Process for the preparation of a human plasma protein methacryl frozen gel to promote healing of diabetic wounds and product thereof

Abstract

The application discloses a preparation method of human plasma protein methacryl freeze gel for promoting diabetic wound healing and a product thereof, and the preparation method comprises the following steps: (1) reacting human plasma protein after thawing with methacrylic anhydride, and obtaining an intermediate product after post-treatment after termination of the reaction; (2) dissolving the intermediate product, an initiator and a catalyst in a PBS buffer solution, and obtaining a precursor solution after uniform mixing; and (3) injecting the precursor solution prepared in the step (2) into a mold, and obtaining the human plasma protein methacryl freeze gel for promoting diabetic wound healing after low-temperature freezing. The freeze gel is synthesized by taking human plasma protein as raw material for the first time, the plasma protein does not denature in the preparation process, the prepared freeze gel has excellent mechanical properties, water absorption, degradability and biocompatibility, and the freeze gel has a significant promoting effect on diabetic wound healing.

Description

Technical Field

[0001] This invention relates to the technical field of gels, and more specifically to a method for preparing a human plasma protein methacryloyl cryogel that promotes the healing of diabetic wounds and the product thereof. Background Technology

[0002] Diabetic wounds are a major complication of diabetes, associated with increased incidence, high prevalence, and socioeconomic burden. Clinical studies have shown that peripheral neuropathy, inflammatory dysfunction, reduced angiogenesis, and uncontrolled bacterial infection are related to the pathogenesis of diabetic wounds, leading to wound damage and even non-healing. Therefore, due to the complexity of diabetic wounds, a comprehensive treatment strategy is urgently needed.

[0003] Currently, the treatment of diabetic wounds mainly focuses on blood sugar control and debridement. However, due to the high cost, complex procedures, and high risk of infection, patients still face significant financial and psychological stress. While wound dressings are an effective treatment method, traditional dressings only isolate the wound and cannot effectively promote healing, and changing them causes pain for patients. Ideal wound dressings should possess good breathability, antibacterial properties, anti-inflammatory effects, and antioxidant properties.

[0004] To effectively control wound healing and infection, the development of multifunctional and high-performance wound dressings has become an urgent priority. Recently, wound dressings including electrospun nanofibers, sponges, and hydrogels have been designed for wound healing. Among them, hydrogels are three-dimensional hydrophilic polymer networks with structures similar to the extracellular matrix. Therefore, hydrogels have great application potential in tissue engineering, drug delivery, and wound dressings. For example, Chinese patent document with application publication number CN 118892573 A discloses a method for preparing a pH-responsive dual-release hydrogel that promotes the healing of diabetic wounds, including: (1) preparing sodium alginate oxide; (2) preparing astilbene liposomes; (3) preparing sodium alginate oxide solution with distilled water, adding astilbene liposomes to obtain OSA@AL solution; dissolving carboxymethyl chitosan and diclofenac sodium in distilled water to prepare CMCS@DS solution; mixing the above two solutions in equal volumes and preparing ALD hydrogel by Schiff base reaction. This technology enables controlled release of drugs in a controlled time and space, allowing the hydrogel to function at all four stages of wound healing, thereby accelerating the healing of diabetic wounds. However, the raw materials used in this technology are mostly antioxidants, but their effects on promoting tissue regeneration and cell growth are limited, and the wound healing rate still needs further improvement. Summary of the Invention

[0005] Based on the aforementioned deficiencies in the existing technology, this invention discloses a method for preparing a human plasma protein methacryloyl cryogel that promotes the healing of diabetic wounds. For the first time, a cryogel is synthesized using human plasma protein as a raw material. During the preparation process, the plasma protein does not denature. The prepared cryogel has excellent mechanical properties, water absorption, biodegradability, and biocompatibility. Furthermore, this cryogel has a significant promoting effect on the healing of diabetic wounds.

[0006] The specific technical solution is as follows:

[0007] A method for preparing a human plasma protein methacryloyl cryogel that promotes wound healing in diabetic patients includes:

[0008] (1) The thawed human plasma protein was reacted with methacrylic anhydride. After the reaction was terminated, the intermediate product was obtained by post-processing and was denoted as PlasmaMA.

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

[0010] (3) The precursor solution prepared in step (2) is injected into the mold and then frozen at low temperature to obtain the human plasma protein methacryloyl cryogel that promotes the healing of diabetic wounds.

[0011] This invention is the first to use human plasma protein as a raw material. After reacting with methacrylic anhydride (MMA), the amino groups on the human plasma protein are converted into amide bonds, and C=C double bonds are introduced into the human plasma protein. Then, an initiator and a catalyst are added, and the human plasma protein methacryloyl cryogel is obtained by polymerization at low temperature.

[0012] Experiments have shown that, compared to hydrogels, cryogels possess higher mechanical strength and pressure resistance, providing a physical barrier for wounds and reducing external environmental irritation or contamination. Cryogels also have excellent water absorption capabilities, absorbing exudate and reducing the risk of wound infection. Simultaneously, cryogels keep wounds moist, promoting healing while preventing over-moistening that can lead to maceration.

[0013] Further comparative experiments showed that the human plasma protein methacryloyl cryogel prepared in this invention has superior pressure resistance and wound healing promotion ability compared with cryogel prepared from bovine serum albumin.

[0014] Preferably, in step (1):

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

[0016] Further optimization yielded a molar ratio of 2.2:1.

[0017] Preferably, in step (1):

[0018] The pH of the reaction solution was maintained at 7.5–8.0 during the reaction process;

[0019] The reaction temperature was room temperature.

[0020] Preferably, in step (1):

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

[0022] The freeze-dried intermediate product should be stored at a temperature not lower than -20°C until use.

[0023] Preferably, in step (2):

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

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

[0026] Further 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] Further optimization is achieved by using a PlasmaMA concentration of 2–4 g / 100 mL.

[0030] Experiments revealed that when the concentration of PlasmaMA was low, the prepared cryogel had poor storage stability and degraded after being placed in PBS buffer at room temperature for several days. When the concentration of PlasmaMA was too high, the mechanical strength of the prepared cryogel was too great, resulting in a decrease in its compressive strength.

[0031] Preferably, in step (2):

[0032] The concentration of the initiator in the precursor solution is (0.4–1.0)% μL / μL;

[0033] The catalyst concentration in the precursor solution is (0.1–0.4)% μL / μL.

[0034] Further preferred, the initiator concentration is 0.5% μL / μL; the catalyst concentration is 0.1% μL / μL. The cryogel prepared under this ratio exhibits more stable shape, texture, and state, and the initiator and reaction catalyst show less toxicity to cells, making it suitable for long-term cell treatment.

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

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

[0037] Experiments have shown that adjusting the freezing temperature affects the pore structure of the prepared cryogel.

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

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

[0040] This invention is the first to synthesize a cryogel using human plasma protein as a raw material. The plasma protein does not denature during the preparation process, and the resulting cryogel has excellent mechanical properties, water absorption, biodegradability, and biocompatibility. Furthermore, by adjusting the raw material ratio, parameters such as pore length, swelling ratio, porosity, and residual rate of the prepared cryogel can be controlled to meet different application requirements.

[0041] Further testing revealed that the human plasma protein methacryloyl cryogel prepared in this invention can release a large amount of growth factor VEGF, which can significantly affect cell growth capacity and promote cell growth and proliferation; it can also promote cell migration more effectively and has excellent antioxidant capacity. This cryogel has a significant promoting effect on the healing of diabetic wounds. Attached Figure Description

[0042] Figure 1 Figure (A) shows the results of the methacrylation degree determination of PlasmaMA prepared in Example 1. Figure (A) shows the samples of raw material Plasma and intermediate product PlasmaMA. 1 (B) The figure shows the degree of substitution of the intermediate product PlasmaMA sample calculated by TNBS; (C) The figure shows the degree of methacrylation of the intermediate product PlasmaMA sample calculated by TNBS.

[0043] Figure 2 The diagram shows the secondary structure of Plasma, the raw material used in Example 1, and the prepared PlasmaMA sample.

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

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

[0046] Figure 5 The cyclic compression performance of the cryogels prepared in Example 1 and Comparative Example 1 are shown in the diagram.

[0047] Figure 6 The swelling ratio (A), porosity (B), residual ratio (C), and pore length (D) of the human plasma protein methacryloyl cryogels prepared in Examples 1-3 are shown in the figure.

[0048] Figure 7 Photographs (a) and (b) of the lyophilized gels prepared in Examples 1-3 are shown.

[0049] Figure 8 The cumulative release of growth factor VEGF (A), PCR results of growth factor VEGF (B), PCR results of growth factor EGF (C), live / dead staining experiment (D), cell proliferation experiment (E) and scratch experiment (F) were performed in the PlasmaMA cryogel prepared in Example 1 and the BSAMA cryogel prepared in Comparative Example 1, respectively.

[0050] Figure 9 Figure (A) shows the results of the in vitro ABTS clearance assay performed in the human plasma protein methacryloyl cryogel prepared in Example 1 and the BSAMA cryogel prepared in Comparative Example 1, and Figure (B) shows the results of the flow cytometry experiment performed in 3D cell culture under hydrogen peroxide stimulation.

[0051] Figure 10 Photographs (A) and wound healing ratios (B) of diabetic wounds treated with PlasmaMA cryogel prepared in Example 1, BSAMA cryogel prepared in Comparative Example 1, and the control group after different treatment times.

[0052] Figure 11Staining of diabetic wound sections treated with PlasmaMA cryogel prepared in Example 1, BSAMA cryogel prepared in Comparative Example 1, and the control group at different treatment times included HE staining and Masson staining (A); wound length on day 14 based on HE section analysis (B) and collagen deposition area on day 14 based on Masson section analysis (C). Detailed Implementation

[0053] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be noted that the following embodiments are intended to facilitate the understanding of the present invention and do not constitute any limitation thereof.

[0054] Example 1

[0055] 1. Thaw 100 mL of human plasma protein (Plasma, pH = 7.4 ± 0.1, Kejing Biotechnology) at 4°C and magnetically stir (600 rpm) at room temperature. After homogeneous mixing, add 1.521 mL of methacrylic anhydride (MAA, 94%; Sigma-Aldrich), with a MAA to Plasma molar ratio of 2.2:1. React at room temperature with 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 hours until the reaction is complete. Filter the solution using 70 mm filter paper, and then dialyze it in distilled water for 4 hours at room temperature using a Pellicon 2 box equipped with a 10 kDa Biomax membrane to remove unreacted MAA and methacrylic acid byproducts. Finally, freeze the collected solution at -20°C for 3 hours and then freeze-dry it at -80°C for 4 days to obtain PlasmaMA, which is stored at -20°C for later use.

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

[0057] Plasma and PlasmaMA samples were dissolved in 0.1M NaOH aqueous solution at a concentration of 1.6 mg / mL. Simultaneously, glycine standard sample solutions (0, 1, 2, 4, 8, 16, 32, 64 μg / mL) were prepared for constructing a standard curve. After complete dissolution, 0.25 mL of each solution was added to the corresponding well, followed by 0.25 mL of 0.1% TNBS (P2297, Sigma) solution. The mixture was incubated at 37°C for 2 hours. After 2 hours, 0.25 mL of 1M HCl aqueous solution and 0.25 mL of 10% w / v SDS aqueous solution were added to each well, and the absorbance was measured at 335 nm. The degree of substitution and the degree of methacrylamide could be calculated from the standard curve.

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

[0059] The secondary structure of Plasma and PlasmaMA samples was evaluated using circular dichroism (CD) spectroscopy. The results are as follows: Figure 2 As shown, the CD spectrum of natural Plasma exhibits two negative bands at 208 nm and 222 nm, which is a typical characteristic of α-helical proteins. PlasmaMA also displays typical protein characteristics. Furthermore, the percentage of α-helices in the natural Plasma and PlasmaMA samples was calculated and listed in Table 1 below. All of the above demonstrates that the PlasmaMA samples obtained using the preparation process described in this invention did not undergo denaturation.

[0060] Table 1

[0061]

[0062] 2. The lyophilized PlasmaMA was dissolved in phosphate-buffered saline (PBS; pH = 7.4; Gibco, Life), and then APS (R113155, 10 wt%, Rhawn) and TEMED (R007178, 20 vol%, Rhawn) were added and mixed thoroughly to obtain the precursor solution. The concentration of PlasmaMA in the precursor solution was 3 g / 100 mL, the concentration of APS was 0.5% μL / μL, and the concentration of TEMED was 0.1% μL / μL.

[0063] 3. The precursor solution was injected into a silicone mold that had been pre-cooled to -20°C, and frozen at -20°C for 2 days to obtain human plasma protein methacryloyl cryogel, denoted as 3% PlasmaMA.

[0064] The PlasmaMA cryogel prepared in this embodiment was transferred to a -80℃ freeze dryer and freeze-dried for 1 day to obtain the lyophilized gel. At 37℃, the initial mass of the lyophilized gel was first weighed and recorded as M0. Then, the lyophilized gel was placed in PBS buffer (pH = 7.4). At different time points (15 min, 30 min, 45 min, 60 min, 90 min, 120 min, 180 min, 240 min), the lyophilized gel, after swelling in the PBS buffer, was removed. The surface moisture of the lyophilized gel was blotted with lint-free paper, and the weight was recorded as M. t The formula for calculating the swelling ratio is as follows:

[0065] Swelling rate (%) = (M) t -M0) / M0×100%, the result is as follows Figure 3 As shown in Figure A, in this invention, all data detection is performed using 3 samples as a group, and the obtained data is the average value of the 3 samples.

[0066] The PlasmaMA cryogel prepared in this example was placed in PBS buffer (pH=7.4) and swollen for 1 day. The next day, the surface moisture was blotted off with lint-free paper and the gel was weighed, denoted as W. t The PlasmaMA cryogel was then placed on paper, with the paper being replaced continuously. Once the external water in the PlasmaMA cryogel was mostly removed, the dehydrated PlasmaMA cryogel was weighed and recorded as W0. The porosity calculation formula is as follows:

[0067] Porosity (%) = (W t -W0) / W t ×100%, the result is as follows Figure 3 As shown in B.

[0068] Observations revealed that the PlasmaMA cryogel prepared in this embodiment exhibited excellent absorption and storage properties for liquids, with a swelling ratio of 1750±50% and a porosity as high as 89±1%.

[0069] The PlasmaMA cryogel prepared in this embodiment was placed in PBS buffer (pH=7.4) to reach equilibrium swelling, and its wet weight was measured and recorded as S0. Then, the PlasmaMA cryogel was placed in 0.01% trypsin-EDTA solution at 37°C and PBS buffer (pH=7.4), respectively. Every hour, it was removed, the surface moisture was blotted off with lint-free paper, and the weight was recorded as S. t Change the enzyme solution or PBS buffer every 2 hours. The remaining percentage is calculated using the following formula:

[0070] Residual rate (%) = S t / S0×100%, the residual rate of trypsin and the residual rate of PBS buffer are respectively as follows: Figure 4 As shown in the left and right images.

[0071] As can be seen from the figure, the cryogel prepared in this invention has good stability and can exist stably 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 sodium bicarbonate and 21.53 g sodium carbonate decahydrate in 1 L distilled water) and stir magnetically at 37 °C (500 rpm). After the BSA is completely dissolved, adjust the pH of the buffer solution to 9 using 5 M sodium hydroxide aqueous solution (NaOH; Sigma-Aldrich). Then add 3.116 mL of methacrylic anhydride (MAA, 94%; Sigma-Aldrich) to the BSA / CB buffer solution and react at 37 °C with magnetic stirring (500 rpm). During the reaction, maintain the pH of the reaction system at 7.5 by adding 5 M NaOH solution. After 1 h of reaction, 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. The solution was filtered sequentially using 70 mm filter paper, and then dialyzed in distilled water for 4–6 hours at room temperature using a Pellicon 2 cartridge equipped with a 10 kDa Biomax membrane to remove unreacted MAA and methacrylic acid byproducts. Finally, the collected solution was frozen at -20°C for 3 hours and then freeze-dried at -80°C for 4 days to obtain BSAMA, which was then stored at -20°C for later use.

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

[0075] The bovine serum albumin cryogel prepared in this comparative example is designated 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 under controlled force mode using a dynamic mechanical analyzer (DMA; Q800; TA Instrument). Data are shown in […]. Figure 5 .

[0077] Observations revealed that the PlasmaMA cryogel prepared in this embodiment exhibited good compression recovery and mechanical stability, showing almost no stress reduction after 10 consecutive loading / unloading cycles at 80% strain. Compared to the BSAMA cryogel prepared in Comparative Example 1, the PlasmaMA cryogel prepared in this invention exhibits greater stress, higher compressive strength, and better stability.

[0078] Example 2

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

[0080] Example 3

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

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

[0083] Table 2

[0084]

[0085] The pore length was measured by placing the PlasmaMA cryogels prepared in each example in PBS buffer (pH=7.4) until they reached equilibrium swelling, removing them, adding Rhodamine staining agent for staining, and then photographing the scaffold structure using a high-resolution laser confocal microscope (WM2016013, Nikon, Japan). The pore size was then counted using ImageJ (75 test pores).

[0086] Comparing the data in Table 2, it can be seen that the preparation method disclosed in this invention can control the swelling rate, porosity, residual rate and pore length of the prepared cryogel, thereby adapting to different application scenarios.

[0087] Figure 7 Photographs (a) of the lyophilized gel and (b) of the impregnated gel of the cryogels prepared in Examples 1-3 are shown. The lyophilized gel was obtained by transferring each cryogel to a freeze dryer at -80°C and freeze-drying for 1 day. The impregnated gel was obtained by naturally thawing the cryogel. Observation revealed that, compared with the cryogels prepared in Examples 1 and 3, the 2% PlasmaMA cryogel prepared in Example 2 had an unstable shape and was more prone to losing mechanical properties, which may be related to its slightly larger pore size.

[0088] Example 5

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

[0090] Tests showed that the PlasmaMA cryogel prepared in this example could promote the healing of diabetic wounds, but it had poor storage stability and degraded after being placed in PBS buffer at room temperature for 5 days.

[0091] Example 6

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

[0093] Tests showed that the PlasmaMA cryogel prepared in this embodiment has the function of promoting the healing of diabetic wounds, but the excessive mechanical strength leads to a decrease in compressive strength.

[0094] Application examples

[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 cryogels were stored in sealed EP tubes at a ratio of 1:1 mL PBS solution (pH = 7.4). Samples were stored at set intervals of 3, 7, and 14 days. Each time, 200 μL of the PBS solution from each gel was removed, and 200 μL of purified PBS solution was added to maintain the 1:1 mL PBS solution system. The collected samples were stored at -20°C. After all samples were collected, the cumulative release of VEGF growth factor was detected using an ELISA-VEGF kit (EK0539, Booster, China). The results are as follows: Figure 8 As shown in Figure A.

[0097] 2. Prepare a sterile cryogel. After placing it in a cell resealable chamber, wash it 3-5 times with sterile PBS to remove initiators and reaction catalysts from the surface and pores. Then, perform 3D cell plating. Place the washed cryogel at the bottom of the plate, aspirate the PBS solution absorbed by the gel during swelling, and add a small amount of complete culture medium to infiltrate the cells. Then, process the cells by adding cell suspension dropwise onto the surface of the cryogel and incubating at 37°C with 5% CO2. After 4 hours, the cells will adhere to the gel, and then more culture medium will be added. Human umbilical vein endothelial cells (HUVECs) (CL-0675, Pricella Biotechnology, China) were cultured using cryogel 3D culture, with a cell count of approximately 7 × 10⁻⁶. 6 The cells were incubated at 37°C in a 5% CO2 incubator. On the third day, cellular RNA was extracted, and after confirming the RNA concentration was within acceptable limits, cDNA templates were amplified. Subsequently, quantitative real-time PCR was performed, and the results are as follows: Figure 8 As shown in B and C.

[0098] observe Figure 8 As shown in A, B, and C, compared to 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 cell growth capacity and promote cell growth and proliferation, and the relative expression levels of the gene are higher than those of the BSAMA cryogel.

[0099] 3. Mouse fibroblasts (L929) (CL-0137, Pricella Biotechnology, China) were cultured in sterile cryogels using 3D chromatography. The cells were then washed 3–5 times with sterile PBS to remove initiators and reaction catalysts from the surface and pores. The cell count was approximately 2 × 10⁻⁶. 4 Cells were incubated at 37°C in a 5% CO2 incubator. Live and dead cell staining was performed on days 1, 3, and 5 of culture. Cells were stained with calcein-AM (2 μmol / L) and PI (4.5 μmol / L) for 15 minutes in the dark at 37°C. Cell morphology was observed, and images were acquired using a high-resolution laser confocal microscope. The results are shown below. Figure 8 D.

[0100] 4. Culture L929 cells in a sterile cryogel for 3D. Wash 3-5 times with sterile PBS to remove initiators and reaction catalysts from the surface and pores. The cell count should be approximately 5 × 10⁻⁶. 4Cells were incubated at 37°C in a 5% CO2 incubator. The effect of cryogel on cell proliferation was determined using the CCK-8 assay on days 1, 3, and 5. Before the assay, the original culture medium was washed away, and complete culture medium containing CCK-8 reagent was added and incubated for 2 hours. The absorbance (OD) was then measured at 450 nm. The results were then subtracted from the control group values. Figure 8 E in Chinese.

[0101] observe Figure 8 As shown in Figures D and E, compared with the BSAMA cryogel prepared in Comparative Example 1, the PlasmaMA cryogel prepared in Example 1 has a more significant function in promoting cell growth and proliferation.

[0102] 5. Pre-seed L929 cells in 24-well plates and perform scratch assays when cells reach approximately 80% confluence. Sterile cryogels were used for the experiments. Three groups were included: Control (comprising cells, serum-free DMEM medium, and transwell chambers), BSAMA (comprising cells, serum-free DMEM medium, transwell chambers, and BSAMA cryogel), and PlasmaMA (comprising cells, serum-free DMEM medium, transwell chambers, and PlasmaMA cryogel). The cells were washed 3-5 times with sterile PBS to remove initiators and reaction catalysts from the surface and pores. After washing, the cryogel was placed in the transwell chambers, with the gel adhering to the bottom. Substance exchange occurred through a filter membrane at the bottom of the chamber. Images were taken at 0h and 24h after the scratching process and analyzed using ImageJ. The experimental results are shown below. Figure 8 The migration ratio obtained by calculating F is shown in the figure on the right.

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

[0104] 6. The antioxidant capacity of BSAMA and PlasmaMA cryogels was evaluated using an antioxidant capacity kit (ABTS). The lyophilized gels of both gels (prepared according to the method used for swelling ratio testing) were immersed in the analytical reagents, and the OD value of the analytical solution was measured at 414 nm. The results are shown below. Figure 9 A.

[0105] 7. Using lyophilized BSAMA and PlasmaMA cryogels, each gel was immersed in a 100 mM H2O2 solution for 3 days. The corresponding H2O2 solutions were then used for experiments. L929 cells were pre-seeded in 6-well plates. When the cells reached approximately 80% confluence, the complete culture medium was mixed with the corresponding leaching solution, and the cells were treated with 1 mM H2O2 per well. After 24 hours of treatment, the cells were stained using the DCFH-DA probe, and the staining was then measured by flow cytometry. ROS indicates fluorescence; if the cells have weak antioxidant capacity, ROS will be significantly elevated. The study was divided into four groups: PBS (containing serum-containing complete culture medium, 2 mL / well), H2O2 (containing 20 μL H2O2 treatment solution and 1980 μL serum-containing complete culture medium), H2O2@BSAMA (containing 20 μL H2O2@BSAMA treatment solution and 1980 μL serum-containing complete culture medium), and H2O2@PlasmaMA (containing 20 μL H2O2@PlasmaMA treatment solution and 1980 μL serum-containing complete culture medium). Results are as follows: Figure 9 B.

[0106] observe Figure 8 The results for samples A and B showed that both had antioxidant capacity, but the antioxidant capacity of PlasmaMA cryogel was significantly stronger than that of BSAMA cryogel.

[0107] 8. Twelve SPF-grade healthy SD rats (female, 8 weeks old, weighing 220–250 g) were purchased from Beijing Vital River Laboratory Animal Co., Ltd. (Beijing, China). All animal experiments were conducted strictly in accordance with the "Regulations on the Breeding and Use of Laboratory Animals" issued by the National Institute of Animal Health and the guidance of the Ethics Committee of the Wenzhou Institute of the Chinese Academy of Sciences (Animal Welfare Protection Code: WIUCAS24072203). After one week of acclimatization in the animal room (temperature 22±2℃, humidity 60±5%, alternating light and dark), a diabetic model was induced in the rats.

[0108] The specific steps are as follows: After one week of adaptive feeding (temperature 20-25℃, humidity 40-70%, 12-hour light-dark cycle), rats were fed a high-sugar, high-fat diet. The rats were fasted for 18 hours the night before injection, but allowed free water. SD rats were anesthetized with isoflurane (R50-22-10, RWD). After 2 minutes of satisfactory anesthesia, STZ (KM6241, KKL) was injected intraperitoneally at a dose determined by the rat's body weight (60 mg / kg). Four hours after injection, glucose solution was administered. The rats were fed for another week, and fasting blood glucose levels were measured. Rats with blood glucose levels higher than 11.1 mmol / L and exhibiting polydipsia, polyuria, and weight loss were identified as having a diabetic model. After successful model establishment, the rats were anesthetized and placed in a prone position. A circular wound, approximately 6 mm in diameter and 1 mm deep, was made in the dorsal skin layer. The rats were randomly divided into three groups: Control group, BSAMA cryogel group, and PlasmaMA cryogel group. The control group treated the wound with PBS buffer, while the other groups treated their wounds with the corresponding cryogels, changing the gels daily. The wound area was recorded and measured on days 0, 3, 7, and 14, and the healing rate was calculated. The wound area on day 0 was denoted as A0, and the area at each subsequent time point was denoted as A... t .

[0109] Wound healing rate % = (A0 - A) t ) / A0×100%;

[0110] Photos of the wound Figure 10 As shown in Figure A, the wound healing ratio diagram is as follows: Figure 10 As shown in Figure B, the specific healing rate data is listed in Table 3 below.

[0111] Table 3

[0112]

[0113]

[0114] 9. Wound Healing Experiment: At each time point, rats were euthanized, and skin tissue from the wound area was obtained and fixed with 4% paraformaldehyde. The fixed tissue samples were embedded in paraffin and cut into 6μm sections. Analysis was performed using hematoxylin-eosin (H&E) and Masson staining techniques. The results are as follows: Figure 11 As shown.

[0115] HE sections revealed changes in cell activity and tissue structure during wound healing. The Control group showed significant inflammatory cell infiltration, low cell density, and a large wound area, indicating slow healing. The BSAMA group showed reduced inflammation and increased cell number, but the tissue structure remained relatively loose. The PlasmaMA group performed best, with high cell proliferation (such as fibroblasts and keratinocytes) in the wound area, near-complete resolution of inflammation, and tightly packed tissue, indicating that the wound had transitioned from the inflammatory phase to the repair phase, and the healing process was significantly accelerated.

[0116] Masson sections reflect the formation of collagen fibers and tissue remodeling. In the Control group, collagen fibers were sparse and randomly distributed, indicating a low repair capacity. In the BSAMA group, collagen deposition was enhanced, but the fiber arrangement was still irregular. In the PlasmaMA group, collagen fiber staining was significantly increased, 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 remodeling were the most active, and the wound healing effect was significantly better than other groups.

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

Claims

1. Use of human plasma protein methacryl freeze gel in preparing a dressing for promoting healing of diabetic wounds, characterized in that: the human plasma protein methacryl freeze gel is prepared by the following method: (1) reacting human plasma protein after thawing with methacrylic anhydride, and then treating the intermediate product obtained after the reaction is terminated by post-treatment, denoted as PlasmaMA; the molar ratio of methacrylic anhydride to human plasma protein is (1-2.5):1; (2) dissolving PlasmaMA prepared in step (1), an initiator and a catalyst in PBS buffer, and mixing uniformly to obtain a precursor solution; the concentration of PlasmaMA in the precursor solution is 2-4 g / 100 mL; the concentration of the initiator in the precursor solution is 0.5% μL / μL; the concentration of the catalyst in the precursor solution is 0.1% μL / μL; the catalyst is selected from tetramethyl ethylenediamine; (3) injecting the precursor solution prepared in step (2) into a mold, and obtaining human plasma protein methacryl freeze gel after low-temperature freezing; the temperature of the low-temperature freezing is -20°C. In step (1):

2. Use of the human plasma protein methacryloyl cryogel according to claim 1 for the preparation of a dressing promoting healing of diabetic wounds, characterized in that, the pH value of the reaction solution is maintained at 7.5-8.0 during the reaction; the reaction temperature is room temperature. In step (1):

3. Use of the human plasma protein methacryloyl cryogel according to claim 1 for the preparation of a dressing for promoting healing of diabetic wounds, characterized in that, the post-treatment includes dialysis, filtration and freeze-drying; the intermediate product after freeze-drying is stored at not less than -20°C for use. In step (2):

4. Use of the human plasma protein methacryloyl cryogel according to claim 1 for the preparation of a dressing for promoting healing of diabetic wounds, characterized in that, the initiator is selected from one or more of ammonium persulfate, sodium persulfate and potassium persulfate. In step (2), PlasmaMA is first added to PBS buffer and mixed uniformly, and then the initiator and the catalyst are added.

5. Use of the human plasma protein methacryloyl cryogel according to claim 1 for the preparation of a dressing for promoting healing of diabetic wounds, characterized in that, ​

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