CN102078636A - Hydrogel dressing containing recombinant human epidermal growth factor and preparation method and application thereof - Google Patents

Abstract

The invention discloses a hydrogel dressing containing recombinant human epidermal growth factor, which is composed of 100 parts by volume of mixed solution A, 2 to 8 parts by volume of a crosslinking agent, and the mass percentage is 2.0 to 5.0% polyvinyl alcohol aqueous solution and mass percent 0.5-2.0% sodium alginate aqueous solution is mixed according to the mass ratio (2-5): 1, and then 0-0.1% sodium benzoate, 5-20 μg/g recombinant human epidermal growth factor and 2.0% mass percentage are added ～10% glycerin to prepare mixed solution A, take 2～8 parts by volume of sodium borate solution and calcium chloride solution and mix the cross-linking agent obtained by dropping into 100 parts by volume of mixed solution A and let it stand for defoaming to obtain a gel dressing . The dressing of the invention has good biocompatibility, can be molded at room temperature, has mild and simple preparation conditions, can effectively maintain the activity of the recombinant epidermal growth factor, and has a controlled release effect on it. The dressing has a good therapeutic effect on wounds of diabetic rats and can accelerate wound healing.

Description

Hydrogel dressing containing recombinant human epidermal growth factor and its preparation method and application

technical field

The invention belongs to the field of biomedical engineering materials, and in particular relates to a hydrogel dressing containing recombinant human epidermal growth factor (rh-EGF) and a preparation method thereof, and its application as a medicine for treating diabetic foot chronic ulcer.

Background technique

Diabetic foot chronic ulcer seriously affects the life safety and quality of life of diabetic patients. It has a high incidence rate, great harm, and expensive treatment costs. Once a foot ulcer occurs, it is very difficult to treat. Studies have shown that decreased levels of growth factors are an important reason for delayed wound healing in diabetes. Since recombinant growth factors can accelerate wound healing, local application of exogenous growth factors is one of the commonly used clinical methods to accelerate the healing of diabetic foot ulcers. However, due to the short half-life of growth factors, they are easily degraded by matrix metalloproteinases (MMPs) when used directly on wounds; spray-type carriers are likely to cause the loss of growth factors, and lack of slow-release and sustained action functions, making local exogenous growth factors Application does not give desired results.

On the basis of traditional treatment, in recent years, a series of new medical dressings that have been proved by clinical observation to promote ulcer healing and avoid amputation have come out, further improving the prevention and treatment of diabetic foot.

The type and function of dressings for diabetic foot ulcers vary according to the type of wound and the stage of ulcer healing. Mainly include film (protect wound surface), foam (protect, fill, absorb), hydrogel (debridement, rehydration, moisturizing), hydrocolloid (debridement, protection), alginate (hemostasis, absorption), plus Medicinal dressing (with anti-bacterial function), etc. Among them, the hydrogel is processed by polymer materials, and the polymer liner is added with glue-linked polymers, which can not only absorb the exudate from the wound, but also keep the exudate in the dressing, so that the wound is slightly moist and slightly moist. acid and hypoxic environment, promote tissue repair and re-epithelialization; in recent years, it has been proven to have a significant healing effect on chronic ulcers.

Chinese patent CN1121876C (authorized announcement date September 24, 2003) and Yoshii et al. (RadiationPhysics and Chemistry, 1999, 55 (2): 1331) prepared polyvinyl alcohol composite hydrogel dressings by electron beam radiation crosslinking . However, the radiation process will lead to the denaturation and inactivation of biological agents, which limits the use of this method in the preparation of growth factor-containing dressings. Chinese patent application 200410051638.4 (published on July 6, 2005) and Chinese patent application 200510036465.3 (published on February 15, 2006) loaded basic fibroblast growth factor into carbomer to prepare gel preparations. However, the main component of the gel matrix is acrylic resin, which has the disadvantages of rapid drug release, difficult degradation, and poor biocompatibility. Therefore, it is of great significance to develop a special gel dressing for diabetic foot chronic ulcer with low price, simple preparation process and good biocompatibility. At present, there are no patents or articles reported on special dressings for diabetic foot chronic ulcers in China.

Contents of the invention

The primary purpose of the present invention is to overcome the shortcoming and deficiency of prior art, provide a kind of with sodium alginate and polyvinyl alcohol as main raw material, take calcium chloride and sodium borate as cross-linking agent, add recombinant human epidermal growth factor (rh -EGF) and preservative sodium benzoate special hydrogel dressing for diabetic foot chronic ulcers.

Another object of the present invention is to provide a method for preparing the above-mentioned hydrogel dressing containing recombinant human epidermal growth factor.

Another object of the present invention is to provide the application of the above-mentioned hydrogel dressing containing recombinant human epidermal growth factor.

The object of the present invention is achieved through the following technical solutions:

The hydrogel dressing containing recombinant human epidermal growth factor is prepared from the following ingredients in parts by volume:

Mixed solution A: 100 parts by volume

Crosslinking agent: 2 to 8 parts by volume;

The mixed solution A is prepared by the following method: at 10-40°C, polyvinyl alcohol (PVA) and sodium alginate (SA) are formulated into an aqueous solution, and 2.0-5.0% by mass of polyvinyl alcohol aqueous solution and mass percent Mix 0.5-2.0% sodium alginate aqueous solution according to (2-5): 1, stir evenly, then add 0-0.1% sodium benzoate and 5-20 μg/g recombinant human epidermal growth factor (rh-EGF) and glycerol with a mass percentage of 2.0% to 10%, to obtain a mixed solution A;

The cross-linking agent is prepared by mixing sodium borate aqueous solution with equal concentration and calcium chloride aqueous solution in equal volume ratio.

The mass fraction of the sodium borate aqueous solution and the calcium chloride aqueous solution is 1.0-2.0%.

The weight average molecular weight of the polyvinyl alcohol (PVA) is 120,000-150,000.

The viscosity of the 1% aqueous solution of sodium alginate is 200-500 mPa·s.

The recombinant human epidermal growth factor is commercially available from CYTOLAB/PEPROTECH ASIA.

The preparation method of the above-mentioned hydrogel dressing containing recombinant human epidermal growth factor comprises the following steps:

(1) At 10-40°C, prepare polyvinyl alcohol and sodium alginate into an aqueous solution, mix the polyvinyl alcohol aqueous solution and sodium alginate aqueous solution at a mass ratio of 1:4 to 4:1, stir evenly, and then Add sodium benzoate, rh-EGF and glycerol to obtain mixed solution A;

(2) At 10-40°C, take 2-8 parts by volume of the cross-linking agent and slowly drop them into 100 parts by volume of the mixed solution A under stirring conditions, and let it stand for defoaming to obtain recombinant human epidermal growth factor-containing Hydrogel dressing.

The obtained hydrogel dressing has the characteristic of shear thinning. Under the condition of 1.0s ^-1 shear rate, the shear viscosity range of dressings with different proportions is 1.2～10Pa·s (37℃); the elastic modulus ranges from 20～ 50Pa (37°C); when the dressing is left at room temperature for 2 weeks, the modulus changes by 0-6%, and the viscosity changes by 0-10%.

The rh-EGF hydrogel dressing had no significant changes in appearance, properties, odor, and pH value at different temperatures (4°C, 25°C, 37°C) and different storage times (1d, 7d, 14d).

The application of the above-mentioned hydrogel dressing containing recombinant human epidermal growth factor, and the application of the hydrogel dressing in the preparation of medicines for treating diabetic foot chronic ulcers.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

(1) The present invention uses sodium alginate and polyvinyl alcohol two kinds of good biocompatibility macromolecular materials as main raw materials, and adopts in-situ cross-linking technology to prepare a new type of special gel dressing for chronic diabetic foot ulcers, The dressing has good biocompatibility.

(2) The hydrogel dressing of the present invention can be molded at room temperature, the preparation conditions are mild, and the process is simple.

(3) The hydrogel dressing of the present invention can effectively maintain the activity of the recombinant epidermal growth factor, and has a controlled release effect on it.

(4) Animal experiments show that the hydrogel dressing of the present invention has a good therapeutic effect on diabetic rat wounds and can accelerate wound healing.

Description of drawings

Fig. 1 is the comparison diagram of the heart, liver and kidney slices of the rats in the high dose group and the normal control group, wherein, A is the heart slices of the rats in the high dose group, B is the liver slices of the rats in the high dose group, and C is the liver slices of the high dose group. Rat kidney slices, D: blank group rat heart slices, E: blank group rat liver slices, F: blank group rat kidney slices.

Figure 2 is a photo of the skin irritation reaction of rats in the intact skin group, where A is before gel administration, B is 48 hours after gel administration, C is 72 hours after gel administration, D is 7 days after gel administration, and E is gel administration. 14d after glue.

Figure 3 is a photo of the skin irritation reaction of rats in the damaged skin group, where A is before gel administration, B is 48 hours after gel administration, C is 72 hours after gel administration, D is 7 days after gel administration, and E is gel administration. 14d after glue.

Fig. 4 is a photograph of the sensitization reaction of rats in the test group, wherein A is before sensitization, B is 24 hours after sensitization, C is 48 hours after sensitization, and D is 72 hours after sensitization.

Fig. 5 is a graph showing the activity detection of rh-EGF after it is released from the dressing.

Figure 6 is a graph showing the effect of different drug treatments on wound healing in rats, where A is the blank control group, B is the hydrogel matrix group, C is the rh-EGF hydrogel group, and D is the rh-EGF aqueous solution group.

Detailed ways

The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

Example 1

The preparation method of the hydrogel dressing containing recombinant human epidermal growth factor comprises the following steps:

(1) At 40°C, polyvinyl alcohol (PVA) (Xilong Chemical Factory, Shantou City, Guangdong Province, molecular weight 12-150,000) was prepared into a 5.0% aqueous solution, and sodium alginate (Dongsheng Chemical Reagent Factory, Wenzhou City, Zhejiang Province, Medium viscosity) is mixed with 2.0% aqueous solution, then polyvinyl alcohol and sodium alginate aqueous solution are mixed by 4: 1 (mass ratio) and are made into the polyvinyl alcohol/sodium alginate mixed solution of 89.95g, stir well, then add sodium benzoate (SB) 0.05g, 1mg rh-EGF (purchased from CYTOLAB/PEPROTECH ASIA), glycerol 10g, mixed and dissolved to obtain mixed solution A;

(2) At 40°C, prepare sodium borate and calcium chloride into 1% solutions respectively, and mix them at a volume ratio of 1:1 to obtain a cross-linking agent. Take 4.0 mL of the cross-linking agent and slowly drop it into 100 mL of mixed solution A. , stir evenly, stand still to remove foam, and obtain the hydrogel dressing containing recombinant human epidermal growth factor.

The obtained dressing has the characteristics of shear thinning. Under the condition of 1.0s ^-1 shear rate, the shear viscosity of the dressing is 5.6Pa·s (37°C); the modulus of elasticity is 38Pa (37°C); the dressing After 2 weeks at room temperature, the elastic modulus changes by 4.2±0.3%, and the viscosity changes by 6.5±0.5%.

The steady-state rheological parameters (apparent viscosity, shear rate and shear stress) and dynamic rheological parameters (elastic modulus) of the hydrogel dressing matrix were measured using the ARES/RFS advanced rheological expansion system produced by TA Instrument Company of the United States. , viscosity modulus, etc.). A cylindrical mold is selected for testing. The outer diameter of the cylinder is 34.0mm, the inner diameter is 32.0mm, and the height of the cylinder is 33.0mm.

After adding rh-EGF, the appearance, properties, odor, and pH of the hydrogel did not change significantly at different temperatures (4°C, 25°C, 37°C) and storage times (1d, 7d, 14d).

Example 2

The preparation method of the hydrogel dressing containing recombinant human epidermal growth factor comprises the following steps:

(1) At 10°C, take polyvinyl alcohol (PVA) (Xilong Chemical Factory, Shantou City, Guangdong Province, molecular weight 120,000 to 150,000) to prepare a 2.0% aqueous solution, and get sodium alginate (Dongsheng Chemical Reagent Factory, Wenzhou City, Zhejiang Province, Medium viscosity) is mixed with 0.5% aqueous solution, then polyvinyl alcohol and sodium alginate aqueous solution are mixed by 5: 1 (mass ratio) and are made into the polyvinyl alcohol/sodium alginate mixed solution of 89.95g, stir well, then add sodium benzoate 0.03g, 1mg rh-EGF (purchased from CYTOLAB/PEPROTECH ASIA), 2.0g glycerin, mixed and dissolved to obtain mixed solution A;

(2) At 10°C, prepare sodium borate and calcium chloride into 2% solutions respectively, and mix them at a volume ratio of 1:1 to obtain a cross-linking agent. Take 2.0 mL of the cross-linking agent and slowly drop it into 100 mL of mixed solution A. , stir evenly, stand still to remove foam, and obtain the hydrogel dressing containing recombinant human epidermal growth factor.

The obtained dressing has the property of shear thinning. Under the condition of 1.0s ^-1 shear rate, the shear viscosity of the dressing is 2.4Pa·s (37°C); the elastic modulus is 30Pa (37°C); the dressing is left at room temperature for 2 weeks , the modulus of elasticity changes by 2.8±0.4%, and the viscosity changes by 3.8±0.7%.

After adding rh-EGF, the appearance, properties, odor, and pH of the hydrogel did not change significantly at different temperatures (4°C, 25°C, 37°C) and storage times (1d, 7d, 14d).

Example 3

The preparation method of the hydrogel dressing containing recombinant human epidermal growth factor comprises the following steps:

(1) At 25°C, take polyvinyl alcohol (PVA) to prepare a 3.0% aqueous solution, take sodium alginate to prepare a 1.0% aqueous solution, and then mix polyvinyl alcohol and sodium alginate aqueous solution at 2:1 (mass ratio) Prepare 89.95g of polyvinyl alcohol/sodium alginate mixed solution, stir evenly, then add 0.04g of sodium benzoate, 1mgrh-EGF (purchased from CYTOLAB/PEPROTECH ASIA), 8.0g of glycerin, mix and dissolve to obtain mixed solution A;

(2) At 25°C, prepare sodium borate and calcium chloride into 1.5% solutions respectively, and mix them at a volume ratio of 1:1 to obtain a cross-linking agent. Take 8.0 mL of cross-linking agent and slowly drop them into 100 mL of mixed solution A. , stir evenly, stand still to remove foam, and obtain the hydrogel dressing containing recombinant human epidermal growth factor.

The obtained dressing has the characteristics of shear thinning. Under the condition of 1.0s ^-1 shear rate, the shear viscosity of dressings with different ratios is 6.8Pa·s (37°C); the modulus of elasticity is 43Pa (37°C); After 2 weeks at room temperature, the elastic modulus changes by 2.9±0.5%, and the viscosity changes by 4.3±0.4%.

After adding rh-EGF, the appearance, properties, odor, and pH of the hydrogel did not change significantly at different temperatures (4°C, 25°C, 37°C) and storage times (1d, 7d, 14d).

Example 4

The preparation method of the hydrogel dressing containing recombinant human epidermal growth factor comprises the following steps:

(1) At 10°C, take polyvinyl alcohol (PVA) (Xilong Chemical Factory, Shantou City, Guangdong Province, molecular weight 120,000 to 150,000) to prepare a 2.0% aqueous solution, and get sodium alginate (Dongsheng Chemical Reagent Factory, Wenzhou City, Zhejiang Province, Medium viscosity) is mixed with 0.5% aqueous solution, then polyvinyl alcohol and sodium alginate aqueous solution are mixed by 5: 1 (mass ratio) and are made into the polyvinyl alcohol/sodium alginate mixed solution of 97.9g, stir well, then add sodium benzoate 0.1g, 0.5mg rh-EGF (purchased from CYTOLAB/PEPROTECH ASIA), glycerol 2.0g, mixed and dissolved to obtain mixed solution A;

(2) At 10°C, prepare sodium borate and calcium chloride into 2% solutions respectively, and mix them at a volume ratio of 1:1 to obtain a cross-linking agent. Take 2.0 mL of the cross-linking agent and slowly drop it into 100 mL of mixed solution A. , stir evenly, stand still to remove foam, and obtain the hydrogel dressing containing recombinant human epidermal growth factor.

The obtained dressing has the property of shear thinning. Under the condition of 1.0s ^-1 shear rate, the shear viscosity of the dressing is 2.4Pa·s (37°C); the elastic modulus is 30Pa (37°C); the dressing is left at room temperature for 2 weeks , the modulus of elasticity changes by 2.8±0.4%, and the viscosity changes by 3.8±0.7%.

After adding rh-EGF, the appearance, properties, odor, and pH of the hydrogel did not change significantly at different temperatures (4°C, 25°C, 37°C) and storage times (1d, 7d, 14d).

Example 5

The preparation method of the hydrogel dressing containing recombinant human epidermal growth factor comprises the following steps:

(1) At 25°C, take polyvinyl alcohol (PVA) to prepare a 3.0% aqueous solution, take sodium alginate to prepare a 1.0% aqueous solution, and then mix polyvinyl alcohol and sodium alginate aqueous solution at 2:1 (mass ratio) Prepare 92g of polyvinyl alcohol/sodium alginate mixed solution, stir evenly, then add 2mg of rh-EGF (purchased from CYTOLAB/PEPROTECH ASIA), 8.0g of glycerin, mix and dissolve to obtain mixed solution A;

(2) At 25°C, prepare sodium borate and calcium chloride into 1.5% solutions respectively, and mix them at a volume ratio of 1:1 to obtain a cross-linking agent. Take 8.0 mL of cross-linking agent and slowly drop them into 100 mL of mixed solution A. , stir evenly, stand still to remove foam, and obtain the hydrogel dressing containing recombinant human epidermal growth factor.

The obtained dressing has the characteristics of shear thinning. Under the condition of 1.0s ^-1 shear rate, the shear viscosity of dressings with different ratios is 6.8Pa·s (37°C); the modulus of elasticity is 43Pa (37°C); After 2 weeks at room temperature, the elastic modulus changes by 2.9±0.5%, and the viscosity changes by 4.3±0.4%.

After adding rh-EGF, the appearance, properties, odor, and pH of the hydrogel did not change significantly at different temperatures (4°C, 25°C, 37°C) and storage times (1d, 7d, 14d).

Embodiment 6 safety test

Test method: Select 20 adult, healthy rats with a weight of 200-300 g (provided by the Animal Experiment Center of Sun Yat-Sen University North Campus, the same below) with a weight of 200-300 g, with half male and half female, and the female should be nulliparous and sterile. According to the preliminary test and the data of structurally related compounds, it is predicted that the hydrogel dressing of the present invention will not produce toxicity, so 20 rats were divided into 2 groups, with 10 rats in each group, half male and half male. Group 1 is a high-dose group, and the hydrogel dressing prepared in Example 1 is given. Group 2 was the blank control group, without any drugs. 24 hours before the administration, the back of the rat was depilated mechanically, and the depilated area was about 20cm ² . After 24 hours of depilation, the hydrogel dressing prepared in Example 1 was evenly applied to the high-dose group rat back depilation area by a dose of 5g/kg, then covered with gauze and cellophane, and then fixed with non-irritating adhesive tape and bandage. The control group was not smeared and served as a blank control. After 24 hours, remove the residual test substance with warm water or a non-irritating solvent, and observe daily for 7 to 14 consecutive days. After administration, attention should be paid to the signs of systemic toxicity and death in animals, including changes in animal weight, skin, hair, eyes and mucous membranes. Changes in breathing, circulation, central nervous system, extremities, etc. In case of dead animals, autopsy and visual observation are required, and pathological examination is required when there are visible lesions. After the experiment, all the rats were sacrificed to take heart, liver and kidney tissues for pathological examination.

Test results: according to the heart, liver and kidney section comparison diagram of the high dose group and the normal control group rats in Fig. Good biosecurity.

Embodiment 7 Rat skin irritation test

Test method: Select 30 adult, healthy, skin-free rats with a body weight of 200-300 g (same source as above), half male and half female, and females should be nulliparous and sterile. Divide 30 rats into intact skin group, damaged skin group and control group, with 10 rats in each group, half male and half male. 24 hours before the test, the two sides of the back and spine were mechanically depilated, and the depilated area was about 20cm ² . For the mice in the damaged skin group, the skin of the depilated area was scratched with a sterilized injection needle until there was bleeding.

After 24 hours of depilation, the hydrogel dressing prepared in Example 1 was evenly applied to the depilatory areas on the back of the rats in the two test groups by a dose of 5g/kg, then covered with gauze and plastic wrap, and then fixed with non-irritating adhesive tape and bandage . The control group was not smeared and served as a blank control. After 24 hours, remove the residual test substance with warm water or a non-irritating solvent, and observe immediately. Then after 1h, 24h, 48h, and 72h, the irritation reaction of the administration site was observed again. Observe daily for 7 to 14 consecutive days.

The observation results of each rat were scored according to Table 1, and the average score was calculated, and then the stimulus intensity was evaluated according to Table 2.

Table 1 Skin irritation score

Average response = total score of erythema formation + total score of edema formation / total number of animals

Table 2 Evaluation of skin irritation intensity

Test results (see Table 3, Figure 2, Figure 3):

Table 3 The average value of skin irritation reaction scores of intact skin group and damaged skin group

Embodiment 8 rat skin sensitization test

Select 20 adult, healthy, skin-free rats with a body weight of 200-300 g (the same source as above), half male and half female, and the female should be nulliparous and sterile. Rats were randomly divided into 2 groups according to body weight, 10 in each group, half male and half male. The first group is the test group, which is given the hydrogel dressing prepared in Example 1, and the second group is the blank control group, which is not given any medicine.

①Sensitization exposure: 24 hours before the test, the rats in the test group were mechanically depilated on both sides of the back, and the depilated area was about 3cm×3cm. Get 0.1 g of the hydrogel dressing prepared in Example 1 and apply it to the depilated area on the right side of the back of the animal, cover it with gauze and plastic wrap, and then fix it with a non-irritating adhesive tape for 6 hours. On the seventh and fourteenth day, repeat in the same way.

②Stimulation contact: 14 days after the last hydrogel dressing sensitization, apply 0.1g of the test substance to the depilatory area on the left side of the back of the rats in the test group, remove the test substance after 6 hours, and observe immediately, and then at 24h, 48h, 72h to observe the skin allergic reaction again. Score according to Table 3 to determine the nature of the allergic reaction of the test substance to the skin. In order to reflect the sensitization intensity of the test substance, the sensitization rate can be judged according to the classification in Table 4. The sensitization rate can be judged according to the classification in Table 4.

Table 4 Scoring criteria for the degree of skin allergic reaction

Table 5 Skin sensitization evaluation label

Test results (see Table 6, Figure 4):

Table 6 Test group rat skin sensitization average value

The release mode and stability observation of embodiment 9rh-EGF in the hydrogel dressing

1. Prepare a hydrogel dressing by adding 10 μg of rh-EGF to each gram of the hydrogel dressing prepared in Example 1.

2. Put 5g of rh-EGF hydrogel dressing into a beaker containing 100ml of PBS solution by inversion method. 0.5ml was sampled at the same location for 24 hours, and the same amount of PBS at the same temperature was added after each sampling.

3. Use the double antibody sandwich ABC-ELISA method for concentration detection (kit source: Shanghai Senxiong Technology Industrial Co., Ltd.):

(1) Establish a standard curve: set standard wells with 8 wells, and add 100 μl of sample diluent (referring to the release product of the gel dressing in the medium in the above experiment (take a certain amount of release liquid and then dilute)) into each well 100μl of rh-EGF of known concentration was added to the first well, after mixing, 100μl was sucked out with a pipette, and transferred to the second well. Repeatedly do double dilution to the seventh well, and finally, suck out 100 μl from the seventh well and discard to make the volume 100 μl. The eighth well is a blank control.

(2) Adding samples: Add sample diluent to each hole of the sample to be tested and each sample obtained at 5min, 10min, 20min, 30min, 1hr, 1.5hr, 2hr, 4hr, 6hr, 8hr, 10hr, 12hr, 24hr. 50 μl.

(3) Mix the reaction plate thoroughly and place it at 37° C. for 120 minutes.

(4) Plate washing: fully wash the reaction plate 4-6 times with washing solution (mass fraction 0.05% Tween20-PBS), and print on filter paper to dry.

(5) Add 50 μl of primary antibody working solution (biotinylated anti-human EGF antibody) to each well, and place the reaction plate at 37° C. for 60 minutes.

(6) Plate washing: Same as before.

(7) Add enzyme-labeled antibody working solution (horseradish peroxidase-labeled Streptavidin 100 μl to each well.

(8) The reaction plate was left at 37° C. for 60 minutes.

(9) Plate washing: Same as before.

(10) Add 100 μl of substrate working solution (horseradish peroxidase substrate OPD) to each well, and react in the dark at 37°C for 5-10 minutes.

(11) Add 1 drop of stop solution (2mol/L sulfuric acid) to each well and mix well.

(12) Measure the absorbance at 492nm.

4. Calculate the cumulative release rate. Cumulative release rate = actual measured concentration / theoretical complete release concentration * 100%

5. Tested 1d, 7d, and 14d after the rh-EGF hydrogel was made, and observed the rh-EGF hydrogel at different temperatures (4°C, 25°C, 37°C) and different storage times ( 1d, 7d, 14d) changes in appearance, properties, odor, and pH value to evaluate the stability of the rh-EGF hydrogel.

6. Repeat the experiment 2 times.

Test results (see Table 7):

Table 7 rh-EGF hydrogel stored for different time after 24 hours cumulative release rate (%)

2. The rh-EGF hydrogel has no obvious changes in appearance, properties, odor and pH value at different temperatures (4°C, 25°C, 37°C) and different storage times (1d, 7d, 14d).

Activity detection after the release of embodiment 10rh-EGF in the hydrogel dressing

experiment method:

1. Take respectively 13 time points of 1d, 7d, and 14d in the above-mentioned embodiment 7, and add 0.4 ml of release solution into 3.6 ml of DMEM medium (Sigma-Aldrich Company) containing 10% (volume fraction) calf serum (containing rh -EGF 1～10ng/ml), filtered with a 0.22μm filter for later use.

2. Balb/c3T3 cells (from the Cell Bank of the Chinese Academy of Sciences) were seeded in 96-well plates after passage.

3. Take 200 μl of the above-mentioned spare release solution (that is, the release solution obtained at 5min, 10min, 20min, 30min, 1hr, 1.5hr, 2hr, 4hr, 6hr, 8hr, 10hr, 12hr, and 24hr) and add the inoculated Balb/c3T3 In the 96-well plate of the cells, a positive control group was set at the same time (the medium contained rh-EGF standard 1-64ng/ml, respectively 1ng/ml, 4ng/ml, 16ng/ml, 64ng/ml, each concentration was set 3 wells) Negative control group (medium without rh-EGF) each with 12 wells. Incubate for 48 hours at 37°C in 5% CO ₂ environment.

4. MTT method was used to detect activity at a wavelength of 492nm: After 48 hours of cell culture, 20 μl of MTT (5g/L) was added, and the culture was continued for 4 hours. The supernatant was removed, 150 μl of DMSO was added, and the absorbance was detected at a wavelength of 492nm.

Test result is shown in Fig. 5, and Fig. 5 is the activity detection result of rh-EGF after releasing in the dressing of embodiment 1, and experimental group (the release solution of the rh-EGF hydrogel after storing 1d, 7d, 14d is respectively 1 day group , 7-day group, 14-day group), the average activity (absorbance) of positive control group and negative control group were 0.90±0.10, 0.86±0.10, 0.80±0.14, 0.87±0.15, 0.55±0.10 respectively. The activities of the experimental group and the positive control group to promote the proliferation of BALB/c3T3 were higher than those of the negative control group (p<0.05). It shows that the dressing matrix can effectively maintain the activity of rh-EGF.

Example 11 Containing rh-EGF dressings to treat wounds in diabetic rats

experiment method:

Fifty-six clean-grade SD rats (purchased from the Experimental Animal Center of Guangzhou University of Traditional Chinese Medicine) with a weight of 250-300 g were taken, and after adaptive feeding for 1 week, 35 mg/kg STZ was injected intraperitoneally for 3 consecutive days to induce diabetes. Three days after the injection, blood was collected from the tail vein to detect the random blood glucose level. If the random blood glucose level of the rat was ≥16.7mmol/L, the diabetes model was considered successful.

Select the diabetic rats with successful modeling to continue feeding. During the feeding period, they can eat and drink freely, monitor their body weight and blood sugar every week, and inject 1-2U/d of low protamine insulin (NPH) subcutaneously according to the blood sugar level, and keep the blood sugar at 16.7mmol/L ~28mmol/L.

Diabetic rats were anesthetized by intraperitoneal injection of pentobarbital 30 mg/kg for 6 weeks, the back was clipped, 75% alcohol was disinfected, and a sterilized template with a side length of 1.3 cm was used as a sign to place a scalpel on the middle of the back of the rat and 1 cm behind the skull. A 1.3cm×1.3cm square wound was formed at the center of the wound, and the excision range was as deep as the fascia. After the wound was formed, each rat was reared in a single cage.

The experimental animals were divided into 4 groups according to the wound treatment factors (see Table 8). Blank control group: no treatment; hydrogel matrix group: external hydrogel matrix ("hydrogel matrix" means that the hydrogel dressing made in Example 1 does not contain rh-EGF) 0.5g/each Wound surface; rh-EGF hydrogel group: rh-EGF hydrogel 0.5g/every wound (containing rh-EGF 5 μg) that external application embodiment 1 makes every day; rh-EGF aqueous solution group: daily external application rh-EGF aqueous solution 0.5ml/each wound (containing rh-EGF 5μg).

Table 8 Grouping of 28 modeled male SD diabetic rats

Respectively after the wound formation (while measuring the size of the wound with medication) 0d, 3d, 5d, 7d, 10d, 14d, the wound was photographed with a digital camera (A610, Canon), and a graduated ruler was placed and marked with the mouse Number. Wound area was calculated with Image J (version 1.36b).

Each measurement was repeated twice, and the average was taken.

The test results are shown in FIG. 6 , comparing the wound healing rates, it was found that the difference in the healing rates among the four groups was statistically significant (p<0.05). The healing rate of the rh-EGF hydrogel group was the highest at each time point. The healing rate of the hydrogel treatment group (B, C) was higher than that of the blank control group on the 3rd and 5th day after wound formation (p<0.05); the healing rate of the hydrogel treatment group (B, C) was better than that of the non-hydrogel treatment group on the 10th day after wound formation (A, D) (p<0.05); on the 14th day, the three treatment groups were all better than the blank control group (p<0.05). Dressings containing rh-EGF can rapidly and effectively promote wound healing in diabetic rats (see Table 9).

％)4 groups of healing rates at different times in table 9 (

%)

Note: *Compared with group A, p<0.05; △Compared with group D, p<0.05.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (6)

1. contain the aerogel dressing of recombinant human epidermal growth factor, it is characterized in that: the composition by following volume parts prepares:

Mixed solution A: 100 parts by volume

Cross-linking agent: 2～8 parts by volume;

Described mixed solution A, prepare by the following method: under 10～40 ℃, with mass percent 2.0～5.0% polyvinyl alcohol water solutions and mass percent 0.5～2.0% sodium alginate aqueous solution according to mass ratio (2～5): 1 mixes, stir, add the sodium benzoate of mass percent 0～0.1%, the recombinant human epidermal growth factor of 5～20 μ g/g and the glycerol of mass percent 2.0～10% then, obtain mixed solution A.

2. according to the described aerogel dressing that contains recombinant human epidermal growth factor of claim 1, it is characterized in that: described cross-linking agent is mixed by isocyatic sodium borate aqueous solution and calcium chloride water equal-volume ratio;

The mass fraction of described sodium borate aqueous solution and calcium chloride water is 1.0～2.0%.

3. according to the described aerogel dressing that contains recombinant human epidermal growth factor of claim 1, it is characterized in that: the weight average molecular weight of described polyvinyl alcohol is 12～150,000.

4. according to the described aerogel dressing that contains recombinant human epidermal growth factor of claim 1, it is characterized in that: described sodium alginate 1% viscosity in aqueous solution is 200～500mPas.

5. the described preparation method that contains the aerogel dressing of recombinant human epidermal growth factor of claim 1 is characterized in that may further comprise the steps:

Under (1) 10～40 ℃, polyvinyl alcohol, sodium alginate are mixed with aqueous solution, with mass percent 2.0～5.0% polyvinyl alcohol water solutions and mass percent 0.5～2.0% sodium alginate aqueous solution according to mass ratio (2～5): 1 mixes, stir, add sodium benzoate, rh-EGF and glycerol according to proportioning then, obtain mixed solution A;

Under (2) 10～40 ℃, the cross-linking agent of getting 2～8 parts by volume under stirring condition slowly splashes in the mixed solution A of 100 parts by volume, leaves standstill de-bubble, promptly gets the aerogel dressing that contains recombinant human epidermal growth factor.

6. the described application of aerogel dressing in preparation treatment diabetic foot chronic ulcer medicine that contains recombinant human epidermal growth factor of claim 1.

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