A machine learning assisted ratiometric fluorescent sensing peptide hydrogel and a preparation method thereof

Abstract

The present application provides a machine learning assisted ratio fluorescent sensing peptide hydrogel and a preparation method thereof, wherein the ratio 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 of the present application is exposed to active oxygen, the thione group is cleaved, resulting in the separation of the fluorescent molecule from the polymer and the aggregation in the water environment. This process leads to the quenching of coumarin 7 and the aggregation of tetraphenylethylene, thereby causing the change of the excitation light intensity ratio and the corresponding change of the fluorescence color, so that the active oxygen level can be monitored in real time and quantitatively through machine learning assistance, and the inflammation degree of the wound can be accurately reflected, thereby providing valuable data for the closed-loop treatment of chronic diabetic wounds.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of preparation and application of hydrogel dressing materials, in particular to a machine learning assisted ratio fluorescent sensing peptide hydrogel and a preparation method thereof. BACKGROUND

[0002] Due to hyperglycemia and metabolic disorders, the inflammation management of diabetic chronic wounds remains a major clinical challenge. The normal wound healing process is usually divided into four consecutive stages: hemostasis, inflammation, proliferation and remodeling. In the inflammation stage, inflammatory cells will clear the 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 can lead to persistent inflammation, forming a pathologic inflammatory microenvironment. Therefore, effective regulation of the inflammatory response has important 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, generate reactive oxygen species (ROS) through NADPH oxidase-mediated superoxide anion (O2 - ) production; at the same time, the secretion of pro-inflammatory cytokines (such as TNF-α and IL-1β) triggers intracellular signal cascades, causing the calcium level of the cell membrane to rise, thereby activating calcium-dependent protein kinases, which in turn induce the formation of ROS. Moreover, studies have found that ROS levels are positively correlated with the inflammatory state, and ROS can be used as a biomarker to assess the inflammatory state of chronic wounds.

[0003] In clinical practice, ROS levels are usually quantified using fluorescent special probes (such as DCFH-DA, Luminol) or electron spin resonance (ESR) technology. The detection process includes sample collection, processing, detection and data analysis, which takes a long time and is difficult to monitor ROS levels in situ and real-time at the wound site. At present, a variety of ROS fluorescent probes have been developed, mainly using modified fluorophores to target ROS. However, most of the ROS detection methods based on fluorescence 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 a standard curve based on intensity suitable for in situ wound monitoring. Compared with the method based on fluorescence intensity, the ratio fluorescence method relies on color change, has anti-interference ability and high sensitivity. In addition, polypeptides have great potential in biomedical applications due to their biocompatibility, adjustable biodegradability, chemical diversity and programmable biological activity. Recent research progress has expanded the application of polypeptides in anti-infection therapy and inflammation regulation, especially in chronic wound and inflammation treatment. Therefore, it is necessary to develop in situ ratio fluorescent materials based on polypeptides to cope with the challenges brought by the complex environment of chronic diabetic wounds, so as to realize real-time monitoring of wound inflammation and provide valuable data for guiding the adjustment of treatment programs. However, there is no technical scheme found at home and abroad about preparing ratio fluorescent sensing peptide hydrogel for real-time monitoring of inflammation in diabetic chronic wounds. SUMMARY

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

[0005] To achieve the above-mentioned purpose, the present application provides a ratio fluorescent sensing peptide hydrogel, which is cross-linked from modified polylysine modified with different fluorescent molecules and polyglutamic acid.

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

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

[0008] The application also provides a method for preparing a ratio fluorescent sensing peptide hydrogel, which is prepared from N6-Cbz-L-lysine and L-glutamic acid-5-benzyl ester raw materials and comprises the following steps:

[0009] S1: N6-Cbz-L-lysine and alpha-pinene are dissolved in anhydrous THF, heated, stirred, and then the reaction solution is poured into n-hexane to precipitate yellow solids, which are dried to obtain (S)-(4-(2,5-dioxooxazolidin-4-yl)butyl) benzyl carbamate, i.e., Lys-NCA;

[0010] S2: L-glutamic acid-5-benzyl ester and alpha-pinene are dissolved in anhydrous THF, heated, stirred, and then the reaction solution is poured into n-hexane to precipitate light yellow solids, which are dried to obtain (S)-3-(2,5-dioxooxazolidin-4-yl) propionic acid benzyl ester, i.e., Glu-NCA;

[0011] S3: Lys-NCA and Glu-NCA are respectively dissolved in anhydrous DMF, and then n-hexylamine / anhydrous DMF solution is added, and the reaction is carried out under negative pressure, after the reaction is completed, the reaction solution is poured into anhydrous ether, centrifuged, and dried under reduced pressure to obtain polylysine with a protective group and polyglutamic acid with a protective group;

[0012] S4: The polylysine with a protective group and the polyglutamic acid with a protective group are respectively dissolved in TFA, and then a hydrogen bromide acetic acid solution with a mass ratio of 30% is added dropwise, sealed and stirred, after the reaction is completed, acetone is added for precipitation, centrifuged, and washed with acetone for 2-3 times until the supernatant is colorless, and after dialysis, the polylysine and the polyglutamic acid are freeze-dried;

[0013] S5: The fluorescent molecules TPE-NH2 and C7 are respectively dissolved in a proper amount of DMSO with the active oxygen response group dithioketone and 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide hydrochloride, and then triethylamine and N,N-dimethylacrylamide are added, and after a period of reaction, polylysine, EDCI, triethylamine and DMPA are continuously added, after the reaction is completed, a proper amount of acetone is added for precipitation to obtain oily precipitates, the remaining organic reagents are removed by rotary evaporation to obtain solid polymers Plys-TK-TPE and Plys-TK-C7;

[0014] S6: The polyglutamic acid, Plys-TK-TPE and Plys-TK-C7 are respectively dissolved in DMSO to obtain PGlu / DMSO, Ply-TPE / DMSO and Ply-C7 / DMSO solutions, and the three solutions are mixed, EDCI is added, the mixture is rapidly and fully mixed, and then poured into a mold to obtain a hydrogel, and the hydrogel and the mold are placed in deionized water for dialysis, and finally most of the hydrogel remains in the mold.

[0015] Further, in step S1, the reaction temperature is 55℃, the stirring time is 45 min, the drying temperature is 40℃, and the drying time is 12 h.

[0016] Further, in step S2, the reaction temperature is 55℃, the stirring time is 3 h, the drying temperature is 40℃, and the drying time is 12 h.

[0017] Further, in step S3, the reaction is carried out under anhydrous conditions, the stirring time is 48 h, the molar ratio of Lys-NCA and Glu-NCA is both 1 / 200, and the volume of anhydrous ether is 15 times that of the reaction solution.

[0018] Further, in step S4, the reaction temperature is 25℃, the stirring time is 2 h, and the volume of acetone is five times that of the reaction solution.

[0019] Further, in step S5, the molar ratio of the fluorescent molecule C7: TK-2COOH: DMPA is 1:1:2.5.

[0020] The molar ratio of the fluorescent molecule C7: triethylamine: DMPA is 1:2.5:2.5, the reaction temperature is 25℃, and the stirring time is 3 h.

[0021] The molar ratio of the fluorescent molecule TPE-NH2 / polylysine / EDCI / triethylamine / DMPA is 1:55:1:1:1, the reaction temperature is 25℃, and the stirring time is 12 h.

[0022] Further, in step S6, the mixed molar ratio of PGlu / DMSO, Ply-TPE / DMSO and Ply-C7 / DMSO solutions is 2:1:1.

[0023] The amount of EDCI is 30% of the total moles of lysine, and the dialysis time is 12 h.

[0024] Compared with the prior art, the present application has the following advantages:

[0025] (1) The ratio fluorescent sensing peptide hydrogel of the present application not only can monitor the inflammatory state in ratio, but also can promote wound healing, and has potential application value in the field of treatment of diabetic chronic wounds.

[0026] (2) The ratio fluorescent sensing peptide hydrogel of the present application mainly relies on color change to monitor the inflammatory state, and has stronger anti-interference ability and high sensitivity compared with methods based on fluorescence intensity.

[0027] (3) The ratio fluorescent sensing peptide hydrogel prepared by the present application has good water solubility and biocompatibility.

[0028] (4) The present application is simple, mild in experimental conditions and easy to operate. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 A schematic diagram of the synthesis process of the ratio fluorescent sensing peptide hydrogel in Example 1 of the present application;

[0030] Figure 2 A scanning electron microscope image of the ratio fluorescent sensing peptide hydrogel in Example 2 of the present application;

[0031] Figure 3 A stress-strain curve of the ratio fluorescent sensing peptide hydrogel in Example 3 of the present application;

[0032] Figure 4 Nuclear magnetic resonance hydrogen spectrum of polylysine before and after modification of two fluorescent molecules in Example 4 of the present application;

[0033] Figure 5 Rheological property diagram of the ratio fluorescent sensing peptide hydrogel in Example 5 of the present application;

[0034] Figure 6 Fluorescence image of the ratio fluorescent sensing peptide hydrogel in Example 6 of the present application under different H2O2 concentrations;

[0035] Figure 7 Emission spectrum diagram of the ratio fluorescent sensing peptide hydrogel co-cultured under different H2O2 concentrations in Example 6 of the present application;

[0036] Figure 8 Characteristic peak intensity change diagram of the ratio fluorescent sensing peptide hydrogel in different groups under 365 nm excitation in Example 6 of the present application;

[0037] Figure 9 Plate coating experiment photo of the ratio fluorescent sensing peptide hydrogel on E. coli in Example 7 of the present application;

[0038] Figure 10 Plate coating experiment photo of the ratio fluorescent sensing peptide hydrogel on S. aureus in Example 7 of the present application;

[0039] Figure 11 Scanning electron microscope image of the bacterial membrane of E. coli (top) and S. aureus (bottom) in Example 7 of the present application;

[0040] Figure 12 Bacterial live and dead staining fluorescence confocal laser scanning microscope image of E. coli (left) and S. aureus (right) treated by the hydrogel in Example 7 of the present application;

[0041] Figure 13Figure for hemolysis test of the ratio fluorescent sensing peptide hydrogel in Example 8 of the present application;

[0042] Figure 14 Figure for cytotoxicity analysis of L929 cells after 24 h of concentration gradient of the ratio fluorescent sensing peptide hydrogel in Example 9 of the present application;

[0043] Figure 15 Figure for total antioxidant capacity clearance of the ratio fluorescent sensing peptide hydrogel in different concentrations of Ply-C7 / TPE solution in Example 10 of the present application;

[0044] Figure 16 Figure for hydroxyl radical clearance of the ratio fluorescent sensing peptide hydrogel in different concentrations of Ply-C7 / TPE solution in Example 10 of the present application;

[0045] Figure 17 Figure for H2O2 clearance of the ratio fluorescent sensing peptide hydrogel in different concentrations of Ply-C7 / TPE solution in Example 10 of the present application;

[0046] Figure 18 Figure for superoxide radical clearance of the ratio fluorescent sensing peptide hydrogel in different concentrations of Ply-C7 / TPE solution in Example 10 of the present application;

[0047] Figure 19 Figure for images of infected wounds at different treatment time intervals in Example 11 of the present application;

[0048] Figure 20 Figure for wound healing and data statistics at different treatment times in Example 11 of the present application;

[0049] Figure 21 Figure for bacterial colony photographs obtained from infected wounds on the 7th day in Example 11 of the present application;

[0050] Figure 22 Figure for mouse skin tissue in the wound area stained with Masson and H&E in Example 11 of the present application;

[0051] Figure 23 Figure for in vivo INOS and CD206 levels detected by immunofluorescence laser confocal scanning microscopy of tissue sections at the 7th day in Example 11 of the present application;

[0052] Figure 24 Figure for training and application of KNN model in Example 12 of the present application. DETAILED DESCRIPTION

[0053] In order to make the purpose, technical scheme and advantages of the present application more clear, the technical scheme of the present application will be further described below.

[0054] Unless otherwise specified, all chemical reagents are commercially available and used directly without further purification. N6-Cbz-L-lysine and L-glutamic acid-5-benzyl ester were purchased from Aladdin Reagent Company. Triphosgene, hexylamine, anhydrous diethyl ether, THF, anhydrous DMF, DMSO, TFA, phosphate buffered saline (PBS, 0.01 M, pH = 7.2-7.4), a-pinene, EDCI and C7 were purchased from Adamas Reagent Co., Ltd. n-hexane was purchased from Tiemai (Shanghai) Chemical Industry Development Co., Ltd. TPE-NH2 was purchased from Xi'an Ziyue Biological Technology Co., Ltd. Mice about 4-6 weeks old were purchased from Shanghai Experimental Animal Center of Chinese Academy of Sciences. Hydrogen peroxide (H2O2) assay kit and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay kit were purchased from Beijing Solabio Technology Co., Ltd. Hydroxyl radical (·OH) assay kit was purchased from Jingkang Biological Engineering Co., Ltd. in Shanghai, China. - O2

[0055] In the present application, 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 (S)-(4-(2,5-dioxo-oxazolidin-4-yl)butyl) benzyl carbamate; Glu-NCA is (S)-3-(2,5-dioxo-oxazolidin-4-yl) propionic acid benzyl ester; ROS is reactive oxygen species; TK-2COOH is dithiol ketal; TPE-TK is tetraphenylethylene dithiol ketal propionic acid; C7-TK is coumarin-benzimidazole dithiol ketal propionic acid; Plys-TK-TPE is aggregation-induced emission luminophore tetraphenylethylene modified polylysine; Plys-TK-C7 is aggregation-induced quenching luminophore coumarin 7 modified polylysine; PGlu is polyglutamic acid; PGlu / DMSO is dimethyl sulfoxide solution of polyglutamic acid; Ply-TPE / DMSO is dimethyl sulfoxide solution of aggregation-induced emission luminophore tetraphenylethylene modified polylysine; Ply-C7 / DMSO is dimethyl sulfoxide solution of aggregation-induced quenching luminophore coumarin 7 modified polylysine.

[0056] Example 1: Preparation method of fluorescent sensing peptide hydrogel.

[0057] The synthesis process is shown in Figure 1 , which specifically includes the following steps:

[0058] S1: Preparation of (S)-(4-(2,5-dioxo-oxazolidin-4-yl)butyl) benzyl carbamate (Lys-NCA).

[0059] Dissolve 10 g N6-Cbz-L-lysine and 20 mL a-pinene in anhydrous THF, react at 55 °C for 45 min, pour the reaction solution into 200 mL of n-hexane, precipitate yellow solid, filter, and dry in vacuum.

[0060] S2: Preparation of (S)-benzyl 3-(2,5-dioxooxazolidin-4-yl)propanoate (Glu-NCA).

[0061] Dissolve 10 g L-glutamic acid-5-benzyl ester and 20 mL a-pinene in anhydrous THF, react at 55 °C for 3 h, pour the reaction solution into 200 mL of n-hexane, precipitate light yellow solid, filter, and dry in vacuum.

[0062] S3: Preparation of protected polyglutamic acid and polylysine.

[0063] In the glove box, weigh 17.8 mmol monomer and dissolve in 20 mL anhydrous DMF. After sealing, take out the solution, inject 1 mL 2% hexylamine / anhydrous DMF solution into the reaction solution. After reacting under negative pressure for 48 h, pour the reaction solution into 300 mL anhydrous ether, and obtain solid after centrifugation. Repeat this step 2 times, and dry under reduced pressure to obtain protected polyglutamic acid (yellow solid) and polylysine (green solid).

[0064] S4: Removal of polyglutamic acid and polylysine protecting groups.

[0065] Dissolve the polymer in step (3) in TFA, then add 30% hydrogen bromide acetic acid solution dropwise. Seal the flask with a rubber plug and balloon, and stir the mixture for 2 h. Add 5 times the amount of acetone to the reaction solution for precipitation, centrifuge, and wash with acetone 2-3 times until the supernatant is colorless. After freeze-drying, white powder is obtained.

[0066] S5: Modification of polymers with fluorescent molecules.

[0067] First, couple the fluorescent molecules TPE-NH2 and C7 with the ROS-responsive group K-2COOH respectively to obtain TPE-TK and C7-TK, and then modify them to the side groups of polylysine. Since the reaction conditions of the two fluorescent molecules are the same, the unified experimental procedure is as follows: dissolve the fluorescent molecules in DMSO, then add 2.5 equivalents of EDCI, triethylamine and DMPA. After reacting at room temperature for 3 h, add 55 equivalents of polylysine, 1 mole of EDCI, triethylamine and DMPA, and react at room temperature overnight. Add 10 times the amount of acetone to the reaction solution for precipitation to obtain oily precipitate. Remove the remaining organic reagents by rotary evaporation to obtain solid polymer.

[0068] S6: Preparation of hydrogel.

[0069] The polymer in step (5) was dissolved in DMSO with a concentration of 100 μΜ / mL. Two portions of PGlu / DMSO, Ply-TPE / DMSO and Ply-C7 / DMSO were mixed respectively, and then EDCI was added, which was equivalent to 30% of the total moles of lysine. The mixture was mixed thoroughly and then poured into the mold. After the reaction overnight, the hydrogel and the mold were placed in deionized water for dialysis for 12 h, and the water was changed every 2 h, and finally physiological saline was used for dialysis. Most of the final prepared hydrogel was left in the mold.

[0070] Example 2: Scanning electron microscope of fluorescent sensing peptide hydrogel.

[0071] The hydrogel obtained in Example 1 was dissolved in ultrapure water to prepare a solution with a concentration of 1 mg / mL, which was dropped onto the surface of a silicon wafer and dried at room temperature to prepare a sample. The sample was detected by a scanning electron microscope at 2 kV. As shown in FIG. 2, the polypeptide hydrogel has a honeycomb-like porous microstructure, which is crucial for fixing hydrophobic fluorescent molecules in the hydrogel network. Figure 2

[0072] Example 3: Compression resistance test of fluorescent sensing peptide hydrogel.

[0073] The hydrogel obtained in Example 1 was tested by using a CTM2050 microcomputer-controlled electronic universal testing machine with a loading speed of 1 mm / min, and the load and displacement data were collected. The prepared cylindrical hydrogel sample had a diameter of 8 mm and a height of 9 mm. As shown in FIG. 3, the polypeptide hydrogel has sufficient compression resistance, which is sufficient to meet the requirements of promoting wound healing. Figure 3

[0074] Example 4: Nuclear magnetic resonance spectrum test of fluorescent sensing peptide hydrogel.

[0075] The hydrogel prepared in Example 1 was dissolved in DMSO-d6, and the spectrum was recorded on a Bruker AV 400 MHz spectrometer at room temperature with TMS as the standard. As shown in FIG. 4, C7 (characteristic peak at about 7.5 ppm) and TPE-NH2 (characteristic peak at about 7.0 ppm) were successfully grafted to the polymer. Figure 4

[0076] Example 5: Rheological property test of fluorescent sensing peptide hydrogel.

[0077] ​​​The hydrogel obtained from Example 1 was used to measure viscoelastic properties using a HAKKE rheometer in oscillation mode. The frequency was set at 1 Hz, and the storage modulus (G') and loss modulus (G") values were recorded by strain sweep experiments from 0.1% to 100%. The gap width used was 5 mm. As shown in Figure 5 , the polypeptide hydrogel has sufficient stability to meet the requirements of wound detection.

[0078] Example 6: In vitro real-time ROS response test of fluorescent sensing peptide hydrogel.

[0079] H2O2 solutions with concentrations of 0, 100, 200, 300, 400, and 500 μΜ were prepared in physiological saline. As shown in Figure 6 , the hydrogel obtained from Example 1 was transferred to the H2O2 solution and incubated for 30 min. After removing the hydrogel, the residual solution was washed off, Figure 6 , group a is the image of the hydrogel before UV light irradiation under the culture of H2O2 with concentrations of 0-500 μΜ; the image was captured under a 356 nm UV lamp, as shown in Figure 6 , group b, a significant color change was observed, which supports our design concept. As shown in Figure 7 , the fluorescence characteristics of the polypeptide hydrogel show the relationship between the characteristic peak intensity of the two fluorescent molecules and the different concentrations of H2O2. When analyzed separately, it was found that under consistent culture conditions, as shown in Figure 8 , group b, the peak intensity of TPE increased with the increase of H2O2 concentration, while the peak intensity of C7 decreased, as shown in Figure 8 , group a. This inverse relationship confirms the mechanism of ROS-triggered fluorescence change.

[0080] Example 7: Antibacterial performance test of fluorescent sensing peptide hydrogel.

[0081] 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 with PBS to 10^6, 10^5, 10^4 (CFU / mL). The hydrogel disc with a diameter of 5.5 mm and a height of 1 mm was cultured with 1 mL of diluted bacterial solution in a 37°C shaking incubator for 4-6 h. 100 μL of bacterial solution was dropped onto the LB AGAR plate, and the bacterial solution was evenly spread on the surface of the medium with a spreader, and incubated in a 37°C incubator for 12 h. The bacterial growth of the control group and the material group was compared. All results were repeated three times. As shown in Figure 9 and Figure 10 , the hydrogel has significant antibacterial effect on Escherichia coli and Staphylococcus aureus.

[0082] To evaluate the ablation effect of hydrogels on bacteria, scanning electron microscopy analysis was also performed. The bacteria were washed with PBS and incubated with hydrogels for 0-4 h, then fixed in 2.5% glutaraldehyde at 4°C for 12 h, followed by dehydration in different concentration gradients of ethanol (25%, 50%, 75% and 100%), and then air-dried. The samples were sputter-coated with gold and observed under a scanning electron microscope (SEM, JEOL, JSM-6360LV) at 10 kV. Figure 11 Compared with the control group of a, Figure 11 In b, the bacteria co-cultured with hydrogels showed obvious damage, such as membrane adhesion and leakage of bacterial contents. In the column of c pictures, it can be clearly observed that the part of the bacteria in contact with the surface of the material are broken and adhered together, which directly reflects that the material has a strong antibacterial effect. Figure 11

[0083] The planktonic bacteria were stained using "LIVE / DEAD BacLight Bacterial Viability Kit", and the live bacteria stained with SYTO-9 were green, while the dead bacteria stained with iodine propyl iodide (PI) were red. 2000 μL of bacterial suspension (E. coli or S. aureus, total number of colonies about 10^9 (CFU) / mL) was centrifuged to remove the LB culture medium, and then 1.0 mL of PBS solution was added, and incubated together at 37°C on a shaking bed for 0-24 h. Then, the mixture dispersion was centrifuged at a speed of 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.

[0084] Again, centrifugation was performed at a speed of 6000 r / min, and the precipitate was resuspended in 300 μL of PBS to obtain the stained sample. Subsequently, 1 μL of the above dispersion was dropped on a clean glass slide, and a cover glass was covered. As Figure 12 This analysis further demonstrates that the polypeptide hydrogel has excellent antibacterial performance.

[0085] Example 8: Hemolysis test of fluorescent sensing peptide hydrogel.

[0086] 2 mL of fresh blood was collected from the eyes of mice, centrifuged (2500 r / min, 10 min), and the red blood cells (RBC) were resuspended in PBS to prepare an RBC suspension. Then the cells were contacted with different weights of hydrogel (0-200 mg) for 2 h (the mixture was gently inverted every 2 h to increase the contact between the gel and the 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 and negative controls were deionized water and PBS (pH = 7.4), respectively. As Figure 13 ​As shown, even at a dose of hydrogel far exceeding the typical level for wound applications (200 mg of hydrogel pieces), the hemolytic activity of the hydrogel was negligible. This indicates that the hydrogel has good blood compatibility and is unlikely to cause damage to red blood cells when applied in vivo.

[0087] Example 9: Cytotoxicity test of fluorescent sensing peptide hydrogel.

[0088] L929 cell line was cultured in MEM containing 10% v / v FBS, 1% v / v penicillin-streptomycin, at 37 °C, 5% CO2. The cytotoxicity of the polypeptides to L929 and L02 cells was evaluated by CCK-8 assay. Then 20 μL of polypeptide solution at different concentrations was added, and incubated with cells for 24 h. After treatment, 20 μL of CCK-8 dye was added to each well, and incubated at 37 °C for another 1 h. Cells treated with PBS were used as a control. Subsequently, the absorbance was detected at 450 nm wavelength using a microplate reader for cell viability analysis. The relative cell viability (%) was determined by comparing the treated and untreated samples. As shown in Figure 14 At all concentrations tested, the hydrogel maintained 80% cell viability 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.

[0089] Example 10: In vitro antioxidant test of fluorescent sensing peptide hydrogel.

[0090] Three representative ROS, H2O2, O2 - and ·OH, were studied to evaluate the ability of the hydrogel to scavenge ROS. The free radical scavenging test was performed using a DPPH kit; the H2O2 assay kit was used to measure the absorbance at 405 nm wavelength, to determine the concentration of remaining H2O2, and to calculate the elimination ability of H2O2. To test the hydrogel's ability to scavenge hydroxyl radicals, the Fenton reaction was first used to generate ·OH in the system, and then salicylic acid was added to different concentrations of the material. Salicylic acid will react with ·OH to form a colored compound, and the ·OH elimination ability of the material was quantified using the ·OH assay kit to measure the absorbance at 550 nm wavelength.

[0091] The results showed that the hydrogel could effectively inhibit DPPH radicals at a concentration as low as 10 μg / mL, and the inhibition rate was more than 80% ( Figure 15 ) when the concentration was 100 μg / mL. In addition, the hydrogel also had a concentration-dependent ROS scavenging activity. The inhibition rate of ·OH by the hydrogel was more than 90% ( Figure 16); the hydrogel showed significant scavenging activity against H2O2 at a concentration of 200 pg / mL Figure 17 . In addition, the hydrogel showed a scavenging rate of O2 - close to 90% at a concentration of 100 pg / mL Figure 18 .

[0092] These results demonstrate the strong antioxidant capacity of the hydrogel, which is crucial for reducing oxidative stress, a major factor in delaying wound healing. In addition, the hydrogel can scavenge low concentrations of ROS, especially H2O2 and O2 - , suggesting that it has the potential to become a ROS sensor in the wound environment. The reactivity of the ROS-responsive groups of the hydrogel with these ROS markers indicates that this material can reliably monitor ROS levels in real time in wounds.

[0093] Example 11: Test of the healing-promoting properties of the fluorescent sensing peptide hydrogel.

[0094] The healing effect of the polypeptide hydrogel dressing on diabetic chronic wounds was evaluated using white BABL / c mice. A 4.50 mm diameter wound was made on the back of each mouse, which was then infected with S. aureus (10^8 CFU / mL). The infected mice (about 20 g) were then randomly divided into three groups (5 mice per group): natural healing group, PBS treatment group, and peptide hydrogel group. The peptide hydrogel group changed the dressing every three days, while the PBS group changed the new PBS solution every three days. The natural healing group received no treatment. The healing process of the wound was monitored daily by taking photos of the wound and calculating the wound area using image analysis software. As shown in Figure 19 , the wound healed significantly faster using the hydrogel dressing, completely closing on the 9th day. According to the time course calculated from the sequential image analysis, the hydrogel group had a relatively significant effect on promoting wound healing Figure 20 .

[0095] 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. The eluted bacteria were then cultured on LB agar plates for colony counting. As shown in Figure 21 , the polypeptide hydrogel group had almost no bacterial colonies, indicating that the hydrogel dressing had a good antibacterial effect at the wound site.

[0096] 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 22Group a is hematoxylin and eosin (H&E) staining of mouse skin tissue in the wound area, and group b is Masson staining of mouse skin tissue in the wound area. Histological analysis showed that the wound of the polypeptide hydrogel group was completely closed, new epidermis was formed, the number of inflammatory cells was the least, and the fibroblasts were arranged in order, which was very similar to the normal tissue structure Figure 22 ), which indicates that the polypeptide hydrogel group has better healing effect.

[0097] After the wound was formed for 4 h (24 h after the application of the hydrogel dressing), the mice in the natural healing group and the hydrogel group were sacrificed, and the immunofluorescence tissue sections were analyzed to evaluate the expression of iNOS and CD206 by fluorescence staining. As shown in Figure 23 , quantitative analysis using ImageJ showed that the natural healing group had a higher density of positive cells compared with the polypeptide hydrogel group, indicating that the polypeptide hydrogel group had a lower level of inflammation.

[0098] Example 12: Sensing performance test of fluorescent sensing peptide hydrogel.

[0099] A digital camera (Nikon D7000, lens focal length 18-105 mm) was used to take JPEG images of the hydrogel. All images were cropped to a standard size of 250 x 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 to match the size of the hydrogel to the size of the solid sample holder (i.e. the hydrogel can be directly measured 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 diabetic infected wound mouse model was established. After 12 h of inoculation with different concentrations of Staphylococcus aureus, the wound was imaged by fluorescence. First, the mouse was anesthetized and the wound was placed upwards. 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, and the distance change was used for feature extraction to obtain the RGB histogram of the feature region to train the KNN model. According to the five-fold cross-validation method, 20% of the images in the data set were used as a validation set (as shown in Figure 24 ). Figure 24 The following is the analysis result of the model for five concentrations (100-500 μM) of samples. The signal distribution curves are discrete, indicating that the model is not simply memorizing the training data, but can generalize to new, unseen data, which means it can accurately process new data. The bar chart shows the comparison of the ground truth and the predicted results of different samples. By comparing the actual value and the algorithm prediction value, the accuracy and reliability of the model were evaluated.

[0100] The above merely describes the preferred embodiments of the present application, and does not limit the present application in any way. Any person skilled in the art, without departing from the scope of the technical solutions of the present application, can make any form of equivalent replacement or modification of the technical solutions and technical contents disclosed by the present application, and such changes still belong to the protection scope of the present application.

Claims

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

2. A method of preparing a ratiometric fluorescent sensing peptide hydrogel, characterized in that, Preparation of the ratio fluorescent sensing peptide hydrogel for machine learning assisted according to claim 1 based on N6-Cbz-L-lysine and L-glutamic acid-5-benzyl ester raw materials, comprising 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 yellow solid, dry, to obtain (S)-(4-(2,5-dioxooxazolidin-4-yl)butyl) benzyl 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 light yellow solid, dry, to obtain (S)-3-(2,5-dioxooxazolidin-4-yl) propionic acid benzyl ester, i.e. Glu-NCA; S3: dissolve the Lys-NCA and the Glu-NCA in anhydrous DMF respectively, then add n-hexylamine / anhydrous DMF solution to both, 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 a protective group and polyglutamic acid with a protective group; S4: dissolve the polylysine with a protective group and the polyglutamic acid with a protective group in TFA respectively, then add hydrogen bromide acetic acid solution with a mass ratio of 30% dropwise to both, seal and stir, after the reaction is completed, add acetone to precipitate, centrifuge, and wash 2-3 times with acetone until the supernatant is colorless, after dialysis, freeze-dry to obtain polylysine and polyglutamic acid; S5: dissolve the fluorescent molecules TPE-NH2 and C7 respectively with the active oxygen response group dicarboxy ketone thioalkoxide and 1-ethyl-(3-dimethylaminopropyl) carbonyldiimidazole hydrochloride in an appropriate amount of DMSO, then add triethylamine and N,N-dimethylacrylamide, continue to add the polylysine, EDCI, triethylamine and DMPA after a period of reaction, after the reaction is completed, add an appropriate amount of acetone to precipitate, to obtain oily precipitate, remove the remaining organic reagents by rotary evaporation, to obtain 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 respectively, mix the three solutions, then add EDCI, mix the mixture thoroughly, then pour it into a mold, to obtain a hydrogel, and put the hydrogel and the mold together into deionized water for dialysis, and finally most of the hydrogel remains in the mold. The mixing molar ratio of the PGlu / DMSO, Ply-TPE / DMSO and Ply-C7 / DMSO solutions in step S6 is 2:1:1; The amount of EDCI is 30% of the total moles of lysine; the dialysis time is 12 h.

3. The method of claim 2, wherein the ratio fluorescent sensing peptide hydrogel is prepared by, In step S1, the reaction temperature is 55℃, the stirring time is 45 min, the drying temperature is 40℃, and the drying time is 12 h.

4. The method of claim 2, wherein the ratio fluorescent sensing peptide hydrogel is prepared by, In step S2, the reaction temperature is 55℃, the stirring time is 3 h, the drying temperature is 40℃, and the drying time is 12 h.

5. The method of claim 2, wherein the ratio fluorescent sensing peptide hydrogel is prepared by, In step S3, the reaction is carried out under anhydrous conditions, the molar ratio of Lys-NCA and Glu-NCA to n-hexylamine is both 1 / 200, and the volume of anhydrous ether is 15 times that of the reaction solution.

6. The method of claim 2, wherein the ratio fluorescent sensing peptide hydrogel is prepared by, In step S4, the reaction temperature is 25℃, the stirring time is 2 h, and the volume of acetone is five times that of the reaction solution.

7. The method of claim 2, wherein the ratio fluorescent sensing peptide hydrogel is prepared by, 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, and the reaction temperature is 25℃; The molar ratio of the fluorescent molecule TPE-NH2 / polylysine / EDCI / triethylamine / DMPA is 1:55:1:1:1, and the reaction temperature is 25℃.

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