Ratio fluorescence sensing peptide hydrogel for machine learning assistance and preparation method thereof

Abstract

The invention provides ratiometric fluorescent sensing peptide hydrogel for machine learning assistance and a preparation method of the ratiometric fluorescent sensing peptide hydrogel. The ratiometric fluorescent sensing peptide hydrogel is obtained by crosslinking modified polylysine modified with different fluorescent molecules and polyglutamic acid; when the ratio fluorescent sensing peptide hydrogel is exposed to active oxygen, the thioketone group is cracked, so that the fluorescent molecules are separated from the polymer and are gathered in a water environment. The process causes quenching of coumarin 7 and aggregation of tetraphenylethylene, thereby causing change of exciting light intensity ratio and corresponding change of fluorescence color, so that the level of active oxygen can be quantitatively monitored in real time through machine learning assistance, and the inflammation degree of a wound can be accurately reflected. Valuable data are provided for closed-loop treatment of chronic diabetes wounds.

Description

Technical Field

[0001] The present invention relates to the field of preparation and application of hydrogel dressing materials, and particularly to a ratio fluorescence sensing peptide hydrogel for machine learning assistance and a preparation method thereof. Background Art

[0002] Due to hyperglycemia and metabolic disorders, the inflammatory management of diabetic chronic wounds remains a major clinical challenge. The normal wound healing process is generally divided into four consecutive stages: hemostasis stage, inflammation stage, proliferation stage, and remodeling stage. In the inflammation stage, inflammatory cells will remove necrotic tissue and debris at the wound site, creating conditions for the subsequent proliferation stage. However, in diabetic chronic wounds, the persistent hyperglycemic state and metabolic disorders will lead to the continuous delay of the inflammatory response, forming a pathological inflammatory microenvironment. Therefore, effectively regulating the inflammatory response is of great clinical significance for promoting diabetic wound healing, reducing the risk of infection, and preventing systemic complications. In the inflammatory state, inflammatory cells, especially neutrophils and macrophages, will generate reactive oxygen species (ROS) through NADPH oxidase-mediated superoxide anion (O2 - ). At the same time, the secretion of pro-inflammatory cytokines (such as TNF-α and IL-1β) will trigger an intracellular signal cascade, increasing the calcium level in the cell membrane, thereby activating calcium-dependent protein kinases, and then inducing the formation of ROS. Moreover, it has been found that the ROS level is positively correlated with the inflammatory state, and ROS can be used as a biomarker for evaluating the inflammatory state of chronic wounds.

[0003] Clinically, fluorescent specific probes (such as DCFH-DA, Luminol) or electron spin resonance (ESR) techniques are usually used to quantify the ROS level. The detection process includes sample collection, processing, detection and data analysis, which is time-consuming and difficult to monitor the ROS level in situ and in real time at the wound site. Currently, a variety of ROS fluorescent probes have been developed, mainly using modified fluorophores to target ROS. However, most fluorescence-based ROS detection methods also require sampling, detection and analysis and cannot be used for real-time detection. In addition, due to the inherent complexity of the microenvironment, it is challenging to establish an intensity-based standard curve suitable for in situ wound monitoring. Compared with fluorescence intensity-based methods, the ratio fluorescence method relies on color changes, has anti-interference ability and high sensitivity. In addition, polypeptides have biocompatibility, tunable biodegradability, chemical diversity and programmable bioactivity, and have great potential in biomedical applications. Recent research progress has expanded the application of polypeptides in anti-infection treatment and regulation of inflammatory responses, especially in the treatment of chronic wounds and inflammation. Therefore, it is necessary to develop polypeptide-based in situ ratio fluorescence materials to address the challenges brought by the complex environment of chronic diabetic wounds, so as to achieve real-time monitoring of wound inflammation and provide valuable data for guiding the adjustment of treatment plans. However, no technical solutions for preparing ratio fluorescence sensing peptide hydrogels for real-time monitoring of chronic diabetic wound inflammation have been found at home and abroad. Summary of the Invention

[0004] The object of the present invention is to provide a ratio fluorescence sensing peptide hydrogel for machine learning assistance, which can monitor inflammation in real time, and a preparation method thereof.

[0005] To achieve the above object, the present invention provides a ratio fluorescence sensing peptide hydrogel, which is obtained by crosslinking modified polylysine and polyglutamic acid modified with different fluorescent molecules.

[0006] Furthermore, the fluorescent molecules are coumarin 7 and tetraphenylethylene; the modified polylysine modified with different fluorescent molecules is Plys-TK-C7 and Plys-TK-TPE.

[0007] Furthermore, the modified polylysine modified with different fluorescent molecules is obtained by dissolving the fluorescent molecule, the ROS-responsive group TK-2COOH and EDCI in an appropriate amount of DMSO, then adding triethylamine and DMPA, reacting for a period of time, adding polylysine, EDCI, triethylamine and DMPA, and after the reaction is completed, adding an appropriate amount of acetone for precipitation to obtain an oily precipitate, and removing the remaining organic reagents by rotary evaporation.

[0008] The present invention also provides a method for preparing a ratio fluorescence sensing peptide hydrogel. Based on N6-Cbz-L-lysine and L-glutamic acid 5-benzyl ester raw materials, the ratio fluorescence sensing peptide hydrogel is prepared, including the following steps: S1: Dissolve N6-Cbz-L-lysine and α-pinene in anhydrous THF, heat, stir, then pour the reaction solution into n-hexane, precipitate a yellow solid, dry to obtain benzyl (S)-(4-(2,5-dioxooxazolidin-4-yl)butyl)carbamate, i.e., Lys-NCA; S2: Dissolve L-glutamic acid 5-benzyl ester and α-pinene in anhydrous THF, heat, stir, then pour the reaction solution into n-hexane, precipitate a pale yellow solid, dry to obtain benzyl (S)-3-(2,5-dioxooxazolidin-4-yl)propionate, i.e., Glu-NCA; S3: Dissolve Lys-NCA and Glu-NCA in anhydrous DMF respectively, then add n-hexylamine / anhydrous DMF solution, react under negative pressure. After the reaction is completed, pour the reaction solution into anhydrous ether, centrifuge, and dry under reduced pressure to obtain polylysine with protecting groups and polyglutamic acid with protecting groups; S4: Dissolve polylysine with protecting groups and polyglutamic acid with protecting groups in TFA respectively, then dropwise add a 30% hydrobromic acid acetic acid solution by mass ratio, seal and stir. After the reaction is completed, add acetone to precipitate, centrifuge, and wash with acetone 2-3 times until the supernatant is colorless. After dialysis, freeze-dry the polylysine and polyglutamic acid; S5: Dissolve the fluorescent molecule TPE-NH2 and the fluorescent molecule C7, respectively, with the reactive oxygen-responsive group dithioketal dicarboxylic acid and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in an appropriate amount of DMSO. Then add triethylamine and N,N-dimethylacrylamide. After reacting for a period of time, continue to add polylysine, EDCI, triethylamine and DMPA. After the reaction is completed, add an appropriate amount of acetone to precipitate to obtain an oily precipitate. Remove the remaining organic reagents by rotary evaporation to obtain the solid polymers Plys-TK-TPE and Plys-TK-C7; S6: Dissolve polyglutamic acid, Plys-TK-TPE and Plys-TK-C7 in DMSO respectively to obtain PGlu / DMSO, Ply-TPE / DMSO and Ply-C7 / DMSO solutions. Mix the three solutions, add EDCI, mix the mixture quickly and thoroughly, then pour it into a mold to obtain a hydrogel, and dialyze it with the mold in deionized water. Finally, most of the hydrogel remains in the mold.

[0009] Further, in step S1, the reaction temperature is 55 °C, the stirring time is 45 min, the drying temperature is 40 °C, and the drying time is 12 h.

[0010] Further, in step S2, the reaction temperature is 55 °C, the stirring time is 3 h, the drying temperature is 40 °C, and the drying time is 12 h.

[0011] Further, in step S3, the reaction is carried out under anhydrous conditions, the stirring time is 48 h, both Lys-NCA and Glu-NCA are 1 / 200, and the volume of anhydrous ether is 15 times that of the reaction solution.

[0012] Further, in step S4, the reaction temperature is 25 °C, the stirring time is 2 h, and the volume of acetone is five times that of the reaction solution.

[0013] Further, in step S5, the molar ratio of the fluorescent molecules C7: TK-2COOH: DMPA is: 1:1:2.5; The molar ratio of the fluorescent molecules C7: triethylamine: DMPA is: 1:2.5:2.5, the reaction temperature is 25 °C, and the stirring time is 3 h; The molar ratio of the fluorescent molecules TPE-NH2 / polylysine / EDCI / triethylamine / DMPA is: 1:55:1:1:1, the reaction temperature is 25 °C, and the stirring time is 12 h.

[0014] Further, in step S6, the mixed molar ratio of the PGlu / DMSO, Ply-TPE / DMSO, and Ply-C7 / DMSO solutions is 2:1:1; The amount of EDCI is 30% of the total molar number of lysine; the dialysis time is 12 h.

[0015] Compared with the prior art, the advantages of the present invention are as follows: (1) The ratiometric fluorescent sensing peptide hydrogel of the present invention can not only monitor the inflammatory state in a ratiometric manner but also promote wound healing, and has potential application value in the field of treating diabetic chronic wounds.

[0016] (2) The ratiometric fluorescent sensing peptide hydrogel of the present invention monitors the inflammatory state mainly relying on color change. Compared with the method based on fluorescence intensity, it has stronger anti-interference ability and high sensitivity.

[0017] (3) The ratiometric fluorescent sensing peptide hydrogel prepared by the present invention has good water solubility and biocompatibility.

[0018] (4) The present invention is simple, the experimental conditions are mild, and it is easy to operate. Description of the Drawings

[0019] Figure 1 It is a schematic diagram of the synthesis process of the ratiometric fluorescent sensing peptide hydrogel in Example 1 of the present invention; Figure 2 Scanning electron microscope image of the ratiometric fluorescent sensing peptide hydrogel in Example 2 of the present invention; Figure 3 Stress-strain curve of the ratiometric fluorescent sensing peptide hydrogel in Example 3 of the present invention; Figure 4 1H nuclear magnetic resonance spectra of polylysine before and after modification with two fluorescent molecules in Example 4 of the present invention; Figure 5 Rheological property diagram of the ratiometric fluorescent sensing peptide hydrogel in Example 5 of the present invention; Figure 6 Fluorescence images of the ratiometric fluorescent sensing peptide hydrogel at different H2O2 concentrations in Example 6 of the present invention; Figure 7 Emission spectra of the ratiometric fluorescent sensing peptide hydrogel co-cultured at different H2O2 concentrations in Example 6 of the present invention; Figure 8 Change diagram of the characteristic peak intensity of the ratiometric fluorescent sensing peptide gel in different groups under 365 nm excitation in Example 6 of the present invention; Figure 9 Photographs of the plate coating experiment of the ratiometric fluorescent sensing peptide hydrogel on Escherichia coli in Example 7 of the present invention; Figure 10 Photographs of the plate coating experiment of the ratiometric fluorescent sensing peptide hydrogel on Staphylococcus aureus in Example 7 of the present invention; Figure 11 Scanning electron microscope images of the biofilms of Escherichia coli (upper) and Staphylococcus aureus (lower) in Example 7 of the present invention; Figure 12 Fluorescence confocal laser scanning microscope images of Escherichia coli (left) and Staphylococcus aureus (right) after treatment with the hydrogel in Example 7 of the present invention; Figure 13 Hemolysis test diagram of the ratiometric fluorescent sensing peptide hydrogel in Example 8 of the present invention; Figure 14 Cytotoxicity analysis diagram of the ratiometric fluorescent sensing peptide hydrogel on L929 cells after 24 h with a concentration gradient in Example 9 of the present invention; Figure 15 Total antioxidant capacity scavenging rate diagram of the ratiometric fluorescent sensing peptide hydrogel in different concentrations of Ply-C7 / TPE solution in Example 10 of the present invention; Figure 16 Hydroxyl radical scavenging rate diagram of the ratiometric fluorescent sensing peptide hydrogel in different concentrations of Ply-C7 / TPE solution in Example 10 of the present invention; Figure 17This is a graph showing the clearance rate of H2O2 in Ply-C7 / TPE solutions of different concentrations using the ratiometric fluorescent sensor peptide hydrogel in Example 10 of the present invention; Figure 18 This is a graph showing the scavenging rate of superoxide radicals of the ratiometric fluorescent sensor peptide hydrogel in Example 10 of the present invention in Ply-C7 / TPE solutions of different concentrations; Figure 19 are images of infected wounds at different treatment time intervals in Example 11 of the present invention; Figure 20 The wound healing and data statistics at different treatment times in Example 11 of the present invention; Figure 21 This is a photo of bacterial colonies obtained from an infected wound on the 7th day in Example 11 of the present invention; Figure 22 The skin tissue diagram of the wound area of ​​mice stained by Masson and H&E in Example 11 of the present invention; Figure 23 This is a graph of INOS and CD206 levels in vivo detected by immunofluorescence laser confocal scanning microscopy of tissue sections on day 7 in Example 11 of the present invention; Figure 24 This is a diagram of the training and application of the KNN model in Example 12 of the present invention. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be further described below.

[0021] Unless otherwise specified, all chemical reagents were commercially available and used directly without further purification. N6-Cbz-L-lysine and L-glutamic acid-5-benzyl ester were purchased from Aladdin Reagents. Triphosgene, hexylamine, anhydrous ether, THF, anhydrous DMF, DMSO, TFA, phosphate buffered saline (PBS, 0.01 M, pH = 7.2-7.4), α-pinene, EDCI, and C7 were purchased from Adamas Reagents Co., Ltd. n-Hexane was purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd. TPE-NH2 was purchased from Xi'an Qiyue Biotechnology Co., Ltd. Mice aged approximately 4-6 weeks were purchased from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences. Hydrogen peroxide (H2O2) detection kit and 2,2-diphenyl-1-picrylhydrazyl (DPPH) detection kit were purchased from Beijing Sora Biotechnology Co., Ltd. Hydroxyl radical (·OH) detection kit was purchased from Shanghai Jingkang Bioengineering Co., Ltd., China. O2 - The detection kit was purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.

[0022] In the present invention, THF is tetrahydrofuran; anhydrous DMF is anhydrous N,N-dimethylformamide; DMSO is dimethyl sulfoxide ； TFA is trifluoroacetic acid; EDCI is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; C7 is coumarin 7; TPE-NH2 is tetraphenylethylene; Lys-NCA is benzyl (S)-(4-(2,5-dioxooxazolidin-4-yl)butyl)carbamate; Glu-NCA is benzyl (S)-3-(2,5-dioxooxazolidin-4-yl)propionate; ROS is reactive oxygen species; TK-2COOH is dithioketal dicarboxylic acid; TPE-TK is tetraphenylethylene dithioether propionic acid; C7-TK is coumarin-benzimidazole dithioether propionic acid; Plys-TK-TPE is polylysine modified with aggregation-induced emission fluorophore tetraphenylethylene; Plys-TK-C7 is polylysine modified with aggregation-induced quenching fluorophore coumarin 7; PGlu is polyglutamic acid; PGlu / DMSO is a dimethyl sulfoxide solution of polyglutamic acid; Ply-TPE / DMSO is a dimethyl sulfoxide solution of polylysine modified with aggregation-induced emission fluorophore tetraphenylethylene; Ply-C7 / DMSO is a dimethyl sulfoxide solution of polylysine modified with aggregation-induced quenching fluorophore coumarin 7.

[0023] Example 1: Preparation method of a fluorescent sensing peptide hydrogel.

[0024] The synthesis process is as Figure 1 shown and specifically includes the following steps: S1: Preparation of benzyl (S)-(4-(2,5-dioxooxazolidin-4-yl)butyl)carbamate (Lys-NCA).

[0025] Dissolve 10 g of N6-Cbz-L-lysine and 20 mL of α-pinene in anhydrous THF, react at 55 °C for 45 min, pour the reaction solution into 200 mL of n-hexane, precipitate a yellow solid, filter, and dry in vacuo.

[0026] S2: Preparation of benzyl (S)-3-(2,5-dioxooxazolidin-4-yl)propionate (Glu-NCA).

[0027] Dissolve 10 g of L-glutamic acid 5-benzyl ester and 20 mL of α-pinene in anhydrous THF, react at 55 °C for 3 h, pour the reaction solution into 200 mL of n-hexane, precipitate a pale yellow solid, filter, and dry in vacuo.

[0028] S3: Preparation of polyglutamic acid and polylysine with protecting groups.

[0029] In the glove box, 17.8 mmol of the monomer was weighed and dissolved in 20 mL of anhydrous DMF. After sealing, the solution was taken out, and 1 mL of 2% hexylamine / anhydrous DMF solution was injected into the reaction solution. After reacting under negative pressure for 48 h, the reaction solution was poured into 300 mL of anhydrous diethyl ether, and the solid was obtained by centrifugation. After repeating this step twice, it was dried under reduced pressure to obtain polyglutamic acid with a protecting group (yellow solid) and polylysine (green solid).

[0030] S4: Removal of the protecting groups of polyglutamic acid and polylysine.

[0031] The polymer in step (3) was dissolved in TFA, and then 30% hydrobromic acid acetic acid solution was added dropwise. The flask was sealed with a rubber stopper and a balloon, and the mixture was stirred for 2 h. 5 times the amount of acetone was added to the reaction solution for precipitation, centrifuged, and washed with acetone 2 - 3 times until the supernatant was colorless. A white powder was obtained after freeze-drying.

[0032] S5: Modifying the polymer with fluorescent molecules.

[0033] First, the fluorescent molecules TPE-NH2 and C7 were respectively coupled with the ROS-responsive group K-2COOH to obtain TPE-TK and C7-TK, and then they were respectively modified onto the side chains of polylysine. Since the reaction conditions of the two fluorescent molecules are the same, the unified experimental steps are as follows: The fluorescent molecule was dissolved in DMSO, and then 2.5 equivalents of EDCI, triethylamine, and DMPA were added. After reacting at room temperature for 3 h, 55 equivalents of polylysine, 1 mole of EDCI, triethylamine, and DMPA were added, and the reaction was carried out overnight at room temperature. 10 times the amount of acetone was added to the reaction solution for precipitation to obtain an oily precipitate. The remaining organic reagents were removed by rotary evaporation to obtain a solid polymer.

[0034] S6: Preparation of the hydrogel.

[0035] The polymer in step (5) was dissolved in DMSO at a concentration of 100 μM / mL. One portion each of two portions of PGlu / DMSO, Ply-TPE / DMSO, and Ply-C7 / DMSO were mixed, and then EDCI equivalent to 30% of the total molar amount of lysine was added. The mixture was quickly and thoroughly mixed and then poured into a mold. After reacting overnight, the hydrogel and the mold were placed in deionized water for dialysis for 12 h, changing the water every 2 h, and finally dialysis was carried out using physiological saline. Most of the finally prepared hydrogel remained in the mold.

[0036] Scanning electron microscope of the fluorescent sensing peptide hydrogel in Example 2.

[0037] The hydrogel obtained in Example 1 was dissolved in ultrapure water to prepare a solution with a concentration of 1 mg / mL. The solution was dropped onto the surface of a silicon wafer and dried at room temperature to obtain a sample. The sample was detected using a scanning electron microscope at 2 kV. As Figure 2 shown, the polypeptide hydrogel has a honeycomb-like porous microstructure, which is crucial for fixing hydrophobic fluorescent molecules in the hydrogel network.

[0038] Example 3: Compressive property test of the fluorescent sensing peptide hydrogel.

[0039] The hydrogel obtained in Example 1 was tested using a CTM2050 microcomputer-controlled electronic universal testing machine at a loading speed of 1 mm / min to collect load and displacement data. The prepared cylindrical hydrogel sample had a diameter of 8 mm and a height of 9 mm. As Figure 3 shown, the polypeptide hydrogel has sufficient compressive properties to meet the requirements for promoting wound healing.

[0040] Example 4: Nuclear magnetic resonance spectroscopy test of the fluorescent sensing peptide hydrogel.

[0041] The hydrogel prepared in Example 1 was dissolved in DMSO-d6 and the spectrum was recorded at room temperature on a Bruker AV 400 MHz spectrometer using TMS as the standard. As Figure 4 shown, both C7 (characteristic peak at approximately 7.5 ppm) and TPE-NH2 (characteristic peak at approximately 7.0 ppm) were successfully grafted onto the polymer.

[0042] Example 5: Rheological property test of the fluorescent sensing peptide hydrogel.

[0043] The hydrogel obtained in Example 1 was used to measure the viscoelastic properties in a strain sweep experiment in oscillatory mode using a HAKKE rheometer. The frequency was set to 1 Hz, and the storage modulus (G') and loss modulus (G'') values were recorded through a sweep test of strain from 0.1% to 100%. The gap width used was 5 mm. As Figure 5 shown, the polypeptide hydrogel has sufficient stability to meet the requirements for wound detection.

[0044] Example 6: In vitro real-time ROS response test of the fluorescent sensing peptide hydrogel.

[0045] H2O2 solutions with concentrations of 0, 100, 200, 300, 400, and 500 μM were prepared in physiological saline. As Figure 6 shown, the hydrogel obtained in Example 1 was transferred into the H2O2 solution and co-incubated for 30 min. After taking out the hydrogel, the residual solution was washed away, Figure 6Group a shows the image of the hydrogel before UV irradiation under the cultivation of H2O2 with a concentration of 0 - 500 μM. The image was captured under a 356 nm UV lamp, as Figure 6 shown in group b, and an obvious color change was observed, which supports our design concept. As Figure 7 shown, the fluorescence characteristics of the polypeptide hydrogel indicate the relationship between the characteristic peak intensities of the two fluorescent molecules and different concentrations of H2O2. When analyzed separately, it was found that under consistent cultivation conditions, as Figure 8 shown in group b, the peak intensity of TPE increases with the increase of H2O2 concentration, while the peak intensity of C7 decreases, as Figure 8 shown in group a. This inverse relationship confirms the mechanism of ROS-triggered fluorescence change.

[0046] Example 7: Antibacterial performance test of the fluorescent sensing peptide hydrogel.

[0047] Escherichia coli and Staphylococcus aureus were cultured in LB medium (0.10 g / mL) at 37 °C for 12 h, and the suspension was diluted to 10^6, 10^5, 10^4 (CFU / mL) with PBS. Hydrogel discs with a diameter of 5.5 mm and a height of 1 mm were cultured with 1 mL of the diluted bacterial solution in a shaker at 37 °C for 4 - 6 h. 100 μL of the bacterial solution was taken and dropped onto an LB AGAR plate, and the bacterial solution was evenly spread on the surface of the medium with a spreader, and then cultured in an incubator at 37 °C for 12 h to compare the bacterial growth in the control group and the material group. All results were repeated three times. The results show that, as Figure 9 and Figure 10 shown, the hydrogel has a significant antibacterial effect on both Escherichia coli and Staphylococcus aureus.

[0048] To evaluate the ablation effect of the hydrogel on bacteria, scanning electron microscopy analysis was also carried out. The bacteria were washed with PBS and cultured with the hydrogel for 0 - 4 h, then fixed in 2.5% glutaraldehyde at 4 °C for 12 h, and then dehydrated in ethanol with different concentration gradients (25%, 50%, 75% and 100%), and then air-dried. Compared with Figure 11 the a control group of Figure 11 the bacteria co-cultured with the hydrogel in b of Figure 11 showed obvious damage, such as membrane adhesion and leakage of bacterial contents. In the c column pictures of

[0049] The planktonic bacteria were stained using the "LIVE / DEAD BacLight Bacterial Viability Kit". The live bacteria stained with SYTO-9 were green, while the dead bacteria stained with propidium iodide (PI) were red. 2000 μL of the bacterial suspension (Escherichia coli or Staphylococcus aureus, with a total colony count of approximately 10^9 (CFU) / mL) was centrifuged to remove the LB medium, and then added to 1.0 mL of PBS solution and incubated together on an oscillating bed at 37 °C for 0 - 24 h. Then, the mixed dispersion was centrifuged at 6000 r / min for 15 min and resuspended in 300 μL of PBS solution containing 0.5 μL of SYTO 9 dye solution and 0.5 μL of PI solution, and the supernatant was removed after 15 min.

[0050] Centrifuge again at 6000 r / min, and resuspend the precipitate in 300 μL of PBS to obtain the stained sample. Subsequently, 1 μL of the above dispersion was dropped onto a clean glass slide and covered with a cover slip in a circle. As Figure 12 shown in this analysis, it further illustrates that the polypeptide hydrogel has excellent antibacterial properties.

[0051] Example 8: Hemolysis test of the fluorescent sensing peptide hydrogel.

[0052] Collect 2 mL of fresh blood from the eyes of mice, centrifuge (2500 r / min, 10 min), and resuspend the red blood cells (RBC) in PBS to prepare an RBC suspension. Then the cells were contacted with hydrogels of different weights (0 - 200 mg) for 2 h (gently invert the mixture every 2 h to increase the contact between the gel and blood cells). Then, the mixture was centrifuged at 4 °C for 5 min, and the absorbance of the supernatant was measured at a wavelength of 560 nm using a microplate reader. The positive control and negative control were deionized water and PBS (pH = 7.4), respectively. As Figure 13 shown, even when the dose of the hydrogel far exceeds the typical wound application level (200 mg of hydrogel fragments), its hemolytic activity is negligible. This indicates that the hydrogel has good blood compatibility and is unlikely to cause damage to red blood cells during in vivo applications.

[0053] Example 9: Cytotoxicity test of the fluorescent sensing peptide hydrogel.

[0054] The L929 cell line was cultured in MEM containing 10% v / v FBS and 1% v / v penicillin-streptomycin at 37 °C and 5% CO2. The cytotoxicity of the polypeptide against L929 and L02 cells was evaluated using the CCK-8 assay. Then, 20 μL of polypeptide solutions at different concentrations were added, and the cells were cultured for another 24 h. After the treatment, 20 μL of CCK-8 dye was added to each well, and the cells were cultured for an additional 1 h at 37 °C. Cells treated with PBS were used as a control. Subsequently, the absorbance was measured at a wavelength of 450 nm using a microplate reader for cell viability analysis. The relative cell viability (%) was determined by comparing the treated and untreated samples. As Figure 14 shown, at all tested concentrations, the hydrogel maintained a cell survival rate of 80% in L929 cells, demonstrating its excellent cell compatibility. This indicates that the hydrogel is not only safe for cells but also supports cell growth and proliferation.

[0055] Example 10: In vitro antioxidant test of the fluorescent sensing peptide hydrogel.

[0056] Three representative ROS, H2O2, O2 - and ·OH, were mainly studied to evaluate the ability of the hydrogel to scavenge ROS. A free radical scavenging assay was performed using a DPPH kit; the absorbance was measured at a wavelength of 405 nm using an H2O2 detection kit to determine the concentration of the remaining H2O2, and the elimination ability of H2O2 was calculated. To test the scavenging ability of the hydrogel against hydroxyl radicals, ·OH was first generated in the system using the Fenton reaction, and then salicylic acid was added to materials at different concentrations. Salicylic acid reacts with ·OH to form a colored compound, and the absorbance was measured at a wavelength of 550 nm using an ·OH detection kit to quantify the ·OH elimination ability of the materials.

[0057] The results showed that the hydrogel could effectively inhibit DPPH radicals at a concentration as low as 10 μg / mL, and the inhibition rate exceeded 80% when the concentration was 100 μg / mL ( Figure 15 ). In addition, the hydrogel also had a concentration-dependent ROS scavenging activity. When the concentration was 100 μg / mL, the inhibition rate of the hydrogel against ·OH exceeded 90% ( Figure 16 ); when the concentration was 200 μg / mL, the hydrogel had a significant scavenging activity against H2O2 ( Figure 17 ). In addition, at a concentration of 100 μg / mL, the scavenging rate of the hydrogel against O2 - was close to 90% ( Figure 18 ).

[0058] These results demonstrate the strong antioxidant capacity of the hydrogel, which is crucial for alleviating oxidative stress, a major factor delaying wound healing. Additionally, the hydrogel can scavenge low concentrations of ROS, especially H2O2 and O2 - , indicating its potential as a ROS sensor in the wound environment. The reactivity of the ROS-responsive groups of the hydrogel with these ROS markers suggests that this material can reliably monitor the ROS levels in wounds in real time.

[0059] Example 11: Test on the wound-healing promoting performance of the fluorescent sensing peptide hydrogel.

[0060] White BABL / c mice were used to evaluate the wound-healing effect of the polypeptide hydrogel dressing on diabetic chronic wounds. A wound with a diameter of 4.50 mm was created on the back of each mouse, and then it was infected with Staphylococcus aureus (10^8 CFU / mL). Then the infected mice (about 20 g) were randomly divided into three groups (5 mice in each group): natural healing group, PBS treatment group, and peptide hydrogel group. The dressing in the peptide hydrogel group was changed every three days, while the PBS group was replaced with a new PBS solution every three days. The natural healing group received no treatment. The wound-healing process was monitored daily by taking pictures of the wounds and calculating the wound area using image analysis software. As Figure 19 shown, the wounds treated with the hydrogel dressing healed significantly faster and were completely closed on the 9th day. According to the time course calculated by continuous image analysis, the hydrogel group had a relatively significant effect on promoting wound healing ( Figure 20 ).

[0061] To evaluate the in vivo antibacterial effect of different treatments, wound tissue samples were collected five days after the initial infection and rinsed with PBS. Then the eluted bacteria were cultured on LB agar plates for colony counting. As Figure 21 shown, there were almost no bacterial colonies in the polypeptide hydrogel group, indicating that the hydrogel dressing had good antibacterial effects at the wound site.

[0062] On the 12th day, the mice in each group were sacrificed, and wound tissue sections were collected for H&E and Masson's trichrome staining. Figure 22 In Figure 22 , group a shows the eosin (H&E) staining of the mouse skin tissue in the wound area, and group b shows the Masson staining of the mouse skin tissue in the wound area. Histological analysis showed that the wounds in the polypeptide hydrogel group were completely closed, with a new epidermis formed, the least number of inflammatory cells, and neatly arranged fibroblasts, which was very similar to the normal tissue structure (

[0063] Four hours after wound formation (24 hours after the application of the hydrogel dressing), the mice in the natural healing group and the hydrogel group were sacrificed, and immunofluorescence tissue sections were analyzed to evaluate the expression of iNOS and CD206 by fluorescence staining. As Figure 23 shown, quantitative analysis using ImageJ showed that the density of positive cells in the natural healing group was higher than that in the polypeptide hydrogel group, indicating that the polypeptide hydrogel group had a lower level of inflammation.

[0064] Example 12: Sensing performance test of the fluorescent sensing peptide hydrogel.

[0065] JPEG images of the hydrogel were taken using a digital camera (Nikon D7000, lens focal length 18 - 105 mm). All images were cropped to a standard size of 250×500 pixels using Python. Fluorescence measurement of the hydrogel was performed using a Hitachi F - 4700 fluorescence spectrophotometer equipped with a solid sample holder. To ensure the stability of the measurement, a mold of appropriate size was designed so that the size of the hydrogel matched the size of the solid sample holder (i.e., the hydrogel could be measured directly without cutting). In addition, the thickness of the hydrogel matched the specified thickness of the holder to prevent the hydrogel from being squeezed when the holder was tightened. A mouse model of diabetic infected wounds was established. Twelve hours after inoculation with different concentrations of Staphylococcus aureus, fluorescence imaging of the wounds was performed. First, the mice were anesthetized and placed with the wound facing up. 5 μL of normal saline was added to the wound, and the hydrogel in the mold was transferred to the wound site. After waiting for 30 min, imaging was performed under a 365 nm ultraviolet lamp. 1500 fluorescence images of the hydrogel with different ROS concentrations were collected, feature extraction was performed using distance variation, the RGB histogram of the feature region was obtained to train the KNN model, and according to the method of five - fold cross - validation, 20% of the images in the dataset were used as the validation set (as Figure 24 shown above). Figure 24 The following are the analysis results of the model for samples of five concentrations (100 - 500 μM). The signal distribution curves are discrete, indicating that the model does not simply memorize the training data but can generalize to new, unseen data, meaning it can accurately process new data. The bar chart shows the comparison of the ground truth and predicted results of different samples. The accuracy and reliability of the model were evaluated by comparing the actual values and the algorithm - predicted values.

[0066] The above are only the preferred embodiments of the present invention and do not impose any limitation on the present invention. Any person skilled in the art within the technical field, without departing from the technical solution of the present invention, making any form of equivalent substitution or modification and other changes to the technical solution and technical content disclosed by the present invention, all belong to the content without departing from the technical solution of the present invention and still fall within the protection scope of the present invention.

Claims

1. A machine learning-assisted ratiometric fluorescence sensing peptide hydrogel, characterized in that, The ratio fluorescence sensing peptide hydrogel is prepared by mixing polylysine Plys-TK-TPE modified with the aggregation-induced emission fluorophore tetraphenylethylene, polylysine Plys-TK-C7 modified with the aggregation-induced quenching fluorophore coumarin 7, and polyglutamic acid PGlu, and then adding the condensing agent EDCI and crosslinking through an amide reaction.

2. A method for preparing a ratio fluorescence sensing peptide hydrogel, characterized in that, Based on the raw materials of N6-Cbz-L-lysine and L-glutamic acid 5-benzyl ester, the ratio fluorescence sensing peptide hydrogel for machine learning assistance described in claim 1 is prepared, including the following steps: S1: Dissolve N6-Cbz-L-lysine and α-pinene in anhydrous THF, heat, stir, and then pour the reaction solution into n-hexane to precipitate a yellow solid. Dry it to obtain (S)-(4-(2,5-dioxooxazolidin-4-yl)butyl)carbamic acid benzyl ester, that is, Lys-NCA. S2: Dissolve L-glutamic acid 5-benzyl ester and α-pinene in anhydrous THF, heat, stir, and then pour the reaction solution into n-hexane to precipitate a pale yellow solid. Dry it to obtain (S)-3-(2,5-dioxooxazolidin-4-yl)propionic acid benzyl ester, that is, Glu-NCA. S3: Dissolve the Lys-NCA and the Glu-NCA in anhydrous DMF respectively, then add a n-hexylamine / anhydrous DMF solution, react under negative pressure conditions. After the reaction is completed, pour the reaction solution into anhydrous diethyl ether, centrifuge, and dry under reduced pressure to obtain polylysine with a protecting group and polyglutamic acid with a protecting group. S4: Dissolve the polylysine with a protecting group and the polyglutamic acid with a protecting group in TFA respectively, then dropwise add a hydrobromic acid acetic acid solution with a mass ratio of 30%, seal and stir. After the reaction is completed, add acetone to precipitate, centrifuge, and wash with acetone 2-3 times until the supernatant is colorless. Dialyze and then freeze-dry the polylysine and polyglutamic acid. S5: Dissolve the fluorescent molecule TPE-NH2 and the fluorescent molecule C7, the reactive oxygen species-responsive group dithioketal dicarboxylic acid, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in an appropriate amount of DMSO respectively. Then add triethylamine and N,N-dimethylacrylamide. After reacting for a period of time, continue to add the polylysine, EDCI, triethylamine, and DMPA. After the reaction is completed, add an appropriate amount of acetone for precipitation to obtain an oily precipitate. Remove the remaining organic reagents by rotary evaporation to obtain the solid polymers Plys-TK-TPE and Plys-TK-C7. S6: Dissolve the polyglutamic acid, Plys-TK-TPE, and Plys-TK-C7 in DMSO respectively to obtain PGlu / DMSO, Ply-TPE / DMSO, and Ply-C7 / DMSO solutions. Mix the three solutions, add EDCI, and mix the mixture quickly and thoroughly. Then pour it into a mold to obtain a hydrogel, and dialyze it with the mold in deionized water. Finally, most of the hydrogel remains in the mold. In step S6, the mixing molar ratio of the PGlu / DMSO, Ply-TPE / DMSO, and Ply-C7 / DMSO solutions is 2:1:1; The amount of EDCI is 30% of the total molar amount of lysine; the dialysis time is 12 h.

3. The method for preparing the ratio fluorescence sensing peptide hydrogel according to claim 2, wherein In step S1, the reaction temperature is 55 °C, the stirring time is 45 min, the drying temperature is 40 °C, and the drying time is 12 h.

4. The method for preparing the ratio fluorescence sensing peptide hydrogel according to claim 2, wherein, In step S2, the reaction temperature is 55 °C, the stirring time is 3 h, the drying temperature is 40 °C, and the drying time is 12 h.

5. The method for preparing a ratio fluorescent sensing peptide hydrogel according to claim 2, characterized in that, In step S3, the reaction is carried out under anhydrous conditions, the stirring time is 48 h, the molar ratios of Lys-NCA and Glu-NCA to n-hexane are both 1 / 200, and the volume of anhydrous diethyl ether is 15 times that of the reaction solution.

6. The method for preparing a ratio fluorescent sensing peptide hydrogel according to claim 2, wherein In step S4, the reaction temperature is 25 °C, the stirring time is 2 h, and the volume of acetone is five times that of the reaction solution.

7. The method for preparing the ratio fluorescent sensing peptide hydrogel according to claim 2, wherein In step S5, the molar ratio of the fluorescent molecule C7: TK-2COOH: DMPA is: 1:1:2.5; The molar ratio of the fluorescent molecule C7: triethylamine: DMPA is: 1:2.5:2.5, the reaction temperature is 25 °C, and the stirring time is 3 h; The molar ratio of the fluorescent molecule TPE-NH2 / polylysine / EDCI / triethylamine / DMPA is 1:55:1:1:1, the reaction temperature is 25 °C, and the stirring time is 12 h.

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