阅读说明：本技术 一种含有绿原酸、芍药苷的透明质酸水凝胶医用敷料 (A medical dressing containing chlorogenic acid and paeoniflorin and hyaluronic acid hydrogel ) 是由 郭建锋 杨浩 于 2021-01-25 设计创作，主要内容包括：本发明公开了一种含有绿原酸、芍药苷的透明质酸水凝胶医用敷料,其组成为：8%透明质酸水凝胶、500μM绿原酸、500μM芍药苷、1%丙二醇,其中透明质酸水凝胶的主要作用为覆盖伤口、保湿、缓释药物；绿原酸的主要作用为促进成纤维细胞增殖、促进成纤维细胞迁移、促进血管内皮细胞增殖；芍药苷的主要作用为促进血管内皮细胞迁移、促进新生血管形成、下调炎症反应；丙二醇的主要作用为防腐、杀菌、保湿。本发明制成可以从多方面促进伤口愈合的医用敷料,该敷料可以促进成纤维细胞增殖,促进血管生成,下调炎症反应,并且有止血、保湿的特性,涵盖了伤口愈合的各个方面,具有广泛的应用前景。(The invention discloses a hyaluronic acid hydrogel medical dressing containing chlorogenic acid and paeoniflorin, which comprises the following components: 8% hyaluronic acid hydrogel, 500 mu M chlorogenic acid, 500 mu M paeoniflorin and 1% propylene glycol, wherein the hyaluronic acid hydrogel mainly has the effects of covering wounds, preserving moisture and slowly releasing medicaments; chlorogenic acid has the main effects of promoting fibroblast proliferation, promoting fibroblast migration and promoting vascular endothelial cell proliferation; the paeoniflorin has the main functions of promoting the migration of vascular endothelial cells, promoting the formation of new blood vessels and reducing inflammatory reaction; the propylene glycol has the main functions of antisepsis, sterilization and moisture preservation. The medical dressing can promote wound healing from multiple aspects, can promote fibroblast proliferation, promote angiogenesis, reduce inflammatory reaction, has the characteristics of hemostasis and moisture retention, covers all aspects of wound healing, and has wide application prospect.)

1. A hyaluronic acid hydrogel medical dressing containing chlorogenic acid and paeoniflorin is characterized by comprising 8% of hyaluronic acid hydrogel, 500 mu M of chlorogenic acid, 500 mu M of paeoniflorin and 1% of propylene glycol, and the preparation method comprises the following specific steps:

s1 deionized water of 20ml is selected, and then 0.5g sodium hyaluronate is dissolved in the deionized water of 20 ml;

s2, adding 3g of adipic acid dihydrazide after uniformly stirring the deionized water and the sodium hyaluronate, and then stirring and standing overnight to obtain a mixed solution;

s3, adjusting the pH value of the mixed solution to 4.7 by using HCl, adding 0.4g of carbodiimide hydrochloride, and fully stirring to ensure that the solution gradually becomes gel;

s4 washing the gel with dehydrated alcohol in an ultrasonic washer for 3 times, and washing with PBS in the ultrasonic washer for 3 times;

s5, freezing the cleaned gel, and freeze-drying in a freeze vacuum drying instrument to prepare a dry gel block for later use;

s6, swelling ratio measurement is carried out on the dried gel block;

s7 performing drug release assay on the dried gel mass;

s8, carrying out external enzymolysis enzyme degradation resistance measurement on the completely swollen hydrogel;

s9 dissolving chlorogenic acid and paeoniflorin in propylene glycol, dissolving the propylene glycol solution containing the dissolved medicine in PBS, and re-dissolving the dried product of hyaluronic acid hydrogel with the solution to obtain the composition: 8% hyaluronic acid hydrogel, 500 μ M chlorogenic acid, 500 μ M paeoniflorin, and 1% propylene glycol.

2. The medical dressing of hyaluronic acid hydrogel containing chlorogenic acid and paeoniflorin of claim 1, wherein the swelling ratio of the dried gel block measured in step S6 comprises the following steps:

s1 dividing the gel blocks into three groups, and weighing respectively;

s2 was swollen in phosphate buffer at pH 7.4;

s3, taking out the rubber blocks every 30min, carefully drying the surface moisture, and measuring the weight of the rubber blocks until the weight of the rubber blocks is unchanged;

s4 the swelling ratio SR is calculated according to the equation: SR ═ w/Wd, where Wd and Ws represent the mass of the hydrogel in the dry and swollen states, respectively.

3. The medical dressing of hyaluronic acid hydrogel containing chlorogenic acid and paeoniflorin of claim 1, wherein the step S7 for measuring drug release of the dried gel block comprises the following specific steps:

s1, weighing the dried gel blocks, and respectively putting the gel blocks into 10mg/mL BSA solution for full swelling for 3 days to make the hydrogel reach swelling and adsorption balance;

s2, taking out the gel block, respectively measuring the volume of the residual BSA solution, measuring the BSA concentration in the BSA solution by using the Nanodrop, and calculating the drug loading rate of the hydrogel;

s3, putting the gel with the drug in 1mL of new PBS solution, and measuring the amount of BSA in the solution by Nanodrop every 2h to obtain the release curve of bovine serum albumin.

4. The medical dressing of claim 3, wherein the hydrogel releases drug over time as characterized by the cumulative release equation: the cumulative amount of drug released (wt%) -Mt/M ∞ where Mt refers to the amount of BSA released from the hydrogel over time t and M ∞ refers to the total amount of BSA loaded into the hydrogel.

5. The medical dressing of hyaluronic acid hydrogel containing chlorogenic acid and paeoniflorin of claim 1, wherein the step S8 of performing the determination of the external resistance to enzymatic degradation of the completely swollen hydrogel comprises the following specific steps:

s1 putting the completely swollen hydrogel into 1mL of 100u/mL hyaluronidase solution;

s2 sampling every 24h, developing with carbazole, measuring absorbance at 530nm, performing linear regression with HAse concentration as abscissa and absorbance as ordinate, and calculating average slope of straight line.

Technical Field

The invention relates to the technical field of biomedical materials, in particular to a hyaluronic acid hydrogel medical dressing containing chlorogenic acid and paeoniflorin.

Background

The skin can protect the body from external damage, sense environmental changes, maintain physiological balance, but is also very easy to be damaged. Certain physiological states (such as age, infection, and severe injury) or underlying medical conditions (such as ischemia, diabetes, and obesity) can interfere with wound healing. Skin repair and regeneration associated with surgical trauma, acute trauma and chronic disease are central issues of health care. The annual medical insurance costs for wound care are estimated to range from $ 281 billion to $ 968 billion, placing a serious burden on healthcare systems worldwide. It is noted that chronic wounds are often accompanied by complications such as inflammation, cellular dysfunction and angiogenesis disorders, which complicates medical care, increases treatment costs, and degrades the quality of life of patients, and thus new treatment strategies are urgently needed.

Wound healing is a dynamic and orderly process involving four phases of hemostasis, inflammation, proliferation and remodeling (maturation). Surgical debridement, antibiotics and wound dressings remain the main means of wound therapy, but most of these are directed to one aspect only and have limited effectiveness. Medical dressings are wound-covering articles, medical materials used to cover sores, wounds, or other injuries. Compared with the traditional medical dressing, the novel medical dressing based on the natural polymer biomaterial has better hemostatic and procoagulant effects, cell proliferation promoting capacity and biocompatibility.

Disclosure of Invention

Based on the technical problems in the background art, the invention provides a drug combination mechanism of chlorogenic acid and paeoniflorin, and combines the chlorogenic acid and paeoniflorin with a hydrogel drug-loading system to prepare the drug-loading hyaluronic acid hydrogel dressing for diabetic skin healing.

The invention provides a hyaluronic acid hydrogel medical dressing containing chlorogenic acid and paeoniflorin, which comprises the following steps:

s1 deionized water of 20ml is selected, and then 0.5g sodium hyaluronate is dissolved in the deionized water of 20 ml;

s2, adding 3g of adipic acid dihydrazide after uniformly stirring the deionized water and the sodium hyaluronate, and then stirring and standing overnight to obtain a mixed solution;

s3, adjusting the pH value of the mixed solution to 4.7 by using HCl, adding 0.4g of carbodiimide hydrochloride, and fully stirring to ensure that the solution gradually becomes gel;

s4 washing the gel with dehydrated alcohol in an ultrasonic washer for 3 times, and washing with PBS in the ultrasonic washer for 3 times;

s5, freezing the cleaned gel, and freeze-drying in a freeze vacuum drying instrument to prepare a dry gel block for later use;

s6, swelling ratio measurement is carried out on the dried gel block;

s7 performing drug release assay on the dried gel mass;

s8, carrying out external enzymolysis enzyme degradation resistance measurement on the completely swollen hydrogel;

s9 dissolving chlorogenic acid and paeoniflorin in propylene glycol, dissolving the propylene glycol solution containing the dissolved medicine in PBS, and re-dissolving the dried product of hyaluronic acid hydrogel with the solution to obtain the composition: 8% hyaluronic acid hydrogel, 500 μ M chlorogenic acid, 500 μ M paeoniflorin, and 1% propylene glycol.

Preferably, the step S6 of measuring the swelling ratio of the dried gel block comprises the following specific steps:

s1 dividing the gel blocks into three groups, and weighing respectively;

s2 was swollen in phosphate buffer at pH 7.4;

s3, taking out the rubber blocks every 30min, carefully drying the surface moisture, and measuring the weight of the rubber blocks until the weight of the rubber blocks is unchanged;

s4 the swelling ratio SR is calculated according to the equation: SR ═ w/Wd, where Wd and Ws represent the mass of the hydrogel in the dry and swollen states, respectively.

Preferably, the step S7 of performing drug release measurement on the dried gel block specifically comprises the following steps:

s1, weighing the dried gel blocks, and respectively putting the gel blocks into 10mg/mL BSA solution for full swelling for 3 days to make the hydrogel reach swelling and adsorption balance;

s2, taking out the gel block, respectively measuring the volume of the residual BSA solution, measuring the BSA concentration in the BSA solution by using the Nanodrop, and calculating the drug loading rate of the hydrogel;

s3, putting the gel with the drug in 1mL of new PBS solution, and measuring the amount of BSA in the solution by Nanodrop every 2h to obtain the release curve of bovine serum albumin.

Preferably, the hydrogel release results over time are characterized by an integrated release equation: the cumulative amount of drug released (wt%) -Mt/M ∞ where Mt refers to the amount of BSA released from the hydrogel over time t and M ∞ refers to the total amount of BSA loaded into the hydrogel.

Preferably, the step S8 of performing the determination of the external enzymolysis resistance of the completely swollen hydrogel includes the following specific steps:

s1 putting the completely swollen hydrogel into 1mL of 100u/mL hyaluronidase solution;

s2 sampling every 24h, developing with carbazole, measuring absorbance at 530nm, performing linear regression with HAse concentration as abscissa and absorbance as ordinate, and calculating average slope of straight line.

According to the invention, the hyaluronic acid hydrogel medical dressing containing chlorogenic acid and paeoniflorin is prepared into a medical dressing capable of promoting wound healing from multiple aspects, the dressing can promote fibroblast proliferation, promote angiogenesis and reduce inflammatory reaction, has the characteristics of hemostasis and moisture retention, covers all aspects of wound healing, and has a wide application prospect.

Drawings

FIG. 1 is a schematic representation of the effect of chlorogenic acid and paeoniflorin on HUVECS angiogenesis;

FIG. 2 is a schematic diagram showing the effect of chlorogenic acid and paeoniflorin on macrophage inflammatory factor expression;

FIG. 3 is a schematic view of a scanning electron microscope (TEM) structure of the hyaluronic acid hydrogel of the present invention;

FIG. 4 is a graph showing the swelling ratio of the hyaluronic acid hydrogel of the present invention;

FIG. 5 is a schematic view showing the sustained release rate of the hyaluronic acid hydrogel of the present invention;

FIG. 6 is a schematic view showing the enzymatic hydrolysis resistance of a hyaluronic acid hydrogel of the present invention;

FIG. 7 is a diagram of the healing rate of the skin of diabetic rats.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.

Example one

Referring to fig. 1 to 7, a hyaluronic acid hydrogel medical dressing containing chlorogenic acid and paeoniflorin, comprises the following steps:

s1 deionized water of 20ml is selected, and then 0.5g sodium hyaluronate is dissolved in the deionized water of 20 ml;

s2, adding 3g of adipic acid dihydrazide after uniformly stirring the deionized water and the sodium hyaluronate, and then stirring and standing overnight to obtain a mixed solution;

s3, adjusting the pH value of the mixed solution to 4.7 by using HCl, adding 0.4g of carbodiimide hydrochloride, and fully stirring to ensure that the solution gradually becomes gel;

s4 washing the gel with dehydrated alcohol in an ultrasonic washer for 3 times, and washing with PBS in the ultrasonic washer for 3 times;

s5, freezing the cleaned gel, and freeze-drying in a freeze vacuum drying instrument to prepare a dry gel block for later use;

s6, swelling ratio measurement is carried out on the dried gel block;

s7 performing drug release assay on the dried gel mass;

s8, carrying out external enzymolysis enzyme degradation resistance measurement on the completely swollen hydrogel;

s9 dissolving chlorogenic acid and paeoniflorin in propylene glycol, dissolving the propylene glycol solution containing the dissolved medicine in PBS, and re-dissolving the dried product of hyaluronic acid hydrogel with the solution to obtain the composition: 8% hyaluronic acid hydrogel, 500 μ M chlorogenic acid, 500 μ M paeoniflorin, and 1% propylene glycol.

In the present invention, the specific steps of measuring the swelling ratio of the dried gel block in step S6 are as follows:

s1 dividing the gel blocks into three groups, and weighing respectively;

s2 was swollen in phosphate buffer at pH 7.4;

s3, taking out the rubber blocks every 30min, carefully drying the surface moisture, and measuring the weight of the rubber blocks until the weight of the rubber blocks is unchanged;

s4 the swelling ratio SR is calculated according to the equation: SR ═ w/Wd, where Wd and Ws represent the mass of the hydrogel in the dry and swollen states, respectively.

In the present invention, the step S7 of performing drug release measurement on the dried gel block specifically comprises the steps of:

s1, weighing the dried gel blocks, and respectively putting the gel blocks into 10mg/mL BSA solution for full swelling for 3 days to make the hydrogel reach swelling and adsorption balance;

s2, taking out the gel block, respectively measuring the volume of the residual BSA solution, measuring the BSA concentration in the BSA solution by using the Nanodrop, and calculating the drug loading rate of the hydrogel;

s3, putting the gel with the drug in 1mL of new PBS solution, and measuring the amount of BSA in the solution by Nanodrop every 2h to obtain the release curve of bovine serum albumin.

In the invention, the hydrogel release result along with time is characterized by an accumulative release equation: the cumulative amount of drug released (wt%) -Mt/M ∞ where Mt refers to the amount of BSA released from the hydrogel over time t and M ∞ refers to the total amount of BSA loaded into the hydrogel.

In the invention, the step S8 of performing the external enzymolysis resistance enzyme degradation determination on the completely swollen hydrogel comprises the following specific steps:

s1 putting the completely swollen hydrogel into 1mL of 100u/mL hyaluronidase solution;

s2 sampling every 24h, developing with carbazole, measuring absorbance at 530nm, performing linear regression with HAse concentration as abscissa and absorbance as ordinate, and calculating average slope of straight line.

Chlorogenic acid can obviously improve the cell activity of mouse fibroblast L929 and the cell activity of human umbilical vein epithelial cell HUVECS, while paeoniflorin has no obvious effect; when chlorogenic acid and paeoniflorin are co-administered, the proportion is not obviously optimal, and is related to the overall administration concentration; both chlorogenic acid and paeoniflorin can promote the migration of HUVECS of human umbilical vein epithelial cells, and can also promote the migration of HUVECS when being used; chlorogenic acid can promote migration of mouse fibroblast L929, paeoniflorin has no obvious effect, and L929 can be promoted to migrate when the chlorogenic acid is used continuously; chlorogenic acid (50 μ M) and paeoniflorin (50 μ M) can promote the formation of blood vessels on matrigel of HUVECS of human umbilical vein epithelial cells, and can also promote the angiogenesis of HUVECS when used together.

Example two

Preparing hyaluronic acid hydrogel, namely adding 0.2g of sodium hyaluronate (1800-2200kDa) into 20ml of deionized water, uniformly stirring, adding 1.4g of adipic dihydrazide, and stirring overnight; adjusting pH to 4.7 with HCl, adding 0.4g carbodiimide hydrochloride, stirring thoroughly, and gradually gelling the solution; washing the gel with deionized water in an ultrasonic cleaner, adjusting the pH value to 7 with NaOH, and precooling in a refrigerator at-20 ℃; freeze-drying in a freeze vacuum drier, and preparing into 8% hydrogel with deionized water.

EXAMPLE III

Cell viability detection, adjusting cell density to be 2 × 104/mL, adding 100 μ L into each well of a 96-well plate except the edge to enable the number of cells in each well to be 2000, shaking and mixing uniformly in a cross manner, and adding 200 μ L of culture medium into the edge well; incubating overnight in an incubator, after the cells are administrated in an adherent manner, adding the cell-inoculated well waste liquid and 1640 complete culture medium solution into wells in sequence from small to large in concentration, adding 180 mu L of 1640 culture medium into each well (5 pairs of wells are set, and one concentration gradient drug is added into each abandoned group), adding 180 mu L of 1640 culture medium containing 10% FBS into a control group, adding 50 mu L of DMSO into a blank group, and incubating for 24h in the incubator. Adding 20 mu of LMTT solution into each well of the dosing group, the control group and the blank group, and tapping the bottom of the plate to enable liquid drops on the wall to fall into the bottom of the well; incubating in an incubator for 4 h; the liquid in each well is sucked out by a syringe and discarded, and 150 microliter of DMSO is added into each well to dissolve the blue-violet formazan; incubating the microplate in a constant temperature oscillator at 37 ℃ and 500rpm for 10 min; detecting the absorbance at 570nm by using an ultraviolet visible spectrophotometer; the increment rate was [ OD administration group-OD blank ]/[ OD control group-OD blank ] × 100%.

Example four

Plate scratch test, 9, adjusting cell density to 4 × 105/mL, adding 1mL into each well of 6-well plate to make the number of cells per well 4 × 105, adding 1mL 1640 medium containing 10% FBS, and cross-shaking and mixing. Culturing in an incubator overnight, after the cells adhere to the wall, washing the cell waste liquid fully creeping on the bottom of the 6-hole plate with 1mL of PBS, and repeating for three times; a scratch is vertically and straightly scribed by using a 1mL gun head; washing with 1mL of PBS, repeating for three times, and washing off the exfoliated cells; respectively adding 2mL of drug solutions with different concentrations, adding 2mL of 1640 culture medium into a control group, wherein each concentration is three pairs of wells; after the incubator is cultured for 24 hours, observing and photographing under a mirror, taking a view field at the middle position, and measuring the width of a scratch by using a caliper of a microscope; mobility ═ W dose/W control × 100%.

EXAMPLE five

A new vessel induction experiment, namely freeze thawing of Matrigel on ice; precooling a gun head, precooling by an EP (EP) tube, uniformly mixing matrigel, taking out 1.2mL of the mixture, subpackaging, operating on ice in the whole process, and storing the rest at the temperature of minus 20 ℃; precooling a 24-pore plate, adding 200 mu L matrigel into each pore, and operating on ice, wherein bubbles are not required;

placing in an incubator for 45min until matrigel is solidified; taking HUVEC cells in logarithmic growth phase, digesting, centrifuging, and resuspending in 1mL 1640 culture medium containing 10% FBS; counting, adjusting the density to 1 × 10⁶Per mL; mu.L of matrigel-plated 24-well plates were added to make the number of cells per well 1X 10⁵A plurality of; adding drugs with different concentrations into the administration group, adding 100 μ L1640 complete culture medium into the control group, each group having 3 auxiliary holes, and gently shaking and mixing; after incubation in an incubator for 4h, the angiogenesis was observed under the microscope.

Example 6

SPF grade 4 week old C57/BL male mice were harvested for BMDM from the femur and tibia and cultured in high glucose DMEM with 10% FBS, 1% double antibody, 20ng/mL M-CSF, 5% CO2, 37 ℃. BMDM was induced to M1-type macrophages on day 5 of culture with high glucose DMEM containing 100ng/mL LPS, 20ng IFN-. gamma.and 20ng/mL M-CSF.

EXAMPLE seven

Establishing a full-thickness skin injury model of a rat, namely, breeding a male Wistar rat with the body weight of 220g at normal temperature in 6 weeks, alternately eating and drinking water day and night freely. Fasting for 12-16 h without water deprivation. Streptozotocin 55mg/kg body weight is injected into the abdominal cavity, the same volume of citrate buffer solution is injected into the abdominal cavity of the animals of the control group, the tail tip is cut off, blood is taken out, and the blood sugar concentration is measured. After 1 week, the diabetic rats showed polydipsia, polyphagia, polyuria and obvious weight loss, and the diabetic animals were listed as diabetic animals with the blood sugar value of more than or equal to 14 mmol/L. On the day of operation, the back of a rat is prepared with skin, washed with warm water and wiped up, 3mL/kg of 10% chloral hydrate is used for intraperitoneal injection anesthesia, a conventional disinfection drape is used, two wound surfaces with the diameter of 2cm are cut off at each side of the spinal column of the back by 1cm, the area of each wound surface is 3.14cm2, the distances of the wound surfaces are 2.0cm, the back has four wound surfaces, the wound surfaces reach the fascia layer deeply but do not damage subcutaneous vascular systems, and the incision is washed with normal saline to wipe off effusion. The wound surface is not bound after being manufactured, the rat is raised in a single-cage standard raising environment in an autoclave padding after being sobered, drinking water is freely taken, the drug corresponding to the wound is given to the rat every day after the model is built, the wound area is measured every day, the healing rate is calculated, the rat is killed in 3 rd, 7 th and 14 th days, a back healing tissue sample including 2mm of complete skin around the rat is taken, and the subsequent molecular biology experiment is carried out.

The invention comprises the following steps: selecting 20ml of deionized water, and then dissolving 0.5g of sodium hyaluronate in 20ml of deionized water; uniformly stirring deionized water and sodium hyaluronate, adding 3g of adipic dihydrazide, and then stirring and standing overnight to obtain a mixed solution; adjusting the pH value of the mixed solution to 4.7 by using HCl, adding 0.4g of carbodiimide hydrochloride, and fully stirring to ensure that the solution gradually becomes gel; washing the gel with dehydrated alcohol in an ultrasonic washer for 3 times, and washing with PBS in the ultrasonic washer for 3 times; freezing the cleaned gel, and freeze-drying in a freeze vacuum drying instrument to obtain dried gel blocks; measuring the swelling ratio of the dried gel block; performing a drug release assay on the dried gel mass; performing external enzymolysis enzyme degradation resistance determination on the completely swollen hydrogel; dissolving chlorogenic acid and paeoniflorin in propylene glycol, dissolving the propylene glycol solution in which the medicine is dissolved in PBS, and re-dissolving the dried hyaluronic acid hydrogel product with the solution to finally obtain a solution with the following components: 8 percent of hyaluronic acid hydrogel, 500 mu M of chlorogenic acid, 500 mu M of paeoniflorin and 1 percent of propylene glycol to prepare the medicine-carrying hyaluronic acid hydrogel.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.