essential oil, namely, a method for preparing frankincense essential oil microcapsules; and third, a method for preparing and applying the pH / glucose dual-responsive chronic wound dressing containing frankincense essential oil to chronic wounds.

[0008] This invention is achieved using the following technical solution:

[0009] A method for preparing a pH / glucose dual-responsive chronic wound dressing containing frankincense essential oil includes the following steps:

[0010] (1) Synthesis of a dual dynamic covalent cross-linked network adhesive hydrogel: A certain amount of high molecular weight and low molecular weight hyaluronic acid were mixed (HA, of which high molecular weight hyaluronic acid HA) H Abbreviated as "H", low molecular weight hyaluronic acid HA L A solution of HA was obtained by completely dissolving 4-carboxymethyl chitosan (CMCS, abbreviated as "C") in PBS; a solution of CMCS was obtained by completely dissolving 4-carboxymethyl chitosan (CMCS, abbreviated as "C") in PBS; and a solution of 4-FPBA (4-formaldehyde-phenylboronic acid, abbreviated as "F") was obtained by completely dissolving 4-FPBA in anhydrous ethanol. The HA, 4-FPBA, and CMCS solutions were then stirred vigorously with a glass rod in that order to form a "CFHh" hydrogel, which was then freeze-dried to obtain a lyophilized hydrogel powder.

[0011] (2) Preparation of frankincense essential oil microcapsules by emulsification and coagulation: At room temperature, a certain amount of chitosan (CS) was added to glacial 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 collected to obtain a chitosan glacial acetic acid solution. A certain amount of Tween-80 was added to the chitosan glacial acetic acid solution as a surfactant, and the mixture was stirred at a certain temperature for 2 hours to obtain a homogeneous mixture solution, i.e., the aqueous phase. A certain amount of frankincense essential oil was dissolved in dichloromethane to obtain a frankincense essential oil dichloromethane solution, i.e., the oil phase. Under homogenization conditions of 13000 rpm in a high-speed shear mill, the oil phase was gradually added dropwise to the aqueous phase to obtain an oil-in-water emulsion. Then, a certain amount of sodium tripolyphosphate (TPP) solution was added dropwise to the stirred emulsion and stirred for a certain time. The microcapsule particles were collected by centrifugation and washing, washed several times with deionized water, and finally ultrasonically treated in an ice bath for 3-5 minutes to obtain a homogeneous suspension. The suspension was then freeze-dried to obtain frankincense essential oil microcapsule freeze-dried powder.

[0012] (3) One-pot stirring and reconstitution to form pH / glucose dual-response hybrid hydrogel dressing coated with frankincense essential oil microcapsules: Take a certain proportion of hydrogel freeze-dried powder and frankincense essential oil microcapsule freeze-dried powder, mix them, add a certain amount of water or PBS and stir to reconstitute to obtain frankincense essential oil microcapsule hybrid hydrogel dressing.

[0013] In the method of this invention, frankincense essential oil is made into microcapsules. Simultaneously, using unmodified hyaluronic acid as a raw material and 4-formaldehyde phenylboronic acid as a crosslinking agent, a pH / glucose dual-responsive hydrogel dressing is prepared. The frankincense essential oil microcapsules are then encapsulated in hydrogel to form a hybrid hydrogel. The stability of the frankincense essential oil is significantly improved under the dual encapsulation of microcapsules and hydrogel. The hydrogel gradually decomposes under the stimulation of the diabetic microenvironment, slowly releasing the frankincense essential oil into chronic diabetic wounds. Currently, the above-mentioned innovative applications of frankincense essential oil have not been disclosed, namely, the application of the dressing prepared by the method of this invention in diabetic chronic skin ulcers and inflammatory skin wounds, and the application of frankincense essential oil in the pH / glucose dual-responsive hybrid hydrogel dressing with frankincense essential oil microcapsules prepared by the method of this invention.

[0014] Preferably, in step (1), the high and low molecular weight HA are raw materials that have not undergone any chemical modification, thus fully retaining the biological activity of HA. The molecular weight ranges of the high and low molecular weight HA are 100,000 to 1,500,000 and 2,000 to 5,000, respectively. More preferably, the molecular weights of the high and low molecular weight HA are 800,000 to 1,500,000 and 3,000, respectively.

[0015] The concentration of the CMCS solution was 5 wt%; the concentration of the 4-FPBA solution was 3 wt%; and the concentration of the HA solution was 8 wt%, with both high and low molecular weight HA solutions having a concentration of 4 wt%. The volume ratio of the HA, 4-FPBA, and CMCS solutions during mixing was 2:0.5:2.

[0016] More preferably, in step (2), the concentration of the CS solution is 1 wt%; the concentration of the TPP solution is 0.4 wt%; the concentration of the glacial acetic acid solution is 1 wt%; and the concentration of the dichloromethane solution of frankincense essential oil is 0.1 g / mL. The weight ratio of CS to frankincense essential oil is 1:0.8. Tween-80 is added and the stirring temperature is 45°C.

[0017] Preferably, in step (3), the mass ratio of the hydrogel freeze-dried powder and the frankincense essential oil microcapsule freeze-dried powder is 1:1, that is, frankincense essential oil and microcapsules are 7wt% each, and the solid mass fraction in the resulting dressing is 14wt%.

[0018] The present invention prepares a dual-dynamic covalently cross-linked network adhesive hydrogel by using 4-FPBA as a cross-linking agent to form pH-responsive Schiff base bonds and glucose-responsive borate ester bonds with CMCS and two different molecular weight HAs, respectively. Simultaneously, unmodified HAs of different molecular weights are introduced into the hydrogel matrix through the formation of borate ester bonds. Frankincense essential oil microcapsules are prepared by emulsification and coagulation. Frankincense essential oil is then encapsulated in the hydrogel using a one-pot rapid remelting synthesis method to finally prepare a pH / glucose dual-responsive microcapsule hybrid hydrogel dressing containing frankincense essential oil. As the hybrid hydrogel dressing slowly decomposes and releases frankincense essential oil in diabetic chronic wounds, a novel application of frankincense essential oil is presented.

[0019] The present invention has the following advantages and benefits:

[0020] First, this invention introduces HA of different molecular weights into dressings without chemical modification, thus fully preserving its biological activity.

[0021] Secondly, the dressing prepared by the present invention exhibits good adhesion and plasticity, and has good wound surface conformability and mechanical properties, that is, it can completely adhere to the skin surface and fill the irregular plane of the wound.

[0022] Third, the present invention exhibits the ability to be removed / soluble in pH and glucose microenvironment, has a sustained-release ability for frankincense essential oil, innovates a way of using essential oil, and provides a new treatment strategy for the repair of diabetic ulcers.

[0023] This invention is rationally designed and uses safe and readily available raw materials to prepare hydrogels. First, it realizes the application of poorly soluble and volatile frankincense essential oil in the field of wound repair by preparing microcapsules, proposing a new application form of frankincense essential oil. Second, it prepares a hydrogel dressing matrix by combining two types of high and low molecular weight hyaluronic acid with different biological activities with chitosan in an unmodified form. This gives the composite dressing anti-inflammatory, antioxidant, antibacterial, and healing-promoting effects. The preparation is rapid and simple, and it has the ability to respond to the acidic / high sugar microenvironment of diabetic wounds, which has important practical application value. Attached Figure Description

[0024] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This describes the chemical reaction principle of hydrogel formation based on the Schiff base reaction and borate ester bond reaction.

[0027] Figure 2 The degradation / acid response of Schiff base bonds in CFHh hydrogels (A) and the degradation / sugar response of borate ester bonds in CFHh hydrogels (B) are represented.

[0028] Figure 3 This is a schematic diagram illustrating the animal experiment process.

[0029] Figure 4 Typical photographs showing the wound healing status of animals under different conditions and at different times.

[0030] Figure 5 This diagram illustrates the results of HE staining of animal wound tissue taken on day 14.

[0031] Figure 6 This diagram illustrates the results of Masson staining of animal wound tissue taken on day 14. Detailed Implementation

[0032] To better understand the above-mentioned objectives, features, and advantages of the present invention, the solutions of the present invention will be further described below. It should be noted that, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

[0033] Many specific details are set forth in the following description in order to provide a full understanding of the invention, but the invention may also be practiced in other ways different from those described herein; obviously, the embodiments in the specification are only some embodiments of the invention, and not all embodiments.

[0034] The specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0035] A method for preparing a pH / glucose dual-responsive chronic wound dressing containing frankincense essential oil includes the following steps: (1) By using 4-formaldehyde phenylboronic acid as a crosslinking agent, Schiff base and borate ester bonds are formed with carboxymethyl chitosan and two different molecular weight hyaluronic acids respectively, a skin surface wound filling and dual dynamic covalent crosslinking adhesive hydrogel is prepared and lyophilized for later use; (2) Frankincense essential oil is made into microcapsules by emulsification coagulation method and lyophilized for later use; (3) The hydrogel and the lyophilized powder of frankincense essential oil microcapsules are mixed in a certain proportion, and deionized water or PBS is added and stirred to reconstitute in one pot to form a pH / glucose dual-responsive hybrid hydrogel dressing coated with frankincense essential oil microcapsules. Example 1

[0036] Step 1: Preparation of CFHh hydrogel

[0037] 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 sonication and vigorous stirring to obtain a CMCS solution (concentration of 5 wt%).

[0038] 2) Secondly, under the same conditions, two different molecular weight hyaluronic acids (HA) were... H HA L 0.08 g of each powder was completely dissolved in 2 mL of 0.01 M PBS (pH 7.4) at a 1:1 ratio to obtain HA solution. H HA L Each concentration was 4 wt%, and the total HA concentration was 8 wt%.

[0039] 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 ultrasonication at room temperature to obtain a 4-FPBA solution (concentration of 3 wt%).

[0040] 4) Finally, the HA solution, 4-FPBA solution, and CMCS solution are added in sequence at a volume ratio of 2:0.5:2, and vigorously stirred with a glass rod to form a C5F3H4h4 hydrogel (the numbers after the letters represent the mass percentages of the components in the material). The structural formula is shown below. Figure 1 As shown in the figure, the hydrogel freeze-dried powder was obtained after freeze-drying.

[0041] Step 2: Preparation of Frankincense Essential Oil Microcapsules

[0042] 1) Preparation of chitosan solution (concentration of 1wt%): Dissolve 0.2g of chitosan in 20mL of glacial acetic acid solution (concentration of 1wt%) and stir overnight to obtain chitosan solution. Centrifuge the chitosan solution at 9000rpm for 30 minutes and take the supernatant to obtain chitosan glacial acetic acid solution (concentration of 1wt%).

[0043] 2) Add 0.225 g Tween-80 as a surfactant to the glacial acetic acid solution of chitosan (20 mL) and stir at 45 °C for 2 h to obtain a homogeneous mixture solution as the aqueous phase.

[0044] 3) Dissolve 0.16g of frankincense oil in 2mL of dichloromethane at a mass ratio of chitosan to frankincense oil of 1:0.8 to obtain a dichloromethane solution of frankincense oil as the oil phase.

[0045] Then, the oil phase was gradually dripped into the mixture solution (20 mL) in step 2) over a homogenization process of 13,000 rpm for 5 minutes to obtain an oil-in-water emulsion.

[0046] 4) Then add sodium tripolyphosphate (TPP) solution (solute mass 0.8g, concentration 4wt%, 20mL) dropwise to the stirred oil-in-water emulsion; stir continuously for 40min.

[0047] 5) The formed microcapsule particles were collected by centrifuging at 9000×g for 30min using a high-speed centrifuge (HC-3018, Anhui Zhongke Zhongjia Scientific Instruments Co., Ltd.); then washed three times with deionized water.

[0048] 6) Finally, the suspension is ultrasonically treated in an ice bath for 5 minutes using an ultrasonic generator to obtain a uniform suspension.

[0049] 7) Freeze-dry using a freeze dryer for 48 hours to obtain frankincense essential oil microcapsule freeze-dried powder.

[0050] Step 3: Mix 50 mg each of the hydrogel lyophilized powder and the frankincense essential oil microcapsule lyophilized powder and add them to 600 μL of water. Rapid reconstitution will yield a novel pH / glucose microenvironment dual-responsive frankincense essential oil microcapsule hybrid hydrogel multifunctional dressing (solid content is 14 wt%). Example 2

[0051] The difference between Example 2 and Example 1 is that 5 mg each of hydrogel lyophilized powder and frankincense essential oil microcapsule lyophilized powder are mixed and added to 1000 μL of water for rapid reconstitution to obtain a pH / glucose dual-responsive chronic wound dressing containing frankincense essential oil (solid content of 1 wt%). Example 3

[0052] The difference between Example 3 and Example 1 is that 10 mg each of the hydrogel freeze-dried powder and the frankincense essential oil microcapsule freeze-dried powder are mixed and added to 1000 μL of water, and the mixture is 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

[0053] The difference between Example 4 and Example 1 is that 125 mg each of the hydrogel freeze-dried powder and the frankincense essential oil microcapsule freeze-dried powder are mixed and added to 1000 μL of water, and the mixture is quickly reconstituted to obtain a novel pH / glucose microenvironment dual-responsive frankincense essential oil microcapsule hybrid hydrogel multifunctional dressing (solid content is 20 wt%).

[0054] The material properties of each embodiment are analyzed as follows:

[0055] I. Degradation / Acid Response of Schiff Base Bonds in CFHh Hydrogel (Simulating the Response of Microbial Metabolic Acid Environment in Ulcer Wounds)

[0056] (1) Take 2 mL of glacial acetic acid solution (pH=5.5) and add it to C5F3H4h4 hydrogel. After standing for 30 s and 2 min, invert the glass bottle to observe whether it liquefies.

[0057] (2) Take 2 mL of dilute hydrochloric acid solution (pH=5.5) and add it to C5F3H4h4 hydrogel. After standing for 30 s and 2 min, invert the glass bottle to observe whether it liquefies.

[0058] (3) Take 2 mL of vitamin C solution (pH=5.5) and add it to C5F3H4h4 hydrogel. After standing for 30 s and 2 min, invert the glass bottle to observe whether it liquefies.

[0059] (4) Take 2 mL of PBS solution (pH=7.4) and add it to C5F3H4h4 hydrogel. After standing for 30 s and 2 min, invert the glass bottle to observe whether it liquefies.

[0060] Analysis results: Figure 2 As shown in Figure A, the addition of inorganic acids (pH 5.5) such as glacial acetic acid and dilute hydrochloric acid significantly increased acid degradation / response compared to adding only PBS solution. The fastest degradation was observed with the organic acid vitamin C at pH 5.5, which completely degraded the CFHh hydrogel in approximately 30 seconds. This is because the vitamin C solution is not only acidic but also has a chemical structure similar to sugars, exhibiting a similar ortho-hydroxyl group that competes with HA to form a borate ester bond.

[0061] II. Degradation of boronic acid ester bonds in CFHh hydrogels / sugar response (simulating the response of high sugar concentration in the microenvironment of diabetic ulcer wounds)

[0062] (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 C5F3H4h4 hydrogel. After standing for 10 s and 30 s, invert the glass bottle to observe whether it liquefies.

[0063] (2) When the glucose concentration is 16.7 mmol / L, 0.0301 g of glucose is dissolved in 10 mL of water. 2 mL of glucose solution is added to C5F3H4h4 hydrogel. After standing for 10 s and 30 s, the glass bottle is inverted to observe whether it liquefies.

[0064] Analysis results: Figure 2As shown in Figure B, adding glucose at two different concentrations, 6.1 mmol / L and 16.7 mmol / L, did indeed result in significant acid degradation / response compared to adding only PBS solution. The fastest degradation rate was observed in the 16.7 mmol / L glucose sample, which completely degraded the CFHh hydrogel in approximately 30 seconds.

[0065] III. Construction and Testing of Mouse Skin Defect Model: Experiment on the Repair Effect of CFHh Hydrogel and Frankincense Oil Microcapsule Hybrid Hydrogel on Diabetic Ulcer Wounds:

[0066] BALB / c mice purchased from Beijing Spaford Biotechnology Co., Ltd. were used, such as Figure 3 A skin defect model of STZ diabetic ulcer was established (8 weeks, 21±0.5g) to observe and evaluate the in vivo chronic wound healing effect of CFHh hydrogel and frankincense essential oil microcapsule hybrid hydrogel (sample prepared in Example 1).

[0067] (1) Establishment of STZ diabetes model:

[0068] 1) Preparation of sodium citrate buffer solution: Add 2.1g of citric acid to 100mL of sterile water to prepare citric acid stock solution, called solution A; add 2.94g of trisodium citrate to 100mL of sterile water to prepare sodium citrate stock solution, called solution B; mix solutions A and B in a ratio of 1:1.32, measure the pH value with a pH test strip, and adjust the pH of the solution to around 4.0. This is the 0.1mol / L sodium citrate buffer solution required for STZ preparation.

[0069] 2) Preparation of STZ solution: Dissolve STZ in 0.1 mol / L sodium citrate buffer to prepare a 10 mg / mL STZ solution, and sterilize by filtration. Prepare in the dark and use immediately.

[0070] 3) Model establishment: Patients fasted for 12 hours before surgery and received an intraperitoneal injection of STZ at a dose of 120 mg / kg. The blank control group received the same dose of sodium citrate buffer. Seven days after injection, blood glucose was measured. A blood glucose level higher than 16.7 mmol / L was selected as a successful diagnosis of type 1 diabetes model and included in the formal experiment.

[0071] (2) Establishment of a skin defect model of STZ diabetic ulcer wound

[0072] 1) To create a skin defect model of STZ diabetic ulcer wound, the hair on the back of the mice was first shaved with an electric razor and then removed with depilatory cream to fully expose the surgical site. After anesthetizing the mice with afodin anesthetic via intraperitoneal injection at a dose of 0.2 mL / 10 g based on their body weight, an ulcer wound was created on the back of each mouse using a punch (6 mm in diameter).

[0073] 2) A blank control group was set up (normal non-diabetic mice, with ulceration wounds created, and no test samples were added).

[0074] 3) A negative control group (STZ diabetic mice, with ulcerated wounds created, and no test samples added) was set up.

[0075] 4) Set up an experimental group (STZ diabetic mice, ulcer wounds were created, and the test samples were added).

[0076] 5) Sample preparation:

[0077] C5F3H4h4 hydrogel group: 100mg C5F3H4h4 hydrogel was reconstituted in 600μL of sterile water in a mold, and then the sample was transferred to the ulceration on the back of a mouse and bandaged with medical gauze and pressure-sensitive tape.

[0078] Frankincense essential oil microcapsule hybrid hydrogel assembly (mass ratio 1:1): 50mg C5F3H4h4 hydrogel + 50mg frankincense essential oil microcapsules were reconstituted in a mold with 600μL of sterile water. The sample was then transferred to the ulceration on the back of a mouse and bandaged with medical gauze and pressure-sensitive tape.

[0079] 6) Closely observe the healing process of the ulcer and take photos of the ulcer on days 1, 3, 7, 10 and 14.

[0080] Analysis results: such as Figure 4 As shown, the wound area in all groups decreased to some extent over time. Compared with the blank control group and the negative control group, the C5F3H4h4 hydrogel group and the frankincense essential oil microcapsule hybrid hydrogel group showed faster wound healing after day 7.

[0081] IV. Pathological Sections

[0082] Wound tissues from mice in the blank control group, negative control group, C5F3H4h4 hydrogel group, and frankincense essential oil microcapsule hybrid hydrogel group on day 14 were collected and pathological sections were prepared for HE staining and Masson staining. The results are as follows: Figure 5 , Figure 6 As shown.

[0083] from Figure 5 , Figure 6 It can be seen that the amount of newly generated 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. Furthermore, the microstructure of the cell lumen in the wound of the frankincense essential oil microcapsule hybrid hydrogel group is richer than that in the hydrogel group and is close to that in the normal group.

[0084] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the present invention. Although detailed descriptions have been provided with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments, and they should all be covered within 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 by: Comprising the following steps: (1) Synthesis of double-dynamic covalent cross-linking network adhesive hydrogel: high and low molecular weight hyaluronic acid is mixed and dissolved in PBS to obtain HA solution, carboxymethyl chitosan is dissolved in PBS to obtain CMCS solution, and 4-formaldehyde phenylboronic acid is completely dissolved in anhydrous ethanol to obtain 4-FPBA solution; wherein the molecular weight range of high and low molecular weight HA is 100-150 million and 2000-5000 respectively; The HA solution, 4-FPBA solution and CMCS solution are mixed and stirred in sequence to obtain CFHh hydrogel, and the hydrogel freeze-dried powder is obtained after freeze-drying; (2) Preparation of frankincense essential oil microcapsules: chitosan is added to glacial acetic acid solution and stirred overnight to obtain chitosan solution, the supernatant is taken after centrifugation, and the chitosan glacial acetic acid solution is obtained, Tween-80 is added as a surfactant to the chitosan glacial acetic acid solution, and a uniform mixture is obtained after stirring, which is the water phase; dissolve frankincense essential oil in dichloromethane to obtain a dichloromethane solution of frankincense essential oil, which is the oil phase; under homogeneous conditions, the oil phase is gradually added to the water phase to obtain an oil-in-water emulsion, then sodium tripolyphosphate solution is added dropwise to the stirring emulsion, and the microcapsule particles are collected after centrifugation and washing, a uniform suspension is obtained after washing and ultrasonic treatment, and the frankincense essential oil microcapsule freeze-dried powder is obtained after freeze-drying; (3) Formation of pH / glucose dual-responsive hybrid hydrogel dressing coated with frankincense essential oil microcapsules: mix the hydrogel freeze-dried powder and the frankincense essential oil microcapsule freeze-dried powder, then add water or PBS and stir to reconstitute, to obtain the frankincense essential oil microcapsule hybrid hydrogel dressing.

2. The method of preparing a pH / glucose dual-responsive hybrid hydrogel dressing coated with microcapsules of frankincense essential oil according to claim 1, characterized in that: In step (1), the molecular weight range of high and low molecular weight HA is 800-150 million and 3000 respectively.

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

2.

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

5. The method of preparing the pH / glucose dual-responsive hybrid hydrogel dressing coated with microcapsules of frankincense essential oil according to claim 4, characterized in that: In step (2), the concentration of CS solution is 1wt%; the concentration of glacial acetic acid solution is 1wt%; the concentration of TPP solution is 0.4wt%; the concentration of 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 after adding Tween-80 is 45℃.

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

1.

7. The method of preparing the pH / glucose dual-responsive hybrid hydrogel dressing coated with microcapsules of frankincense essential oil according to claim 6, characterized in that: In step (3), the mass ratio of hydrogel lyophilized powder and frankincense essential oil microcapsule lyophilized powder was 1:1, i.e. 7wt% of frankincense essential oil and microcapsule, and the solid mass fraction in the obtained dressing was 14wt%.

8. The use of frankincense essential oil in the pH / glucose dual-responsive hybrid hydrogel dressing coated with frankincense essential oil microcapsules prepared by the method of any one of claims 1-7.

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