Preparation method of human plasma protein methacryloyl frozen gel for promoting healing of diabetic wounds and product of human plasma protein methacryloyl frozen gel

Abstract

The invention discloses a preparation method of human plasma protein methylacryloyl frozen gel for promoting healing of diabetic wounds and a product of the human plasma protein methylacryloyl frozen gel, the preparation method comprises the following steps: (1) carrying out reaction on thawed human plasma protein and methacrylic anhydride, and after the reaction is ended, carrying out post-treatment to obtain an intermediate product; (2) dissolving the intermediate product, an initiator and a catalyst in a PBS buffer solution, and uniformly mixing to obtain a precursor solution; and (3) injecting the precursor solution prepared in the step (2) into a mold, and performing low-temperature freezing to obtain the human plasma protein methacryloyl frozen gel for promoting healing of the diabetic wound. The frozen gel is synthesized by taking the human plasma protein as a raw material for the first time, the plasma protein is not denatured in the preparation process, and the prepared frozen gel has excellent mechanical properties, water absorption, degradability and biocompatibility and has a remarkable promotion effect on healing of diabetic wounds.

Description

Technical Field

[0001] The present invention relates to the technical field of gels, and specifically relates to a preparation method and a product of a human plasma protein methacryloyl cryogel for promoting the healing of diabetic wounds. Background Art

[0002] Diabetic wounds are a major complication of diabetes, associated with increased morbidity, high incidence, and socioeconomic burden. Clinical studies have shown that peripheral neuropathy, inflammatory dysfunction, reduced angiogenesis, and uncontrolled bacterial infections are related to the pathogenesis of diabetic wounds and lead to the damaged or even non-healing state of diabetic wounds. Therefore, due to the complexity of diabetic wounds, there is an urgent need to develop a comprehensive treatment strategy.

[0003] Currently, the treatment of diabetic wounds mainly focuses on methods such as blood glucose control and debridement. However, due to the high treatment cost, complex operation, and easy infection, patients still face great economic and mental pressure. Although wound dressings are effective treatment methods, traditional dressings can only isolate the wound, cannot effectively promote healing, and cause pain to patients during replacement. An ideal wound dressing should have good breathability, antibacterial property, anti-inflammatory property, and antioxidant property.

[0004] To effectively control wound healing and infection, it has become an urgent task to develop wound dressings with multifunctionality and high performance. Recently, wound dressings including electrospun nanofibers, sponges, and hydrogels have been designed for wound healing. Among them, hydrogel is a three-dimensional hydrophilic polymer network, and its structure is similar to the extracellular matrix. Therefore, hydrogels have great application potential in tissue engineering, drug delivery, and wound dressings. For example, a Chinese patent document with the application publication number of CN 118892573 A discloses a preparation method of a pH-responsive dual-release hydrogel for promoting the healing of diabetic wounds, including: (1) preparing oxidized sodium alginate; (2) preparing astilbin liposomes; (3) obtaining an OSA@AL solution by preparing an oxidized sodium alginate solution with distilled water and adding astilbin liposomes; dissolving carboxymethylated chitosan and diclofenac sodium in distilled water to prepare a CMCS@DS solution; mixing the above two solutions in equal volume and preparing an ALD hydrogel through a Schiff base reaction. This technical solution can achieve the spatial and temporal controlled release of drugs, enabling the hydrogel to play a role in all four stages of wound healing, thereby accelerating the healing of diabetic wounds. However, most of the raw materials used in this technical solution are raw materials with antioxidant functions, but their effects on promoting tissue regeneration and cell growth are limited, and the wound healing rate still needs to be further improved. Summary of the Invention

[0005] Based on the above-mentioned defects existing in the prior art, the present invention discloses a preparation method of a methacryloyl cryogel of human plasma protein for promoting diabetic wound healing. For the first time, a cryogel is synthesized with human plasma protein as the raw material. During the preparation process, the plasma protein will not denature. The prepared cryogel has excellent mechanical properties, water absorption, degradability and biocompatibility, and this cryogel has a significant promoting effect on diabetic wound healing.

[0006] The specific technical solution is as follows:

[0007] A preparation method of a methacryloyl cryogel of human plasma protein for promoting diabetic wound healing, comprising:

[0008] (1) React the thawed human plasma protein with methacrylic anhydride, and after the reaction is terminated, perform post-treatment to obtain an intermediate product, denoted as PlasmaMA;

[0009] (2) Dissolve the PlasmaMA, initiator and catalyst prepared in step (1) in PBS buffer solution, and mix evenly to obtain a precursor solution;

[0010] (3) Inject the precursor solution prepared in step (2) into a mold, and obtain the methacryloyl cryogel of human plasma protein for promoting diabetic wound healing after low-temperature freezing.

[0011] For the first time, the present invention uses human plasma protein as the raw material. After reacting with methacrylic anhydride (MMA), the amino group on the human plasma protein is converted into an amide bond, and a C═C double bond is introduced into the human plasma protein. Then, an initiator and a catalyst are added, and a methacryloyl cryogel of human plasma protein is polymerized at low temperature.

[0012] It has been found through experiments that compared with hydrogels, cryogels have higher mechanical strength and pressure resistance, can provide a physical barrier for wounds, and reduce the irritation or contamination of the wound by the external environment. Cryogels also have good water absorption capacity, can absorb exudate, and reduce the risk of wound infection. At the same time, cryogels can keep the wound moist, contribute to wound healing, and also avoid the maceration phenomenon caused by excessive moisture.

[0013] Through further comparative experiments, the methacryloyl cryogel of human plasma protein prepared by the present invention has more excellent compressive capacity and wound healing promotion ability compared with the cryogel prepared with bovine serum albumin as the raw material.

[0014] Preferably, in step (1):

[0015] The molar ratio of methacrylic anhydride to human plasma protein is (1-2.5):1;

[0016] More preferably, the molar ratio is 2.2:1.

[0017] Preferably, in step (1):

[0018] During the reaction process, the pH value of the reaction solution is maintained at 7.5 - 8.0;

[0019] The reaction temperature is room temperature.

[0020] Preferably, in step (1):

[0021] The post-treatment includes dialysis, filtration and freeze-drying;

[0022] The intermediate product after freeze-drying is stored at no less than -20 °C for later use.

[0023] Preferably, in step (2):

[0024] The initiator is selected from one or more of ammonium persulfate, sodium persulfate, potassium persulfate;

[0025] The catalyst is selected from one or more of tetramethylethylenediamine, N,N-dimethylethylenediamine, triethylenetetramine, ethylenediamine.

[0026] More preferably, the initiator is selected from ammonium persulfate (APS), and the catalyst is selected from tetramethylethylenediamine (TEMED).

[0027] Preferably, in step (2):

[0028] In the precursor solution, the concentration of PlasmaMA is 1 - 15 g / 100 mL;

[0029] More preferably, the concentration of PlasmaMA is 2 - 4 g / 100 mL.

[0030] It has been found through experiments that when the concentration of PlasmaMA is low, the storage stability of the prepared cryogel is poor, and degradation will occur after being placed in PBS buffer at room temperature for several days; when the concentration of PlasmaMA is too high, the mechanical strength of the prepared cryogel is too large, resulting in a decrease in compressive capacity.

[0031] Preferably, in step (2):

[0032] In the precursor solution, the concentration of the initiator is (0.4 - 1.0)% μL / μL;

[0033] In the precursor solution, the concentration of the catalyst is (0.1 - 0.4)% μL / μL.

[0034] More preferably, the concentration of the initiator is 0.5% μL / μL; the concentration of the catalyst is 0.1% μL / μL. Under this ratio, the shape, texture and state of the cryogel prepared are more stable, and the initiator and reaction catalyst have less toxicity to cells and can be used for long-term cell treatment.

[0035] To improve the uniformity of the low-temperature polymerization reaction, preferably, in step (2), PlasmaMA is first added to the PBS buffer solution and mixed evenly, and then the initiator and catalyst are added.

[0036] Preferably, in step (3), the temperature of the low-temperature freezing is -80 to -20 °C.

[0037] It has been found through experiments that with the adjustment of the temperature of the low-temperature freezing, the pore structure of the prepared cryogel will be affected.

[0038] The present invention also discloses a human plasma protein methacryloyl cryogel for promoting diabetic wound healing prepared according to the above method.

[0039] Compared with the prior art, the present invention has the following advantages:

[0040] The present invention first synthesizes a cryogel using human plasma protein as a raw material. During the preparation process, the plasma protein will not denature. The prepared cryogel has excellent mechanical properties, water absorption, degradability and biocompatibility; and by adjusting the raw material ratio, parameters such as the pore size length, swelling rate, porosity and residue rate of the prepared cryogel can be regulated to meet different application requirements.

[0041] Through further tests, it is found that the human plasma protein methacryloyl cryogel prepared by the present invention can release a large amount of growth factor VEGF, which can significantly affect the growth ability of cells and promote cell growth and proliferation; it can promote cell migration more strongly and has excellent antioxidant ability. This cryogel has a significant promoting effect on diabetic wound healing. Description of the Drawings

[0042] Figure 1 It is a diagram showing the determination result of the methacrylation degree of PlasmaMA prepared in Example 1. (A) Diagram is the 1 1H-NMR spectrum of the raw material Plasma and the intermediate product PlasmaMA samples; (B) Diagram is the substitution degree of the intermediate product PlasmaMA sample calculated by TNBS; (C) Diagram is the methacrylation degree of the intermediate product PlasmaMA sample calculated by TNBS;

[0043] Figure 2 It is the secondary structure diagram of the raw material Plasma and the prepared PlasmaMA sample used in Example 1;

[0044] Figure 3 Swelling rate graph (A) and porosity graph (B) of the human plasma protein methacryloyl cryogel prepared in Example 1;

[0045] Figure 4 Residual rate curves of the human plasma protein methacryloyl cryogel prepared in Example 1 placed in trypsin-EDTA and PBS buffer respectively;

[0046] Figure 5 Cyclic compression performance graphs of the cryogels prepared in Example 1 and Comparative Example 1 respectively;

[0047] Figure 6 Swelling rate (A), porosity (B), residual rate (C) and pore diameter length (D) graphs of the human plasma protein methacryloyl cryogels prepared in Examples 1 to 3 respectively;

[0048] Figure 7 Lyophilized gel photos (a) and infiltrated gel photos (b) of the cryogels prepared in Examples 1 to 3 respectively;

[0049] Figure 8 Cumulative release of growth factor VEGF (A), PCR experimental results of growth factor VEGF (B), PCR experimental results of growth factor EGF (C), live / dead staining experiment (D), cell proliferation experiment (E) and scratch experiment (F) carried out in the environment of the PlasmaMA cryogel prepared in Example 1 and the BSAMA cryogel prepared in Comparative Example 1 respectively;

[0050] Figure 9 Results graph of ABTS in vitro scavenging ability determination (A) and results graph of flow cytometry experiment carried out under hydrogen peroxide stimulation in 3D cell culture (B) in the environment of the human plasma protein methacryloyl cryogel prepared in Example 1 and the BSAMA cryogel prepared in Comparative Example 1;

[0051] Figure 10 Photos of diabetic wounds (A) and wound healing ratio graphs (B) after treatment with the PlasmaMA cryogel prepared in Example 1, the BSAMA cryogel prepared in Comparative Example 1 and the control group respectively for different times;

[0052] Figure 11The diabetic wound sections after treatment with the PlasmaMA cryogel prepared in Example 1, the BSAMA cryogel prepared in Comparative Example 1, and the control group for different times were stained, including HE staining and Masson staining (A); and the wound length on the 14th day analyzed according to the HE sections (B) and the collagen deposition area on the 14th day analyzed according to the Masson sections (C). Detailed implementation mode

[0053] The present invention will be further described in detail below in conjunction with the drawings and embodiments. It should be noted that the following embodiments are intended to facilitate the understanding of the present invention and do not limit it in any way.

[0054] Example 1

[0055] 1. Thaw 100 mL of human plasma protein (Plasma, pH = 7.4 ± 0.1, Kejing Biotech) at 4°C, and perform magnetic stirring (600 rpm) at room temperature. After uniform mixing, add 1.521 mL of methacrylic anhydride (MAA, 94%; Sigma Aldrich). The molar ratio of MAA to Plasma is 2.2:1. At room temperature, react under magnetic stirring (600 rpm). During the reaction, maintain the pH of the reaction system at around 8 by adding 5M NaOH aqueous solution. React for 4 h until the reaction is complete. Filter the solution using a 70 mm filter paper, and then dialyze it in distilled water for 4 hours at room temperature using a tangential flow filtration (TFF) system equipped with a Pellicon 2 cassette containing a 10 kDa Biomax membrane to remove unreacted MAA and methacrylic acid by-products. Finally, the collected solution was frozen at -20°C for 3 hours and then freeze-dried at -80°C for 4 days to obtain PlasmaMA, which was stored at -20°C for later use.

[0056] PlasmaMA is synthesized by the direct reaction between MAA and the free lysine amino group in the Plasma molecule. The degree of methacrylation (DM) and substitution degree (DS) in the PlasmaMA sample are quantitatively determined by the 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay method.

[0057] Take the raw material Plasma and PlasmaMA samples, dissolve them in 0.1M NaOH aqueous solution at a concentration of 1.6mg / mL. At the same time, prepare glycine standard sample solutions (0, 1, 2, 4, 8, 16, 32, 64μg / mL) for making the standard curve. After the solutions are completely dissolved, take 0.25mL of each solution and add them into the corresponding wells respectively. Add 0.25mL of 0.1% TNBS (P2297, sigma) solution to each well and incubate at 37°C for 2h. After 2h, add 0.25mL of 1M HCl aqueous solution and 0.25mL of 10% w / v SDS aqueous solution to each well, and measure the absorbance at 335nm. The degree of substitution and the degree of methacrylation can be calculated by substituting into the standard curve.

[0058] Figure 1 Figure for the determination result of the methacrylation degree of PlasmaMA prepared in this example. (A) shows the 1 1H-NMR spectra of the raw material Plasma and the intermediate product PlasmaMA samples; (B) shows the degree of substitution DS% ~ 89.5% of the intermediate product PlasmaMA sample calculated by TNBS; (C) shows the degree of methacrylation DM ~ 57.3mmol / g of the intermediate product PlasmaMA sample calculated by TNBS; in (A), a is the acrylic proton of the methacrylamide graft (about 5.4ppm and 5.7ppm), b is the methylene proton of the unreacted lysine group (about 3.0ppm); c is the methyl proton of the methacrylamide graft (about 1.9ppm).

[0059] Use circular dichroism (CD) spectroscopy to evaluate the secondary structures of Plasma and PlasmaMA samples. The results are as Figure 2 shown. The CD spectrum of natural Plasma has two negative bands at 208nm and 222nm, which are typical characteristics of α-helical structure proteins. PlasmaMA also has typical protein characteristics. In addition, the percentages of α-helix of natural Plasma and PlasmaMA samples are further calculated and listed in Table 1 below. All of the above indicate that the PlasmaMA sample obtained through the preparation process in the present invention has not denatured.

[0060] Table 1

[0061]

[0062] 2. Dissolve the freeze-dried PlasmaMA in phosphate buffer (PBS; pH = 7.4; Gibco, Life), then add APS (R113155, 10 wt%, Rhawn) and TEMED (R007178, 20 vol%, Rhawn), and mix evenly to obtain a precursor solution; the concentration of PlasmaMA in the precursor solution is 3 g / 100 mL, the concentration of APS is 0.5% μL / μL, and the concentration of TEMED is 0.1% μL / μL.

[0063] 3. Inject the precursor solution into a silicone mold pre-cooled at -20 °C, and freeze it at -20 °C for 2 days to obtain a human plasma protein methacryloyl cryogel, denoted as 3% PlasmaMA.

[0064] Transfer the PlasmaMA cryogel prepared in this example to a freeze-dryer at -80 °C and freeze-dry it for 1 day to obtain a dried gel. At 37 °C, first weigh the original mass of the dried gel, denoted as M 0 , then place the dried gel in PBS buffer (pH = 7.4), and take out the dried gel swollen in PBS buffer at different set time points (15 min, 30 min, 45 min, 60 min, 90 min, 120 min, 180 min, 240 min), use lint-free paper to absorb the moisture on the surface of the dried gel and weigh it, denoted as M t . The swelling ratio calculation formula is as follows:

[0065] Swelling ratio (%) = (M t - M 0 ) / M 0 ×100%, and the results are as shown in Figure 3 A. In this invention, all data detections are carried out with 3 samples as a group, and the obtained data takes the average value of 3 samples.

[0066] Place the PlasmaMA cryogel prepared in this example in PBS buffer (pH = 7.4) and swell it for 1 day. The next day, use lint-free paper to absorb the surface moisture and weigh it, denoted as W t , then place the PlasmaMA cryogel on paper, continuously replace the new paper during this period, wait until the external water of the PlasmaMA cryogel is basically removed, and weigh the dehydrated PlasmaMA cryogel, denoted as W 0 . The porosity calculation formula is as follows:

[0067] Porosity (%) = (W t - W 0 ) / W t ×100%, and the results are as shown in Figure 3 B.

[0068] It was observed that the PlasmaMA cryogel prepared in this example exhibited good performance in absorbing and storing liquids, with a swelling ratio reaching 1750 ± 50%; and a porosity as high as 89 ± 1%.

[0069] After the PlasmaMA cryogel prepared in this example reached equilibrium swelling in PBS buffer (pH = 7.4), the wet weight was measured and denoted as S 0 . Then the PlasmaMA cryogel was placed in 0.01% trypsin-EDTA solution and PBS buffer (pH = 7.4) at 37°C respectively. It was taken out every hour, the surface moisture was blotted with lint-free paper and weighed, denoted as S t . The enzyme solution or PBS buffer was changed every 2 h. The remaining rate calculation formula is as follows:

[0070] Remaining rate (%) = S t / S 0 × 100%, and the results of the remaining rate in trypsin and in PBS buffer are shown in the left and right figures in Figure 4 respectively.

[0071] It can be seen from observing this figure that the cryogel prepared by the present invention has better stability and can stably exist in PBS buffer.

[0072] Comparative Example 1

[0073] 1. Dissolve BSA (V900933; Sigma - Aldrich) at 10 g / 100 mL in 200 mL of 0.25 M carbonate - bicarbonate (CB) buffer (14.65 g of sodium bicarbonate and 21.53 g of sodium carbonate decahydrate in 1 L of distilled water), and perform magnetic stirring (500 rpm) at 37 °C. After the BSA is completely dissolved, use 5 M aqueous sodium hydroxide solution (NaOH; Sigma - Aldrich) to adjust the pH of the buffer solution to 9. Then add 3.116 mL of methacrylic anhydride (MAA, 94%; Sigma Aldrich) to the BSA / CB buffer solution, and carry out the reaction under magnetic stirring (500 rpm) at 37 °C. During the process, maintain the pH of the reaction system at 7.5 by adding 5 M NaOH solution. After reacting for 1 h, terminate the reaction by adjusting the pH of the solution to 7.4 using 6 M hydrochloric acid solution (HCl; Sigma - Aldrich) or 5 M NaOH solution. Filter the solution successively through a 70 - millimeter filter paper, and then dialyze it in distilled water for 4 - 6 hours at room temperature using a tangential flow filtration (TFF) system equipped with a Pellicon 2 cassette containing a 10 kDa Biomax membrane to remove unreacted MAA and methacrylic acid by - products. Finally, the collected solution is frozen at - 20 °C for 3 hours and then freeze - dried at - 80 °C for 4 days to obtain BSAMA, which is stored at - 20 °C for later use.

[0074] Steps 2 - 3 are exactly the same as those in Example 1.

[0075] The bovine serum albumin cryogel prepared in this comparative example is denoted as BSAMA cryogel.

[0076] The cyclic compression properties of the human plasma protein methacryloyl cryogel prepared in Example 1 and the BSAMA cryogel prepared in Comparative Example 1 were studied using a dynamic mechanical analyzer (DMA; Q800; TA Instrument) in the controlled - force mode. The data are shown in Figure 5 .

[0077] It was observed that the PlasmaMA cryogel prepared in this example exhibited good compression recovery and mechanical stability. When continuously loaded / unloaded 10 times at 80% strain, there was almost no stress reduction. Compared with the BSAMA cryogel prepared in Comparative Example 1, the PlasmaMA cryogel prepared in the present invention had a greater stress, higher compressive capacity, and better stability.

[0078] Example 2

[0079] The preparation process is basically the same as that of Example 1, except that in Step 2, the concentration of PlasmaMA in the precursor solution is replaced with 2 g / 100 mL, denoted as 2% PlasmaMA.

[0080] Example 3

[0081] The preparation process is basically the same as that of Example 1, except that in Step 2, the concentration of PlasmaMA in the precursor solution is replaced with 4 g / 100 mL, denoted as 4% PlasmaMA.

[0082] The human plasma protein methacryloyl cryogels prepared in Examples 1 to 3 were tested respectively, specifically including measurement of the swelling ratio, porosity, residual ratio, and pore size length. The measurement results are as Figure 6 shown, and the specific data are listed in Table 2 below.

[0083] Table 2

[0084]

[0085] Among them, for the test of the pore size length, the PlasmaMA cryogel prepared in each example was placed in PBS buffer (pH = 7.4) to reach equilibrium swelling and then taken out, rhodamine staining agent was added for staining. After staining, the scaffold structure was photographed by a high-resolution laser confocal microscope (WM2016013, Nikon, Japan), and then the pore size was statistically analyzed by ImageJ (the number of test pores was 75).

[0086] Comparing the data in Table 2, it can be seen that the swelling ratio, porosity, residual ratio, pore size length, etc. of the prepared cryogel can be regulated by using the preparation method disclosed in the present invention, so as to adapt to different application scenarios.

[0087] Figure 7 Photographs of the freeze-dried gels (a) and infiltrated gels (b) of the cryogels prepared for Examples 1 to 3; among them, the freeze-dried gels were obtained by freeze-drying the cryogels in a -80 °C freeze-dryer for 1 day; the infiltrated gels were obtained by natural thawing of the cryogels. It was observed that compared with the cryogels prepared in Examples 1 and 3, the shape of the 2% PlasmaMA cryogel prepared in Example 2 was unstable and more likely to lose mechanical properties, which may be related to its slightly larger pore size.

[0088] Example 5

[0089] The preparation process is basically the same as that of Example 1, except that in Step 2, the concentration of PlasmaMA in the precursor solution is replaced with 1 g / 100 mL.

[0090] After testing, the cryogel of PlasmaMA prepared in this example has the function of promoting the healing of diabetic wounds, but its storage stability is poor and it will degrade after being placed in PBS buffer at room temperature for 5 days.

[0091] Example 6

[0092] The preparation process is basically the same as that of Example 1, except that in step 2, the concentration of PlasmaMA in the precursor solution is replaced with 15 g / 100 mL.

[0093] After testing, the cryogel of PlasmaMA prepared in this example has the function of promoting the healing of diabetic wounds, but its mechanical strength is too high, resulting in a decrease in compressive capacity.

[0094] Application Example

[0095] The application performance of the PlasmaMA cryogel prepared in Example 1 and the BSAMA cryogel prepared in Comparative Example 1 was evaluated as follows:

[0096] 1. The cryogel was stored in a sealed ep tube at a ratio of 1:1 mL of PBS solution (pH = 7.4), and samples were stored at set times of 3 days, 7 days, and 14 days. Each time, 200 μL of the PBS solution soaked by each gel was taken away, and 200 μL of pure PBS solution was added to maintain the 1:1 mL PBS solution system. Each collected sample was stored at -20 °C. After all samples were collected, the cumulative release of the growth factor VEGF was detected uniformly through an Elisa-VEGF kit (EK0539, Boster, China). The results are as Figure 8 shown in A.

[0097] 2. Sterile cryogel was made, brought into the cell ultra-clean bench, and washed 3 - 5 times with sterile PBS to wash away the initiator and reaction catalyst on the surface and in the pores. Then, 3D cell plating was carried out. The washed cryogel was placed at the bottom of the plate, the PBS solution absorbed by the gel swelling itself was aspirated, and a small amount of complete medium was added to moisten it. Then, the cells were treated, and the cell suspension was dropped on the surface of the cryogel and sent to an incubator at 37 °C and 5% CO 2 to culture. When culturing human umbilical vein endothelial cells (HUVEC) (CL-0675, PricellaBiotechnology, China) using the cryogel in 3D, the number of cells was about 7×10 6 cells, and they were incubated in an incubator at 37 °C and 5% CO 2 . On the third day, cell RNA was extracted. After the RNA concentration was measured to be qualified, a cDNA template was amplified, and then a fluorescence quantitative PCR experiment was carried out. The results are as Figure 8As shown in B and C.

[0098] Observation Figure 8 As can be seen from A, B, and C, compared with the BSAMA cryogel prepared in Comparative Example 1, the PlasmaMA cryogel prepared in Example 1 can release a large amount of growth factor VEGF. VEGF can promote angiogenesis and is required for cell growth and proliferation; it can significantly affect the growth ability of cells, promote cell growth and proliferation, and the relative gene expression levels are all higher than those of the BSAMA cryogel.

[0099] 3. Use a sterile cryogel to 3D culture mouse fibroblasts (L929) (CL-0137, Pricella Biotechnology, China), and wash them 3 - 5 times with sterile PBS to wash away the initiator and reaction catalyst on the surface and in the pores. The number of cells is about 2×10 4 cells, and incubate them in an incubator at 37°C and 5% CO 2 2. Perform live / dead cell staining on the first, third, and fifth days of culture. Stain with calcein-AM (2 μmol / L) and PI (4.5 μmol / L) for 15 minutes in the dark at 37°C. Observe the cell morphology and obtain images using a high-resolution laser confocal microscope. The results are as shown in Figure 8 D.

[0100] 4. Use a sterile cryogel to 3D culture L929, and wash it 3 - 5 times with sterile PBS to wash away the initiator and reaction catalyst on the surface and in the pores. The number of cells is about 5×10 4 cells, and incubate them in an incubator at 37°C and 5% CO 2 2. On the first, third, and fifth days of culture, use the CCK-8 method to determine the effect of the cryogel on cell proliferation. When measuring, first wash away the original culture medium, add complete culture medium mixed with CCK-8 reagent and incubate for 2 h, and then measure the absorbance OD at 450 nm. Subtract the values of the blank group from all the obtained values. The results are as shown in Figure 8 E.

[0101] Observation Figure 8 As can be seen from D and E, compared with the BSAMA cryogel prepared in Comparative Example 1, the PlasmaMA cryogel prepared in Example 1 has a more obvious function of promoting cell growth and proliferation.

[0102] 5. Pre-seed L929 cells in a 24-well plate. When the cells grow to about 80%, perform a scratch assay. Use a sterile cryogel for the experiment. There are three groups, namely Control (the components include cells, DMEM medium without serum, and a transwell insert), BSAMA (the components include cells, DMEM medium without serum, a transwell insert, and BSAMA cryogel), and PlasmaMA (the components include cells, DMEM medium without serum, a transwell insert, and PlasmaMA cryogel). Wash with sterile PBS 3 - 5 times to wash away the initiator and reaction catalyst on the surface and in the pores. After washing, place the cryogel in the transwell insert. The gel adheres to the bottom of the insert, and the insert exchanges substances through the filter membrane at the bottom. Take pictures at the 0 h and 24 h time points of the scratch, and use ImageJ to analyze and process the pictures. The experimental results are as Figure 8 shown in F in the figure, and the calculated migration ratio diagram is as shown in the right figure.

[0103] Observe Figure 8 from F in the figure that compared with the BSAMA cryogel prepared in Comparative Example 1, the PlasmaMA cryogel prepared in Example 1 has a higher migration ratio, indicating that it has a stronger ability to promote cell migration and is more conducive to promoting wound healing.

[0104] 6. Use an antioxidant capacity kit (ABTS) to evaluate the antioxidant capacity of the BSAMA cryogel and the PlasmaMA cryogel. Use the lyophilized gels of both (the preparation of the lyophilized gels refers to the method used for testing the swelling rate), immerse them in the analytical reagent, and then measure the OD value of the analytical solution at 414 nm. The results are as Figure 9 shown in A in the figure.

[0105] 7. Use the lyophilized gels of the BSAMA cryogel and the PlasmaMA cryogel, and soak them separately in an H 2 O 2 solution with a content of 100 mM for 3 days. Then, take the corresponding leached H 2 O 2 solution for the experiment. Pre-seed L929 cells in a 6-well plate. When the cells grow to about 80%, conduct the experiment. Mix the complete medium and the corresponding leachate, and treat the cells at a concentration of 1 mM / well of H 2 O 2 . After treatment for 24 hours, stain with a DCFH-DA probe, and then measure the cell staining situation by flow cytometry. ROS represents the fluorescence situation. If the antioxidant capacity of the cells is weak, ROS will increase significantly. There are four groups in total, namely PBS (the components include complete medium with serum, 2 mL / well), H 2 O 2(The components include 20 μL of H 2 O 2 treatment solution and 1980 μL of complete medium containing serum), H 2 O 2 @BSAMA (The components include 20 μL of H 2 O 2 @BSAMA treatment solution and 1980 μL of complete medium containing serum), H 2 O 2 @PlasmaMA (The components include 20 μL of H 2 O 2 @PlasmaMA treatment solution and 1980 μL of complete medium containing serum). The results are as shown in Figure 9 B below.

[0106] Observation Figure 8 in A and B below shows that both have antioxidant capacity, but the antioxidant capacity of the PlasmaMA cryogel is significantly stronger than that of the BSAMA cryogel.

[0107] Eight and twelve SPF - level healthy SD rats (female, 8 - week - old, body weight 220 - 250 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All animal experiments were carried out strictly in accordance with the "Regulations on the Breeding and Use of Laboratory Animals" of the National Animal Health Research Institute and the guidance of the Ethics Committee of Wenzhou Institute, Chinese Academy of Sciences (Animal Welfare Assurance Number: WIUCAS24072203). After the rats were adapted for one week under animal house conditions (temperature 22 ± 2 °C, humidity 60 ± 5%, light - dark cycle), a diabetes model was induced in the rats.

[0108] The specific steps are as follows: After 1 week of adaptive feeding (temperature 20 - 25°C, humidity 40 - 70%, 12-hour light-dark cycle), the rats were given a high-sugar and high-fat diet. They were fasted for 18 hours without water the night before injection, and the SD rats were anesthetized with isoflurane (R50 - 22 - 10, RWD). After 2 minutes, when the anesthesia was satisfactory, STZ (KM6241, KKL) was injected intraperitoneally, and the injection dose was determined according to the weight of each rat (60 mg / kg). Glucose water needed to be supplemented 4 hours after the injection. After the injection, they were continuously fed for 1 week, and the fasting blood glucose level was measured. Rats with a blood glucose level higher than 11.1 mmol / L and showing polydipsia, polyuria, and weight loss were judged as diabetic models. After confirming the successful establishment of the model, the rats were anesthetized and placed in the prone position. A tool was used to punch a hole in the back skin of the rats, with a circular wound approximately 6 mm in diameter and 1 mm deep. The wound was in the back skin layer. The rats were randomly divided into three groups: Control group, BSAMA cryogel group, and PlasmaMA cryogel group. The wound surface of the Control group was treated with PBS buffer, and the wound surfaces of the other groups were treated with the corresponding cryogels and changed daily. Record and measure the area of the wound surface at days 0, 3, 7, and 14, and calculate the healing rate. Denote the wound area on day 0 as A 0 , and the area at each subsequent time point is denoted as A t .

[0109] Wound healing rate % = (A 0 - A t ) / A 0 × 100%;

[0110] The wound photos are as shown in A in Figure 10 , and the wound healing proportion diagram is as shown in B in Figure 10 . The specific healing proportion data are listed in Table 3 below.

[0111] Table 3

[0112]

[0113]

[0114] 9. At each time point of the wound healing experiment when collecting samples, the rats were euthanized, the skin tissue in the wound area was obtained, and fixed with 4% paraformaldehyde. The fixed tissue samples were embedded in paraffin and cut into 6-μm sections. Analysis was performed by hematoxylin-eosin staining (H&E) and Masson staining techniques, and the results are as shown in Figure 11 .

[0115] HE sections showed the cellular activities and histological structure changes during wound healing. In the Control group, obvious infiltration of inflammatory cells was observed, with a relatively low cell density and a wide wound area, indicating slower healing. In the BSAMA group, inflammation was alleviated and the cell number increased, but the histological structure was still relatively loose. The PlasmaMA group showed the best performance, with a high degree of cell hyperplasia (such as fibroblasts and keratinocytes) in the wound area, almost complete resolution of inflammation, and closely arranged tissues, indicating that the wound had transitioned from the inflammatory phase to the repair phase, and the healing process was significantly accelerated.

[0116] Masson sections reflected the formation of collagen fibers and tissue reconstruction. In the Control group, sparse and disorderly distributed collagen fibers were observed, indicating a low repair ability. In the BSAMA group, collagen deposition was enhanced, but the fiber arrangement was still irregular. In the PlasmaMA group, the staining of collagen fibers increased significantly, with the blue area covering most of the wound, and the fiber arrangement was more compact, indicating that the formation of new collagen fibers and tissue reconstruction were the most active, and the wound healing effect was significantly better than that of other groups.

[0117] The wound length in Figure B and the collagen accumulation in Figure C were both obtained after quantitative analysis using ImageJ.

Claims

1. A method for preparing human plasma protein methacryloyl cryogel for promoting diabetic wound healing, characterized in that: include: (1) reacting thawed human plasma protein with methacrylic anhydride, and after the reaction is terminated, post-processing to obtain an intermediate product, which is recorded as PlasmaMA; (2) dissolving the PlasmaMA, initiator and catalyst prepared in step (1) in PBS buffer and mixing them evenly to obtain a precursor solution; (3) injecting the precursor solution prepared in step (2) into a mold, and freezing it at low temperature to obtain the human plasma protein methacryloyl cryogel for promoting diabetic wound healing.

2. The method for preparing human plasma protein methacryloyl cryogel for promoting diabetic wound healing according to claim 1, characterized in that: In step (1): The molar ratio of methacrylic anhydride to human plasma protein is (1-2.5):

1.

3. The method for preparing human plasma protein methacryloyl cryogel for promoting diabetic wound healing according to claim 1, characterized in that: In step (1): During the reaction, the pH value of the reaction solution is maintained at 7.5-8.0; The reaction temperature is room temperature.

4. The method for preparing human plasma protein methacryloyl cryogel for promoting diabetic wound healing according to claim 1, characterized in that: In step (1): The post-treatment includes dialysis, filtration and freeze drying; The freeze-dried intermediate product is stored at not less than -20°C until use.

5. The method for preparing human plasma protein methacryloyl cryogel for promoting diabetic wound healing according to claim 1, characterized in that: In step (2): The initiator is selected from one or more of ammonium persulfate, sodium persulfate, and potassium persulfate; The catalyst is selected from one or more of tetramethylethylenediamine, N,N-dimethylethylenediamine, triethylenetetramine, and ethylenediamine.

6. The method for preparing human plasma protein methacryloyl cryogel for promoting diabetic wound healing according to claim 1, characterized in that: In step (2): In the precursor solution, the concentration of PlasmaMA is 1 to 15 g / 100 mL; In the precursor solution, the concentration of the initiator is (0.4-1.0)% μL / μL; In the precursor solution, the concentration of the catalyst is (0.1-0.4)% μL / μL.

7. The method for preparing human plasma protein methacryloyl cryogel for promoting diabetic wound healing according to claim 1, characterized in that: In step (2), PlasmaMA is first added to PBS buffer and mixed evenly, and then the initiator and catalyst are added.

8. The method for preparing human plasma protein methacryloyl cryogel for promoting diabetic wound healing according to claim 1, characterized in that: In step (3), the low-temperature freezing temperature is -80 to -20°C.

9. The method for preparing the human plasma protein methacryloyl cryogel for promoting diabetic wound healing according to any one of claims 1 to 8, characterized in that: In step (2): In the precursor solution, the concentration of PlasmaMA is 2 to 4 g / 100 mL; In the precursor solution, the concentration of the initiator is 0.5% μL / μL; In the precursor solution, the concentration of the catalyst is 0.1% μL / μL.

10. A human plasma protein methacryloyl cryogel for promoting diabetic wound healing prepared according to the method of any one of claims 1 to 9.

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