CN115403502B

CN115403502B - Ionic liquid-near infrared fluorescent probe hydrogel and preparation method and application thereof

Info

Publication number: CN115403502B
Application number: CN202210866953.0A
Authority: CN
Inventors: 李春涯; 宗媛鸽; 王炎英; 查如艳
Original assignee: South Central University for Nationalities
Current assignee: South Central Minzu University
Priority date: 2022-07-22
Filing date: 2022-07-22
Publication date: 2024-01-30
Anticipated expiration: 2042-07-22
Also published as: CN115403502A

Abstract

The invention discloses an ionic liquid-near infrared fluorescent probe hydrogel and a preparation method and application thereof. The hydrogel has good antifreezing performance, mechanical performance and biocompatibility, has conductivity similar to that of skin tissues, has excellent adhesion performance on the skin, and can be easily removed without secondary damage to the skin; the hydrogel provided by the invention has an obvious inhibition effect on escherichia coli, staphylococcus aureus and bacillus, and can be used for detecting the HClO level at a wound in real time while treating the wound.

Description

Ionic liquid-near infrared fluorescent probe hydrogel and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biomedical material preparation, relates to an ionic liquid-near infrared fluorescent probe hydrogel, a preparation method and application thereof, and in particular relates to a multifunctional conductive, antifreeze and non-release antibacterial hydrogel with a near infrared fluorescent probe combined with ionic liquid, a preparation method and application thereof in promoting diabetic wound healing.

Background

Diabetes Mellitus (DM) is a common chronic disease characterized by persistent hyperglycemia. Currently, diabetics account for approximately 19-34% (approximately 4.5 billion) of the global population, and the number of diabetics is increasing daily (Yang Yuan, daidi Fan, shihong Screen, xiaoxuan Ma, chemical Engineering Journal,2022, 133859:1385-8947). In particular type II diabetic wounds, which are persistent inflammation, impaired angiogenesis, etc. due to the production of large amounts of reactive oxygen species (Reactive oxygen species, ROS) in the body. Hypochlorous acid (HClO) is a major high reactive ROS that when in excess in vivo concentrations causes oxidative stress and destroys intracellular nucleic acids and proteins, thereby triggering inflammation and slowing wound healing (Xiaoli Qian, hui Yu, wenchao Zhu, xufeng Yao, wangwang Liu, shikui Yang, fangyuan Zhou, yi Liu, dyes and Pigments, volume 188,2021, 109218:0143-7208). Diabetic wounds, one of the most common complications for diabetics, have now become a major challenge for the global healthcare system. In order to monitor HClO levels in vivo, detection methods have been developed in the past few decades, such as colorimetry, chromatography, electrochemistry and fluorescence. Among these methods, the fluorescence method is widely used because of its unique advantages of easy operation and real-time analysis (Yan Zhang, haiyan Yang, mingxin Li, shaigong, jie Song, zhonglong Wang, shift Wang, dyes and Pigments, volume 197,2022,109861,0143-7208). Near infrared fluorescent probes are a chemical dye that is excited by NIR fluorescence and rapidly visualizes and quantifies the analyte after biological imaging. The NIR fluorescence region (650-900 nm) has the advantages of high tissue penetration depth, weak photodamage to living beings, capability of avoiding complex background autofluorescence interference and the like (Chengg, fan J, sun W, chemical Communications,2013,50 (8): 1018-1020). HClO levels in diabetic wounds are often elusive and susceptible to interference by other ROS competitors due to its short life cycle and high activity. Therefore, the development of the hydrogel auxiliary material which can promote the healing of the diabetic wounds and can rapidly and sensitively monitor the HClO level of the wounds in real time has great significance.

Disclosure of Invention

In order to improve the technical problem that the hydrogel auxiliary material can promote the healing of diabetic wounds and monitor the HClO level at the wounds in real time, the invention provides a multifunctional conductive, antifreeze and non-release antibacterial hydrogel combining a near infrared fluorescent probe with ionic liquid, and a preparation method and application thereof. Compared with the reported polyionic liquid functionalized hydrogel (Chao Zhou, chengju shaping, linglingao, jiaguo, peng Li, bo Liu, volume 413,2021, 127429:1385-8947), the ionic liquid-near infrared fluorescent probe hydrogel disclosed by the invention can be used for monitoring the HClO level in a diabetic wound in real time and visually while promoting the healing of the diabetic wound. Diabetic wounds are chronic wounds with abnormally high HClO concentrations, and excessive HClO can gradually lower, or even quench, the fluorescence value of ionic liquid-near infrared fluorescent probe hydrogels. In order to rapidly quantify and visually image the HClO level of the diabetes wound, the ionic liquid-near infrared fluorescent probe hydrogel can be coated on the diabetes wound, and the change condition of fluorescence of the hydrogel can be seen through near infrared fluorescent living body imaging, so that the change value of the HClO concentration of the diabetes wound can be estimated through the change of the fluorescence value of the hydrogel. The ionic liquid-near infrared fluorescent probe hydrogel disclosed by the invention is simple in preparation process and low in cost, and has good mechanical property, in-vitro adhesion property, antifreezing property, hemostatic property and non-release antibacterial property. Compared with commercial auxiliary materials (3M Tegaderm) and a blank control group, the ionic liquid-near infrared fluorescent probe hydrogel can remarkably accelerate healing of diabetic wounds.

In order to achieve the above purpose, the invention adopts the following technical scheme:

the invention provides a compound with a structure shown in a formula I:

the invention also provides a preparation method of the compound with the structure shown in the formula I, which comprises the steps of reacting the compound 3 with thiomorpholine to prepare the compound with the structure shown in the formula I;

the compound 3 has the following structure:

according to an embodiment of the invention, the molar ratio of thiomorpholine to compound 3 is 1: (1-5), exemplary are 1:1, 1:3, 1:5.

According to an embodiment of the present invention, the preparation method may be performed in the presence of a solvent such as an organic solvent. For example, the organic solvent may be selected from DMF.

According to an embodiment of the invention, the preparation method further comprises the step of separating the solid product from the reacted mixture after the reaction is completed. For example, the reaction solution is poured into anhydrous diethyl ether, suction-filtered, washed with anhydrous diethyl ether, and the filter cake is collected and dried to obtain the compound of the structure shown in formula I.

Preferably, the compound of formula I is synthesized as follows:

according to an embodiment of the present invention, the compound 3 is prepared by a process comprising: reacting the compound 1 with the compound 2 to obtain the compound 3;

The compound 1 has the following structure:

the compound 2 has the following structure:

according to one embodiment of the present invention, the above reaction may be carried out in the presence of a solvent such as an organic solvent. For example, the organic solvent may be selected from ethanol.

According to an embodiment of the invention, the molar ratio of compound 1 to compound 2 is 1: (1-5), exemplary are 1:1, 1:2, 1:5.

According to an embodiment of the present invention, a base is preferably added to the reaction of compound 1 with compound 2.

Preferably, the molar ratio of base to compound 2 is 1: (0.5-2), exemplary are 1:0.5, 1:1, 1:2.

Preferably, the base is selected from one, two or more of potassium carbonate, sodium tert-butoxide, potassium phosphate, sodium acetate.

According to an embodiment of the invention, the preparation method further comprises the step of separating the solid product from the reacted mixture after the reaction is completed. For example, the solvent is dried by spinning to give a solid product. Further, the preparation method comprises the step of purifying the product. For example, the purification may be performed by column chromatography. Preferably, the eluent of the column chromatography column separation is dichloromethane/methanol= (40-60): 1 (v/v), and the exemplary eluent is 40:1, 50:1, 60:1.

Preferably, the synthetic route of compound 3 is as follows:

according to an embodiment of the invention, the compound 2 is prepared by a process comprising: 2, 3-trimethyl-3H-indole and p-bromobenzoic acid are reacted to obtain a compound 2.

Preferably, the molar ratio of the 2, 3-trimethyl-3H-indole to the p-bromobenzoic acid is 1: (1-2), exemplary are 1:1, 1:1.2, 1:2.

Preferably, the preparation method of the compound 2 may be performed in the presence of a solvent such as an organic solvent. For example, the organic solvent may be selected from acetonitrile.

Preferably, the preparation method of the compound 2 further comprises the step of separating a solid product from the mixture after the reaction is finished. For example, the reaction solution is poured into anhydrous diethyl ether, suction-filtered, washed with anhydrous diethyl ether, and the cake is collected and dried to give compound 2.

According to an embodiment of the present invention, the compound 1 is prepared by a process comprising: cyclohexanone and phosphorus oxychloride are reacted to give compound 1.

Preferably, the molar ratio of cyclohexanone to phosphorus oxychloride is 1: (0.5-2), exemplary are 1:0.5, 1:1, 1:2.

preferably, the preparation method of the compound 1 may be performed in the presence of a solvent such as an organic solvent. For example, the organic solvent may be selected from DMF.

Preferably, the preparation method of the compound 1 further comprises the step of separating a solid product from the mixture after the reaction is finished. For example, the reaction solution is poured into crushed ice, suction-filtered, washed with ethyl glacial acetate, the filter cake is collected, and dried to give compound 1.

Preferably, the synthetic route of compound 1 is as follows:

according to an embodiment of the present invention, the preparation method of the compound having the structure shown in formula I comprises the following steps:

(1) Synthesizing a compound 1 by taking DMF as a solvent from cyclohexanone and phosphorus oxychloride; the structural formula of the compound 1 is as follows:

(2) Synthesizing a compound 2 by taking acetonitrile as a solvent from 2, 3-trimethyl-3H-indole and p-bromobenzoic acid; the structural formula of the compound 2 is as follows:

(3) Under the action of alkali, synthesizing a compound 3 by taking ethanol as a solvent from the compound 1 and the compound 2; the structural formula of the compound 3 is as follows:

(4) Synthesizing a compound of the formula I by using a compound 3 and thiomorpholine and DMF as a solvent;

the reaction scheme for the compounds of formula I is as follows:

the invention also provides application of the compound with the structure shown in the formula I in preparation of fluorescent probes.

The invention also provides a compound with a structure shown in a formula II:

wherein: m, n, x, y and z are each a number greater than or equal to 1, for example m, n, x, y and z are each independently selected from a number of 1 to 100, preferably each independently selected from a number of 40 to 60, and are exemplified by 40, 50, 60.

The invention also provides a preparation method of the compound with the structure shown in the formula II, which comprises the following steps: the compound with the structure shown in the formula I and the poly IL-NH ₂ The ionic liquid (PIL) generates amide bond reaction to obtain near infrared fluorescent probe grafted poly-IL-NH ₂ The ionic liquid and modified hyaluronic acid (HA-QA-ALD) undergo Schiff base reaction and amide bond reaction to prepare a compound with a structure shown as II;

the poly IL-NH ₂ The ionic liquid (PIL) has the structural formula:

wherein: m and n are each a number of 1 or more, for example, m and n are each independently selected from a number of 1 to 100, preferably each independently selected from a number of 40 to 60, and exemplified by 40, 50, 60.

According to an embodiment of the invention, the compound of the structure of formula I is reacted with a poly-IL-NH ₂ The mass ratio of the ionic liquid is 1 (4-6), and the ionic liquid is 1:4, 1:5 and 1:6.

According to an embodiment of the invention, the modified hyaluronic acid (HA-QA-ALD) is grafted with a near infrared fluorescent probe of a poly-IL-NH ₂ The mass ratio of the ionic liquid is (1-4): 5, and the exemplary mass ratio is 1:5, 2:5 and 4:5.

According to an embodiment of the present invention, the ratio of HA-Qa in the modified hyaluronic acid (HA-QA-ALD) is 46% to 53%, and exemplary is 46%, 50%, 53%.

According to an embodiment of the invention, the poly IL-NH ₂ An ionic liquid (PIL) is prepared by a process comprising: reacting the compound 4 with acrylamide under the action of an initiator to obtain the poly-IL-NH ₂ Ionic Liquids (PILs);

the compound 4 has the following structure:

preferably, the reaction mass ratio of the compound 4 to the acrylamide is 1: (1-2), exemplary are 1:1, 1:1.4, 1:2.

Preferably, the reaction mass ratio of the compound 4 to the initiator is (10-15): 1, exemplary are 10:1, 12.5:1, 15:1. For example, the initiator is selected from ammonium persulfate.

Preferably, the poly IL-NH ₂ The reaction of the ionic liquid (PIL) may be carried out in the presence of a solvent. For example, the solvent may be selected from buffer solutions.

Preferably, the reaction may be carried out at a temperature of 20 ℃ to 100 ℃.

Preferably, the poly IL-NH ₂ The synthetic route for ionic liquids (PIL) is as follows:

according to an embodiment of the invention, the compound 4 is prepared by a process comprising: 1- (3-aminopropyl) -imidazole was reacted with 4-chloromethylstyrene, followed by ZnCl ₂ ZnCl in ethanol solution ₃ ^- Coordination and Cl ^- The displacement of the coordination gives compound 4.

Preferably, the reaction molar ratio of the 1- (3-aminopropyl) -imidazole to the 4-chloromethyl styrene is 1: (0.5-2), exemplary are 1:0.5, 1:1, 1:2.

Preferably, the reaction of 1- (3-aminopropyl) -imidazole with 4-chloromethylstyrene may be carried out in the presence of a solvent such as an organic solvent. For example, the organic solvent may be selected from methanol.

Preferably, the preparation method of the compound 4 further comprises the step of separating to obtain a crude product after the reaction is finished. For example, the solvent is dried by filtration to give the crude product. Preferably, the preparation method further comprises the step of purifying the crude product to give compound 4. For example, the purification may be performed using a column chromatography column to give compound 4. Preferably, the eluent separated by the column chromatography column is dichloromethane and methanol= (90-110): 1 (v/v), and the exemplary eluent is 90:1, 100:1, 110:1.

Preferably, the synthetic route of compound 4 is as follows:

according to an embodiment of the present invention, the modified hyaluronic acid (HA-QA-ALD) HAs the structural formula:

wherein: x, y and z are each a number greater than or equal to 1, for example, x, y and z are each independently selected from a number from 1 to 100, preferably each independently selected from a number from 40 to 60, and are exemplified by 40, 50, 60.

According to an embodiment of the present invention, the modified hyaluronic acid (HA-QA-ALD) is prepared by a process comprising: first through NaIO ₄ Oxidizing hyaluronic acid to obtain a compound with a structure shown in a formula III, and reacting with a Ji Laer special reagent T to obtain modified hyaluronic acid (HA-QA-ALD);

the structural formula of the compound shown in the formula III is as follows:

wherein: x and y are each a number greater than or equal to 1, for example, x and y are each independently selected from a number of 1 to 100, preferably each independently selected from a number of 40 to 60, and are exemplified by 40, 50, 60.

Preferably, the hyaluronic acid and NaIO ₄ The reaction mass ratio of (2) is 1: (0.4-1), and 1:0.4, 1:0.6, and 1:1 are exemplified.

Preferably, the reaction mass ratio of the compound with the structure shown in the formula III and the Ji Laer specific reagent T is 5: (1-3).

Preferably, the reaction of the compound of the structure shown in formula iii with Ji Laer T reagent T may be performed in a solvent, which may be a buffer solution.

Preferably, the reaction of the compound of formula III with Ji Laer T reagent T is carried out under acidic conditions. For example at a pH of 4.5 to 5.

Preferably, the reaction of the compound of the structure of formula III with Ji Laer T reagent T may be carried out at room temperature.

Preferably, the method for preparing the modified hyaluronic acid (HA-QA-ALD) further comprises purifying the product after the reaction is finished. For example, dialysis is performed with a NaCl solution having ph=7 for 2 days, and then with a buffer solution for 2 days.

Preferably, the method for preparing the modified hyaluronic acid (HA-QA-ALD) further comprises drying the purified product.

Preferably, the modified hyaluronic acid (HA-QA-ALD) is synthesized as follows:

the buffer solutions used in the present invention were all 0.01mol/L PBS buffer solutions at ph=7.4, unless otherwise specified.

The invention also provides application of the compound with the structure shown in the formula II in preparation of fluorescent probes. Preferably in the preparation of medicaments for repairing skin wounds.

The invention also provides a fluorescent probe which comprises a compound with a structure shown in a formula II.

The invention also provides a kit comprising the fluorescent probe.

The invention also provides a biosensor which comprises the fluorescent probe.

The invention also provides application of the fluorescent probe, the kit and/or the biosensor in an animal living body imaging system. Preferably in a near infrared fluorescence I region animal living imaging system.

The invention also provides application of the fluorescent probe, the kit and/or the biosensor in detecting HClO level.

The invention also provides application of the fluorescent probe, the kit and/or the biosensor in preparing a medicament for promoting wound healing. Preferably in the preparation of a medicament for promoting the healing of skin wounds caused by diabetes.

The concept and principle of the invention are described as follows: the bonds formed by physical crosslinking are weaker, but the speed of bond formation and dynamic equilibrium reestablishment is faster. The mechanical strength of hydrogels formed by physical crosslinking is weak and unsuitable for dynamic wounds. While stronger bonds can be formed by chemical crosslinking, the rate of bond formation and dynamic equilibrium reestablishment is slower. Thus, the introduction of chemical crosslinking can increase the mechanical strength of the hydrogels. According to the invention, by utilizing the characteristic that an amino group and a carboxyl group can form a physical crosslinking-amido bond, and simultaneously, an aldehyde group and an amino group are easy to generate chemical crosslinking-Schiff base reaction, firstly, a NIR fluorescent probe containing carboxyl functionalization and an ionic liquid containing amino functionalization are prepared, the ionic liquid is firstly mixed with an NIR probe with fluorescence quenching property of HClO reaction after polymerization, and then modified hyaluronic acid is added into the mixture for generating physical crosslinking (amido bond reaction) and chemical crosslinking (Schiff base reaction), so that the NIR fluorescent probe is combined with the polyionic liquid through the amido bond, the modified hyaluronic acid is connected with the polyionic liquid through the amido bond and the Schiff base reaction to form a hydrogel with a 3D three-dimensional network structure, and the hydrogel can visualize and quantify the HClO level of a wound under a physiological state through NIR fluorescent imaging, so that the novel multifunctional ionic liquid and NIR imaging hydrogel is obtained.

The invention has the beneficial effects that:

fluorescent probes are useful for quantitative and qualitative analysis of various analytes. Near Infrared (NIR) fluorescent probes are widely used because of their low background interference, high tissue penetration, and low tissue damage. However, most NIR probes reported in the prior art exhibit small stokes shifts (typically less than 30 nm) and low fluorescence quantum yields (below 0.1 in aqueous solution), the contrast and spatial resolution of biological imaging are severely limited. The near infrared fluorescent probe SCy-7 of the invention has an excitation wavelength of 680nm, an emission wavelength of 778 nm, a Stokes shift of up to 98nm, and a fluorescence quantum yield of 0.18 in methanol. The imaging device is used for imaging living cells and mice, and has the advantages of strong contrast, good spatial resolution and satisfactory tissue imaging depth. Meanwhile, the ionic liquid is a fused salt with excellent chemical and thermal stability, is classified as a green solvent, can undergo polymerization reaction to form a polyionic liquid, and has good ionic conductivity due to the existence of anionic and cationic electrolyte groups on repeated units of the polyionic liquid. Meanwhile, both ionic liquids and polyionic liquids have been shown to have good antibacterial properties against gram-positive and gram-negative bacteria and fungi. Hyaluronic Acid (HA) is a polymer, is a high-grade polysaccharide composed of units D-glucuronic acid and N-acetylglucosamine, is nontoxic, colorless and odorless, can stimulate epithelial cell migration, enhance angiogenesis and reduce inflammation, and is an ideal material for developing hydrogels. The invention is realized by After polymerizing the 1- (3-aminopropyl) -3- (4-vinylbenzyl) imidazole salt ionic liquid containing positive charge active groups, mixing with a NIR fluorescent probe with a dicarboxyl function, grafting the NIR fluorescent probe on the polyionic liquid through an amide bond, simultaneously adding a modified hyaluronic acid solution, and forming a 3D network polymer through chemical and physical crosslinking (Schiff base and amide bond connection), thereby preparing the novel multifunctional ionic liquid-NIR probe hydrogel PIL-HA. The hydrogel has good antifreezing performance, mechanical performance and biocompatibility, has conductivity similar to that of skin tissues, has excellent adhesion performance on the skin, and can be easily removed without secondary damage to the skin; meanwhile, under the condition of no additional antibiotics, the hydrogel has obvious inhibition effect on escherichia coli, staphylococcus aureus and bacillus, and has better treatment results on acute wounds and diabetic skin wounds than commercial Tegaderm ^TM The film and the NIR fluorescence living body imaging result show that the hydrogel can also be used for detecting the HClO level at the wound in real time while treating the wound. Specifically:

(1) The NIR fluorescent probe hydrogel PIL-HA can detect the HClO level at a wound in real time through living body imaging so as to reduce the probability of converting an acute wound into a chronic wound, thereby achieving the aim of synchronously carrying out wound treatment and detection.

(2) The NIR fluorescent probe hydrogel PIL-HA provided by the invention HAs excellent antibacterial performance, and the poly PIL-NH thereof ₂ The positive charge groups contained in the ionic liquid can generate electrostatic interaction with the anionic groups on the cell walls of bacteria to destroy the integrity of the bacteria, so that a good bactericidal effect is achieved.

(3) The aldehyde group and the carboxyl group of the modified hyaluronic acid can be subjected to chemical reaction with amino groups of skin tissues, and meanwhile, the positive charge groups in the modified hyaluronic acid and the hydrogel are mutually combined with the negatively charged sialic acid groups on the viscous liquid membrane and the phospholipid of the cell membrane, so that the hydrogel is endowed with good adhesive property.

(4) The hydrogel disclosed by the invention has a stable structure, short gel time (within 3 min), excellent adhesive property, and good hemostatic property, and can be firmly adhered to skin and a wound part, and can be easily removed under the condition of not causing secondary injury to the wound.

(5) The hydrogel of the invention is associated with poly PIL-NH ₂ The increase in ionic liquid content (2 wt% to 8 wt%) also increases the crosslink density of the hydrogel gradually, and the conductivity of the hydrogel increases from 0.04mS/cm to 0.67mS/cm. The hydrogel has similar conductivity to skin tissues, and thus has great application potential in the processes of transferring bioelectric signals and accelerating wound healing.

(6) The hydrogel provided by the invention has good antifreezing capability, can still have good conductivity, stretching and compression properties under the condition that the temperature reaches-10 ℃, can quickly recover the original shape within 3s after stretching and compression, and has excellent flexibility, so that tissues can be protected from potential injury.

(7) The NIR fluorescent probe hydrogel PIL-HA of the invention HAs far better healing rate of wounds, thickness of granulation tissues, content of pro-inflammatory factors, tumor necrosis factors and interleukins in the treatment of normal and diabetic mice full-thickness skin injury models ^TM Therapeutic effects of the film.

Drawings

FIG. 1a is a schematic diagram of an NIR fluorescent probe SCy-7 ¹ The H NMR spectrum, FIG. 1b is the infrared spectrum of NIR fluorescent probe SCy-7, FIG. 1c is the mass spectrum of NIR fluorescent probe SCy-7, and FIG. 1d is the ultraviolet fluorescence spectrum of NIR fluorescent probe SCy-7.

FIG. 2a is a graph of pH stability of NIR fluorescent probe SCy-7, FIG. 2b is a graph of selectivity experiment results of NIR fluorescent probe SCy-7, FIG. 2c is a graph of fluorescence intensity variation of NIR fluorescent probe SCy-7 versus HClO, and FIG. 2d is a graph of linear relationship between NIR fluorescent probe SCy-7 and HClO concentration.

FIG. 3 is IL-NH ₂ Of ionic liquids ¹ H NMR spectrum.

FIG. 4 is an infrared spectrum of modified hyaluronic acids HA-QA-ALD, hA-ALD and HA.

FIG. 5a is a flow chart of the preparation of a PIL-HA-2 hydrogel gel, and FIG. 5b is a schematic diagram of the gel formation of a PIL-HA-2 hydrogel; fig. 5c is a schematic illustration of the adhesion of PIL-HA-x (x=1.2.3.4) hydrogels to the back of the hand; fig. 5d is a schematic gel time diagram of PIL-HA-x (x=1.2.3.4) hydrogels.

Fig. 6a is an infrared spectrum of a PIL-HA-x (x=1, 2, 3, 4) hydrogel, fig. 6b is an XRD pattern of the PIL-HA-x hydrogel, fig. 6c is a thermogravimetric image of the PIL-HA-x hydrogel, and fig. 6d is a Zeta point bitmap of the PIL-HA-2 hydrogel.

FIG. 7a is an SEM image of a PIL-HA-1 hydrogel, FIG. 7b is an SEM image of a PIL-HA-2 hydrogel, FIG. 7c is an SEM image of a PIL-HA-3 hydrogel, FIG. 7d is an SEM image of a PIL-HA-4 hydrogel, and FIG. 7e is an aperture cross-sectional area image of a PIL-HA-x (x=1.2.3.4) hydrogel.

Fig. 8a is a tensile test plot of the PIL-HA-x (x=1.2.3.4) hydrogel, fig. 8b is a compression test plot of the PIL-HA-x hydrogel, fig. 8c is a schematic representation of the rapid recovery of the PIL-HA-2 hydrogel within 3s after compression test, and fig. 8d is a plot of the splicing tower cut experiment performed on pigskin of the PIL-HA-x hydrogel.

Fig. 9a is a graph comparing the mass of the PIL-HA-x (x=1.2.3.4) hydrogel before and after swelling, fig. 9b is a bar graph comparing the mass of the PIL-HA-x hydrogel before and after swelling, and fig. 9c is a bar graph comparing the bottom area of the PIL-HA-x hydrogel before and after swelling.

FIG. 10a is a graph of the antimicrobial ability of the PIL-HA-x (x=1.2.3.4) hydrogels prepared according to the present invention evaluated by the disc method, FIG. 10b is a statistical graph of the size of the area of the bacteria inhibition produced by the hydrogels evaluated qualitatively by the disc method, FIG. 10c is a graph of the antimicrobial ability of the PIL-HA-x hydrogels prepared according to the present invention evaluated quantitatively, and FIG. 10d is a statistical graph of the bacterial inhibition of the hydrogels evaluated by the colony counting method.

FIG. 11a is a graph showing the absorption performance of the PIL-HA-x (x=1.2.3.4) hydrogel prepared according to the present invention to proteins, FIG. 11b is a water content histogram of the PIL-HA-x hydrogel prepared according to the present invention, and FIG. 11c is a graph showing the maximum tensile deformation of the PIL-HA-x hydrogel prepared according to the present invention. FIG. 11d is a bar graph of statistical elongation of PIL-HA-x hydrogels.

FIG. 12a is a staining chart of live dead cells after 1 and 2 days of co-culturing the NIR fluorescent probe SCy-7 prepared in the present invention with the fibroblast L929, FIG. 12b is a staining chart of live dead cells after 1 and 2 days of co-culturing the NIR fluorescent probe SCy-7 prepared in the present invention with the embryonic fibroblast NIH-3T3, FIG. 12c is a staining chart of live dead cells after 1 and 2 days of co-culturing the PIL-HA-x (x=1.2.3.4) hydrogel prepared in the present invention with the fibroblast L929, and FIG. 12d is a staining chart of live dead cells after 1 and 2 days of co-culturing the PIL-HA-x hydrogel prepared in the present invention with the embryonic fibroblast NIH-3T 3.

FIG. 13a is a statistical graph showing the number of cell survival after 1, 2 and 3 days of co-culturing the NIR fluorescent probe SCy-7 prepared in the present invention with the fibroblast L929, FIG. 13b is a statistical graph showing the number of cell survival after 1, 2 and 3 days of co-culturing the NIR fluorescent probe SCy-7 prepared in the present invention with the embryonic fibroblast NIH-3T3, FIG. 13c is a statistical graph showing the number of cell survival after 1, 2 and 3 days of co-culturing the PIL-HA-x (x=1.2.3.4) hydrogel prepared in the present invention with the fibroblast L929, and FIG. 13d is a statistical graph showing the number of cell survival after 1, 2 and 3 days of co-culturing the PIL-HA-x hydrogel prepared in the present invention with the embryonic fibroblast NIH-3T 3.

FIG. 14a is a graph showing the hemolytic potential of PIL-HA-x (x=1.2.3.4) hydrogels prepared according to the present invention; FIG. 14b is a test chart of a cell adhesion test of PIL-HA-x hydrogels prepared according to the present invention to blood.

Fig. 15a is a graph showing the conductivity of the PIL-HA-x (x=1.2.3.4) hydrogels prepared according to the present invention, and fig. 15b is a schematic diagram showing the conductivity of the PIL-HA-2 hydrogels.

FIG. 16a is a low temperature conductivity graph of a PIL-HA-2 hydrogel made in accordance with the present invention, FIG. 16b is a stretch-recovery graph of the PIL-HA-2 hydrogel at low temperature, and FIG. 16c is a compression-recovery graph of the PIL-HA-2 hydrogel at low temperature.

FIG. 17a is a graph showing in vitro hemostatic capacities of a blank and a PIL-HA-2 hydrogel prepared according to the present invention, FIG. 17b is a graph showing in vivo hemostatic capacities of a blank and a PIL-HA-2 hydrogel prepared according to the present invention, and FIG. 17c is a statistical graph showing hemostatic capacities of a blank and a PIL-HA-2 hydrogel prepared according to the present invention.

FIG. 18a is a graph showing the adhesion of PIL-HA-2 hydrogel prepared by the present invention to pigskin, FIG. 18b shows that PIL-HA-2 hydrogel can be easily peeled off skin, and FIG. 18c shows the principle of adhesion of PIL-HA-2 hydrogel to different articles and the adhesion graph on different articles.

FIG. 19a is a microscopic image of L929 cell scratches before and after the blank and the PIL-HA-x (x=1.2.3.4) hydrogel treatments 6, 12 and 18 hours prepared in accordance with the present invention, FIG. 19b is a quantitative statistical image of the distance between the blank and the L929 cell scratches before and after the PIL-HA-x hydrogel treatments 6, 12 and 18 hours prepared in accordance with the present invention, FIG. 19c is a microscopic image of NIH-3T3 cell scratches before and after the blank and the PIL-HA-x hydrogel treatments 6, 12 and 18 hours prepared in accordance with the present invention, and FIG. 19d is a quantitative statistical image of the distance between the blank and the NIH-3T3 cell scratches before and after the PIL-HA-x hydrogel treatments 6, 12 and 18 hours prepared in accordance with the present invention.

FIG. 20a is an inverted fluorescence imaging microscope of the NIR fluorescent probe SCy-7 and L929 cells prepared in the present invention, FIG. 20b is an inverted fluorescence imaging microscope of the NIR fluorescent probe SCy-7 and NIH-3T3 cells prepared in the present invention, FIG. 20c is an inverted fluorescence imaging microscope of the NIR fluorescent probe hydrogel PIL-HA-2 and L929 cells prepared in the present invention, and FIG. 20d is an inverted fluorescence imaging microscope of the NIR fluorescent probe hydrogel PIL-HA-2 and NIH-3T3 cells prepared in the present invention.

FIG. 21a is a photograph of an NIR fluorescence biopsy taken on a wound of a diabetic mouse with different blood sugar levels of the NIR fluorescence probe hydrogel PIL-HA-2 prepared by the present invention, FIG. 21b is a graph of quantifying and plotting fluorescence values of adhesion hydrogels on a diabetic mouse with different blood sugar levels, FIG. 21c is a graph of an NIR fluorescence biopsy taken on a wound of a diabetic mouse with different blood sugar levels of the NIR fluorescence probe hydrogel PIL-HA-2 prepared by the present invention, and FIG. 21d is a graph of quantifying and plotting fluorescence values on a wound of a diabetic mouse with different blood sugar levels.

FIG. 22a is a photograph of an in vivo NIR fluorescence image of a wound of a normoglycemic mouse with an increase in PMA content at the wound of the NIR fluorescence probe hydrogel PIL-HA-2 prepared according to the present invention; FIG. 22b is a graph of fluorescence values of adhered hydrogels on mice as a function of PMA content at the wound site; FIG. 22c is a photograph of an in vivo NIR fluorescence image of a wound of a normoglycemic mouse with the NIR fluorescence probe hydrogel PIL-HA-2 prepared according to the present invention as the HClO content at the wound increases; FIG. 22d is a graph showing fluorescence values of adhered hydrogels on mice as a function of HClO content at the wound site.

Fig. 23a is a fluorescent biopsy image of acute wounds of skin treated with different protocols on days 0, 3, 7, 10 and 12, fig. 23b is a bar graph of acute wound healing of skin treated with different protocols on days 0, 3, 7, 10 and 12, fig. 23c is a fluorescent biopsy image of chronic wounds of diabetic skin treated with different protocols on days 0, 3, 7, 10 and 14, and fig. 23d is a bar graph of chronic wound healing of diabetic skin treated with different protocols on days 0, 3, 7, 10 and 14.

Fig. 24 is a hematoxylin eosin (H & E) staining image of organs of diabetic mice after wound healing was completed.

FIG. 25a is a graph of H & E staining after 12 days of acute wound treatment with the PIL-HA-2 hydrogel prepared according to the present invention; FIG. 25b is a map of Masson staining after 12 days of acute wound treatment with the PIL-HA-2 hydrogel prepared according to the present invention; FIG. 25c is a graph of quantitative analysis of blood vessel number at an acute wound; FIG. 25d is a graph of quantitative analysis of collagen index at acute wounds; fig. 25e is a graph of quantitative analysis of epidermal thickness at an acute wound.

FIG. 26a is a 3M Tegaderm passing through a commercial film ^TM And PIL-HA-x (x=1.2.3.4) hydrogel-treated diabetic mice skin wound H&E, a dyeing chart; FIG. 26b is a 3M Tegaderm passing through a commercial film ^TM And Malson staining of skin wounds of diabetic mice treated with PIL-HA-x (x=1.2.3.4) hydrogel; FIG. 26c is a graph of quantitative analysis of the number of blood vessels at skin wounds in diabetic mice; fig. 26d is a graph of quantitative analysis of collagen index at skin wound of diabetic mice, and fig. 26e is a graph of quantitative analysis of epidermis thickness at skin wound of diabetic mice.

FIG. 27a is a schematic illustration of a PIL-HA-2 hydrogel and a commercial film 3M tergaderm prepared by the present invention ^TM Immunofluorescence staining pattern for IL-6 12 days after treatment of acute wounds; FIG. 27b is a schematic illustration of a PIL-HA-2 hydrogel and a commercial film 3M tergaderm prepared by the present invention ^TM Immunofluorescence staining pattern of TNF- α 12 days after treatment of acute wounds; FIG. 27c is a 3M Tegaderm passing through a commercial film ^TM And PIL-HA-x (x=1.2.3.4) Immunofluorescence staining pattern of IL-6 after hydrogel treatment of skin wound of diabetic mice; FIG. 27d is a 3M Tegaderm passing through a commercial film ^TM And immunofluorescence staining pattern of TNF- α after treatment of skin wounds in diabetic mice with PIL-HA-x (x=1.2.3.4) hydrogels.

FIG. 28 is a schematic diagram of the NIR fluorescent probe PIL-HA hydrogel prepared by the invention to promote the healing of diabetic wounds and to rapidly and visually monitor the HClO level of the diabetic wounds.

Detailed Description

The technical scheme of the invention will be further described in detail below with reference to specific embodiments. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.

Unless otherwise indicated, the starting materials and reagents used in the following examples were either commercially available or may be prepared by known methods.

Example 1

The preparation method of the multifunctional ionic liquid and NIR imaging hydrogel PIL-HA comprises the following steps:

(1) Preparation of NIR fluorescent probes SCy-7:

10mL of DMF was placed in a 50mL single-neck flask, and phosphorus oxychloride solution (6.8 mL,7.5 mmol) was stirred and slowly added under ice-bath conditions, stirred for 30min, warmed to room temperature, 2mL of cyclohexanone was added, and the temperature was raised to 50℃to continue the reaction for 6h. After the reaction, the reaction mixture was poured into 100g of crushed ice in portions, stirred for 5 hours, a yellow precipitate was precipitated, suction-filtered, washed 3 times with ethyl glacial acetate, and the obtained yellow powder (compound 1) was refrigerated for use.

2, 3-trimethyl-3H-indole (5.0000 g, 29.06 mmol) and p-bromobenzoic acid (7.5300 g, 35.02 mmol) were placed in a 50mL flask under nitrogen atmosphere, 100mL acetonitrile was added, and the mixture was heated to 81℃for 10H, cooled to room temperature, the reaction mixture was poured into 100mL anhydrous diethyl ether in portions, immediately precipitated, filtered off with suction, the filter cake was collected, and dried under vacuum to give 4.8630g of the product (Compound 2).

Compound 1 (3.0000 g, 17.4 mmol), compound 2 (10.7613 g,36.6 mmol) and anhydrous sodium acetate (3.0023 g,36.6 mmol) were placed in a 150mL flask under nitrogen atmosphere, 120mL of ethanol was added, the mixture was heated to 70 ℃ for reaction for 2 hours, cooled to room temperature, extracted with dichloromethane (4×100 mL), the upper liquid was collected, the solvent was rotary evaporated, the column chromatography was separated and purified (eluent: dichloromethane: methanol=50:1, v/v), the product was collected, the solvent was rotary evaporated, and vacuum-dried to obtain 2.7487g of a dark green product (compound 3) having metallic luster.

Under nitrogen atmosphere, compound 3 (1.0000 g, 1.25 mmol) and thiomorpholine (388 mg, 3.76 mmol) are placed in a 50mL flask, 15mL DMF is added, the reaction is stirred at room temperature for 2h, the reaction solution is poured into a large amount of anhydrous diethyl ether in batches, precipitation is generated immediately, suction filtration is carried out, washing is carried out for 3 times by using the anhydrous diethyl ether, filter cakes are collected, and vacuum drying is carried out, thus 0.8152g of product is obtained, namely the NIR fluorescent probe SCy-7.

(2) Poly IL-NH ₂ Preparation of ionic liquid:

(a)IL-NH ₂ preparation of ionic liquid:

after 8.76g of 1- (3-aminopropyl) -imidazole (0.07 mol) was sufficiently dissolved in 80mL of methanol, 10.68g of 4-chloromethylstyrene (0.07 mol) was added. After stirring vigorously at room temperature for 12h, the mixture was stirred with saturated ZnCl ₂ Is stirred for 24 hours at 30 ℃ to carry out ZnCl ₃ ^- Coordination and Cl ^- And (3) coordination displacement. After stirring, the solution was filtered, the filtered solution was spin-dried by vacuum rotary evaporator, and the residual product was purified by silica gel column chromatography using dichloromethane and methanol (100/1, v/v) to give a yellow product which was dried in a vacuum oven at 60 ℃ to give a yellow product of 1- (3-aminopropyl) -3- (4-vinylbenzyl) imidazolium ionic liquid, designated as IL-NH ₂ And (3) an ionic liquid.

(b) Poly IL-NH ₂ Preparation of ionic liquid:

into a 10mL round bottom flask was added 1mL of 0.01mol/L PBS (pH=7.4) buffer solution, to which was added 0.7g of acrylamide and 0.04g of ammonium persulfate, followed by 0.5g of IL-NH obtained in step (a) ₂ Stirring the ionic liquid for 5min to obtain the poly-IL-NH ₂ Ionic liquids, abbreviated as PIL.

(3) Preparation of modified hyaluronic acid:

(a) Preparation of modified hyaluronic acid HA-QA: 1g of hyaluronic acid (MW=340,000 Da) powder was fully dissolved in a beaker containing 100mL of 0.01mol/L PBS (pH=7.4) buffer solution, and the beaker was wrapped with tinfoil paper, and 0.6g of sodium periodate was added after obtaining a transparent solution, and stirred at room temperature for 12 hours. The clear solution was then dialyzed against NaCl solution at ph=7 for 3 days and then against deionized water for 2 days (mw=10 000 da). Finally, freeze-drying the dialyzed product in a vacuum freeze dryer to obtain a white cotton-like product HA-ALD.

(b) Preparation of modified hyaluronic acid HA-QA-ALD by dissolving HA-QA obtained in step (a) in deionized water to obtain 1% (w/w) aqueous solution of HA-aLD, adding 0.6g Girard ^‘s Reagent T, after the pH value of the solution is regulated to 4.5-5, stirring the reaction at room temperature for 26 hours, dialyzing with NaCl solution with pH=7 for 2 days, dialyzing in deionized water for 2 days, and freeze-drying the dialyzed product by a vacuum freeze dryer to obtain a white cotton-like product HA-QA-ALD.

(4) Preparation of novel multifunctional ionic liquid-NIR imaging hydrogel PIL-HA:

after the NIR fluorescent probe SCy-7 obtained in the step (1) was solubilized with 100. Mu.L of methanol, 0.01mol/L PBS (pH=7.4) was added to set the probe concentration to 10 ^-5 g/mL to obtain NIR fluorescent probe solution;

the poly IL-NH obtained in the step (2) is reacted with ₂ The ionic liquid is dissolved in 0.01mol/L PBS (pH=7.4) to prepare the poly IL-NH with the concentration of 2wt%,4wt%,6wt% and 8wt% in sequence ₂ An ionic liquid solution;

dissolving the modified hyaluronic acid HA-QA-ALD prepared in the step (3) in 0.01mol/L PBS (pH=7.4) to obtain a modified hyaluronic acid solution with a concentration of 5 wt%;

200 mu L of the mixture is taken to have the concentration of 10 ^-5 The g/mL NIR fluorescent probe solution was dissolved with 1mL of 0.01mol/L PBS (pH=7.4) at a concentration of 2wt%,4wt%,6wt%,8wt% of poly IL-NH, respectively ₂ Mixing ionic liquid solutions, and respectively addingUniformly mixing 5wt% modified hyaluronic acid solution, and cross-linking at 60deg.C for 1-3min to obtain multifunctional ionic liquid&NIR probe hydrogels PIL-HA-1, PIL-HA-2, PIL-HA-3, PIL-HA-4, abbreviated as PIL-HA-x (where wt% is addition polymerized IL-NH) ₂ The mass percent of the ionic liquid solution).

The synthetic route for the preparation of NIR fluorescent probe SCy-7 in this example is as follows:

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poly IL-NH was prepared in this example ₂ The synthetic route of the ionic liquid is as follows:

the synthetic route for the preparation of HA-QA-ALD in this example is shown below:

the synthetic route for the PIL-HA hydrogel prepared in this example is shown below:

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wherein: m, n, x, y and z are each a number from 40 to 60.

The schematic diagram of the PIL-HA hydrogel prepared by the invention combined with the NIR fluorescent probe for promoting wound healing of diabetics and simultaneously monitoring HClO level at the wound in real time is shown in figure 28.

The PIL-HA hydrogel prepared in this example was characterized and tested for performance, and the results are shown in FIGS. 1-27:

FIG. 1a is a nuclear magnetic resonance hydrogen spectrum of NIR fluorescent probe SCy-7, which is obtained by chemical shift and attribution analysis of corresponding hydrogen atoms in molecular structure, and the invention successfully synthesizes the NIR fluorescent probe.

FIG. 1b is a Fourier infrared spectrum of NIR fluorescent probe SCy-7, 3460cm ^-1 The absorption peak at 2925cm, which confirms the presence of carboxyl groups ^-1 Is CH ₂ ＝CH ₂ Stretching vibration peak of C-H in (4-153cm) ^-1 、1378 cm ^-1 And 1165cm ^-1 Is the skeleton vibration peak of benzene ring, 925cm ^-1 、798cm ^-1 And 752cm ^-1 The stretching vibration peak of C-N, the stretching vibration peak of C-S and the stretching vibration peak of C-C are respectively shown. From the above results, it was found that the present invention has successfully produced a SCy-7 near infrared fluorescent probe.

FIG. 1c is a mass spectrometric characterization of NIR fluorescent probe SCy-7, showing that the M/z= 791.4787 ([ M+H ] +) of SCy-7 is consistent with the formula weight of the target compound, thereby demonstrating the successful preparation of NIR fluorescent probe SCy-7 according to the present invention.

FIG. 1d is an ultraviolet fluorescence spectrum of NIR fluorescent probe SCy-7, characteristic absorption peak at 778nm indicating that the probe belongs to the near infrared fluorescence region (650 nm-900 nm).

FIG. 2a is a graph of the pH stability of NIR fluorescent probe SCy-7, investigating the effect of different pH on HClO response. The fluorescence spectrum of the NIR fluorescent probe SCy-7 solution was barely changed in the pH range of 4.0 to 8.0 without the addition of HClO; however, when HClO (80. Mu.M) was added, the fluorescence intensity of the NIR fluorescent probe SCy-7 solution rapidly decreased in the pH range of 4.0 to 8.0. The results in the figures show that: the probe of the invention can sensitively detect HClO in any wound environment.

FIG. 2b is a graph of the results of a selective assay for NIR fluorescent probe SCy-7, testing the selective specificity of the NIR fluorescent probe SCy-7 of the present invention for HClO to detect the possibility of use of the probe in complex biochemical environments. The experimental results show that: when HClO (80 μm) was added, the fluorescence intensity of NIR fluorescent probe SCy-7 was significantly reduced; to which 80. Mu.M of an inorganic salt (Ca) ²⁺ 、Fe ²⁺ 、Fe ³⁺ 、Zn ²⁺ 、K ⁺ 、Br ^- 、Cl ^- 、I ^- 、Mg ²⁺ 、 Na ⁺ ) Amino acids (cysteine, blood homocysteine), reducing substances (GSH, AA), ROS (H) ₂ O ₂ ) And RNS (H) ₂ S、NO、ONOO ^- ) When the fluorescence intensity of the NIR fluorescent probe SCy-7 was hardly changed. The results showed that: the NIR fluorescent probe SCy-7 can realize the specific detection of HClO.

FIG. 2c is a graph of fluorescence intensity of NIR fluorescent probe SCy-7 versus HClO. When SCy-7 interacted with varying concentrations of HClO (0-100. Mu.M), the fluorescence intensity of the NIR fluorescent probe SCy-7 at 778nm was measured. The results in the figures show that: as the concentration of HClO increases, the fluorescence intensity of NIR fluorescent probe SCy-7 decreases and even quenches.

FIG. 2d is a graph of the linear relationship between fluorescence intensity of NIR fluorescent probe SCy-7 and HClO concentration. The fluorescence intensity values of NIR fluorescent probes SCy-7 were determined when NIR fluorescent probes SCy-7 interacted with varying concentrations of HClO (0-100. Mu.M). And plotted on the ordinate with fluorescence intensity values and on the abscissa with added HClO concentration. The results in the figures show that: the HClO concentration can be quantitatively detected by the change of the fluorescence intensity of the probe.

FIG. 3 is IL-NH ₂ The nuclear magnetic resonance hydrogen spectrogram of the invention can be obtained by chemical displacement and attribution analysis of corresponding hydrogen atoms in a molecular structure, and the invention successfully synthesizes the IL-NH ₂ And (3) an ionic liquid.

FIG. 4 is a Fourier infrared (FT-IR) spectrum of HA-QA-ALD, hA-ALD and HA, showing the results: compared with the raw material HA, the FT-IR spectrum of HA-ALD is 1650cm ^-1 There shows a new characteristic peak due to the hydrazide group. For HA-QA-ALD, we can easily observe that the aldehyde group is present at 1747cm ^-1 Characteristic peaks and quaternary ammonium groups at 1470cm ^-1 And 1491cm ^-1 The characteristic peaks of (2) demonstrate that the invention successfully prepares two modified hyaluronic acids HA-ALD and HA-QA-ALD.

FIG. 5a is a flow chart of the preparation of a PIL-HA hydrogel gel. 1g of modified HA-QA-ALD was first dissolved in 5mL of 0.01mol/L PBS (pH=7.4) buffer solution and designated as solution 1; then 0.2g of acrylamide, 0.4g of ammonium persulfate and 200 muL NIR fluorescent probe SCy-7 and 0.5mL IL-NH ₂ Mix well in 3ml of 0.01mol/L PBS (ph=7.4) buffer solution, designated solution 2; 1mL of solution 1 and 1mL of solution 2 are mixed and placed in a water bath kettle at 60 ℃ for 3min to prepare the PIL-HA hydrogel.

FIG. 5b is a gel diagram of PIL-HA-2 hydrogel. After 1mL of 1mol/L hydroxylamine was added to 0.5g of PIL-HA hydrogel, the hydrogel was placed at 37℃and the gel-to-sol transition was accomplished within 30 min. This is due to the competition of hydroxylamine for PIL-NH ₂ Thereby breaking the schiff base and amide bonds of the hydrogel.

Fig. 5c is a schematic illustration of the adhesion of PIL-HA-x (x=1.2.3.4) hydrogels to the back of the hand. The results in the figures show that the PIL-HA hydrogels have good adhesion to the skin.

Fig. 5d is a schematic gel time diagram of a PIL-HA-x (x=1.2.3.4) hydrogel, which was placed in a 60 ℃ water bath, taken out at 1min intervals to observe the gel condition and photographed for recording. The results in the graph show that the PIL-HA-1 gel time is at least 3min and the PIL-HA-4 gel time is at least 1min. Thus, it was demonstrated that the PIL-HA hydrogels of the present invention can form gels within 3 minutes at 60 ℃.

FIG. 6a is a Fourier infrared spectrum of PIL-HA-x (x=1.2.3.4) hydrogel, which can be seen at 3440cm ^-1 There is a broad and strong peak due to stretching vibration of the hydroxyl group; 2926cm ^-1 The characteristic absorption peak at this point is due to the stretching vibration of the hydrocarbon bond between the schiff base and the amide bond. Due to IL-NH ₂ Is shown to be 1627cm ^-1 Characteristic absorption peak at the site, and the intensity of the absorption peak is dependent on IL-NH ₂ Increased content, which demonstrates IL-NH ₂ Is successfully introduced into PIL-HA hydrogels.

Fig. 6b is an XRD pattern of a PIL-HA-x (x=1.2.3.4) hydrogel, where a peak appears at 2θ=24.5°, indicating that the PIL-HA-x (x=1.2.3.4) hydrogel is a semi-crystalline polymer.

FIG. 6c is a thermogravimetric analysis of PIL-HA-x (x=1.2.3.4) hydrogel, as can be seen by direct comparison, with poly-IL-NH in the hydrogel ₂ Ion liquid concentration increase, fourThe thermal stability constants of the seed PIL-HA hydrogels increased from 106.9 ℃ to 289.7 ℃, 327.6 ℃ and 330.6 ℃, probably because the increase in crosslink density resulted in an increase in the thermal stability constant of the hydrogels.

FIG. 6d is a Zeta potential plot of a PIL-HA-2 hydrogel, from which it can be seen that the Zeta potential of the PIL-HA-2 hydrogel is 38.31mV, indicating that the surface of the hydrogel is positively charged, which demonstrates the antimicrobial properties of the PIL-HA-2 hydrogel.

FIG. 7a is an SEM image of a PIL-HA-1 hydrogel, FIG. 7b is an SEM image of a PIL-HA-2 hydrogel, FIG. 7c is an SEM image of a PIL-HA-3 hydrogel, and FIG. 7d is an SEM image of a PIL-HA-4 hydrogel. As can be seen from the field emission scanning electron microscope, all PIL-HA hydrogels showed interconnected 3D porous structures and with poly-IL-NH ₂ Increasing the dosage of ionic liquid and poly-IL-NH ₂ The opportunity of the amino group of the ionic liquid to react with the aldehyde group and the carboxyl group in the modified hyaluronic acid HA-QA-ALD is increased, and the pore size of the prepared hydrogel is from (5000+/-1500) mu m ² (PIL-HA-1 hydrogel) was reduced to (800.+ -.200) μm ² (PIL-HA-4 hydrogel) to form a more compact network structure. Such a 3D porous structure as described above is advantageous for breathability of the wound and growth survival of cells, and for transport of nutrients and absorption of metabolic waste.

Fig. 8a is a tensile test plot of PIL-HA-x (x=1.2.3.4) hydrogels. The testing method comprises the following steps: the PIL-HA-x (x=1.2.3.4) hydrogels were cut into rectangular hydrogels (10 mm×20mm×10 mm) as tensile samples, and the tensile properties of the four hydrogels were tested using a film tensile machine (instron 5943, usa) with the tensile rate kept constant at 10mm/min, giving a linear relationship between tensile force and stress. As can be seen from the figure, with poly IL-NH ₂ The use amount of the ionic liquid is increased, and the tensile stress of the hydrogel is increased from 0.18MPa to 0.43MPa, 0.44MPa and 0.82MPa. The tensile stress of the PIL-HA-x (x=1.2.3.4) hydrogel prepared by the method is 30% -95% better than the extensibility of human skin.

FIG. 8b is a compression performance test result of PIL-HA-x (x=1.2.3.4) hydrogel, which was fabricated into a cylindrical shape (radius=10 mm, height=40 mm), the compression properties of the four hydrogels were tested by an electronic universal material tester (AI-7000M, china) at a compression test rate of 5mm/min, giving a linear relationship between compression and stress. As can be seen from the figure, with poly IL-NH ₂ The stress of the hydrogel at 80% compression increases from 0.21MPa to 0.42MPa, 0.55MPa and 0.74MPa due to the increase in the amount of ionic liquid.

FIG. 8c shows that the PIL-HA-2 hydrogel can quickly recover its original form within 3s after compression to 80%. The PIL-HA hydrogel disclosed by the invention HAs better mechanical properties with human skin, so that the PIL-HA hydrogel can resist external force better, and potential damage to tissues is avoided.

Fig. 8d is a splice-tower cut experiment using pigskin to evaluate the ability of PIL-HA-x (x=1.2.3.4) hydrogels to adhere to skin tissue. The specific experimental method is as follows: fresh pigskin was purchased from the market, cut into rectangles 5cm long and 2cm wide after removing excess fat, and re-soaked in 10ml of 0.01mol/L PBS (ph=7.4) solution before the experiment to ensure that the pigskin is always in a moist environment in order to prevent the pigskin from drying out. To test the adhesion properties of the hydrogels to the pigskin, two rectangular pieces of pigskin were removed and the surface residue of the PBS solution was blotted off with filter paper. Four rectangular parallelepiped (20 mm. Times.10 mm. Times.2 mm) PIL-HA-x (x=1.2.3.4) hydrogel samples were adhered between two pigskin pieces at room temperature, and the upper and lower pigskin pieces were clamped by a film pulling machine, respectively, using a 100N weighing sensor for 10mm min ^-1 The strain rate of (c) was measured and the results are shown in fig. 8 d. As can be seen from the figure, the tensile stress of all hydrogels showed adhesive strength of (3.21±0.45) KPa to (24.36±0.84) KPa. Compared to the clinically established Fibringlue adhesive strength (about 3 KPa), the PIL-HA-x (x=2.3.4) hydrogels of the present invention are 3-8 times their adhesive strength.

The comparison of the front-to-back changes of the PIL-HA-x (x=1.2.3.4) hydrogels after reaching the swelling limit in 3ml of 0.01mol/L PBS (ph=7.4) solution is shown in fig. 9a, from left to right. As can be seen from the figures: the volume of the swelled hydrogel is obviously enlarged, so that the PIL-HA hydrogel HAs better water absorption.

Fig. 9b is a mass comparison of PIL-HA-x (x=1.2.3.4) hydrogels before and after swelling, as can be seen from the figure: the PIL-HA-x (x=1.2.3.4) hydrogels of the present invention can absorb 6-11 times more water than their own weight.

Fig. 9c is a plot of the bottom area of the PIL-HA-x (x=1.2.3.4) hydrogel before and after swelling, as can be seen from the plot. The basal area of the PIL-HA-x (x=1.2.3.4) hydrogels of the present invention increased 6-11 times after swelling compared to before swelling.

Fig. 10a is a qualitative assessment of the antimicrobial ability of PIL-HA-x (x=1.2.3.4) hydrogels using the disc method as follows: 500. Mu.L of each of the E.coli, staphylococcus aureus and Bacillus subtilis suspensions (1X 10) ⁸ CFU/mL) was spread uniformly on LB medium (30 mL) by ball method, then PIL-HA-x (x=1.2.3.4) hydrogel (1 g) after sterilization was spread uniformly on the surface of the medium, and incubated at 37℃for 24 hours, and bacterial growth around the hydrogel was observed. As can be seen from the figure, with poly IL-NH ₂ The area of the antibacterial zone around the hydrogel is increased along with the increase of the dosage of the ionic liquid, and the surface of the hydrogel does not grow any colony, so that the non-releasing antibacterial performance of the PIL-HA hydrogel is verified from the side.

Fig. 10b is a graph depicting the evaluation of antimicrobial ability of hydrogels using a disc method to evaluate the size of the zone of inhibition of four hydrogels. From the figures it can be seen that: different poly IL-NH ₂ The PIL-HA hydrogel with the ionic liquid content increases the area of the bacteriostasis zone generated by the hydrogel along with the increase of the dosage of the ionic liquid. This is due to the quaternary ammonium cation and poly-IL-NH on HA-QA-ALD in the PIL-HA hydrogel ₂ The positively charged imidazole groups can interact electrostatically with the anionic phosphate groups on the bacterial cell wall, and then the lipophilic moieties are inserted into the phospholipid bilayer of the bacteria to destroy the cell membrane and cause the bacteria to lyse and die.

Fig. 10c uses a colony counting method to quantitatively evaluate the antibacterial ability of PIL-HA-x (x=1.2.3.4) hydrogels by the following method: the PIL-HA-x (x=1.2.3.4) hydrogel is sterilized by ultraviolet rays in advance, and then the concentration is 1×10 ⁸ CFU/mL200. Mu.L of E.coli, staphylococcus aureus and Bacillus subtilis, were transferred to sterilized LB medium (10 mL), respectively, and then sterilized PIL-HA-x (x=1.2.3.4) hydrogels (1 g) were uniformly spread on the surface of the medium, respectively, and incubated at 37℃on a shaker at 120rpm, after 24 hours the bacterial suspension was diluted to the original concentration of 10 with LB medium ^-6 The diluted bacterial suspension was taken in 20. Mu.L, uniformly dispersed on LB solid medium by ball method, and incubated at 37℃for 24 hours. As can be seen from the figure, with poly IL-NH ₂ The antibacterial capability of the hydrogel is improved due to the increase of the dosage of the ionic liquid, which indicates that the PIL-HA hydrogel HAs the characteristic of non-releasing antibacterial property.

FIG. 10d is a graph of the number of Colony Forming Units (CFU) counted separately, each colony being tested repeatedly for more than three independent times, and bacterial suspension without hydrogel added being used as a control for the experiment (control, antibacterial capacity was considered as 0, and the antibacterial rate of the other groups was calculated on the basis of this). In comparison with PIL-HA-1 hydrogel, poly-IL-NH ₂ The antibacterial activity of the PIL-HA-2, PIL-HA-3 and PIL-HA-4 hydrogels prepared by increasing the dosage of the ionic liquid is obviously improved, and particularly the antibacterial activity of the PIL-HA-2, PIL-HA-3 and PIL-HA-4 hydrogels on staphylococcus aureus and bacillus subtilis is obviously improved. In addition, PIL-HA-2, PIL-HA-3 and PIL-HA-4 hydrogels can inhibit about 90% of E.coli and more than 95% of Staphylococcus aureus and Bacillus subtilis. The above results further demonstrate the potent antimicrobial activity of the PIL-HA hydrogels of the present invention against escherichia coli, staphylococcus aureus and bacillus subtilis, as well as demonstrating their great potential in the treatment of bacterial infections.

Fig. 11a is a test result of the adsorption capacity of PIL-HA-x (x=1.2.3.4) hydrogel to Bovine Serum Albumin (BSA). The method comprises the following steps: four kinds of hydrogels of PIL-HA-x (x=1.2.3.4) were cut into rectangles (weight of about 1 g), sterilized by 75% medical alcohol for 1 hour, and then placed under an ultraviolet lamp for sterilization. Soaking in PBS (pH=7.4, 0.01 mol/L) to reach swelling equilibrium, then adding the treated PIL-HA-x (x=1.2.3.4) hydrogel samples into 5mL of 10mg/mL Bovine Serum Albumin (BSA) solution respectively, and incubating at 37deg.C for 24h with shaking table, wherein each sample is heavyThe adsorption capacity of the hydrogel material was evaluated by measuring the ultraviolet intensity of the solution at 595nm with Shimadzu UV-2550 to calculate the residual BSA in the solution, and using a Bovine Serum Albumin (BSA) solution without the addition of the hydrogel sample as a control group, based on the BSA concentration measured by absorptiometry. The calculation formula is as follows:where Co and Cx are the concentration of BSA (mg/mL) after adsorption of the control and added PIL-HA-x (x=1.2.3.4) hydrogels, respectively, W is the weight of PIL-HA hydrogel and V is the volume of added BSA. The adsorption amounts of PIL-HA-1, PIL-HA-2, PIL-HA-3 and PIL-HA-4 to BSA were (123.64.+ -. 7.83) mg/g, (236.97.+ -. 13.24) mg/g, (368.69.+ -. 17.93) mg/g and (503.84.+ -. 15.05) mg/g, respectively. Of these, PIL-HA-4 HAs the best adsorption capacity for BSA, and its adsorption capacity is not only related to the pore structure in the hydrogel, but electrostatic interaction between the positively charged PIL and BSA also helps to increase the adsorption capacity of the hydrogel for BSA.

Fig. 11b is a bar graph of water content of PIL-HA-x (x=1.2.3.4) hydrogels. At a temperature of < -90 ℃, vacuum degree: in the case of < 5Pa, the PIL-HA-x (x=1.2.3.4) hydrogels were vacuum freeze-dried for 24 hours and then re-weighed and recorded, and the water content of the four hydrogels was calculated by calculating the water loss after freeze-drying. The calculation formula is as follows:wherein W is ₀ And W is _x The quality of the hydrogel before and after lyophilization, respectively. As can be seen from the figures: the water content of the PIL-HA-x (x=1.2.3.4) hydrogels of the present invention is about (80±3)%, so that it can provide a moist environment for the wound to slow down the pain of the wound and promote wound healing.

Fig. 11c is a graph of maximum tensile deformation rates of four PIL-HA-x (x=1.2.3.4) hydrogels cut into equal volumes of cuboids (12 mm×5mm×3 mm) and stretched with both hands to record the PIL-HA-x (x=1.2.3.4). As can be seen from the figure, with poly IL-NH ₂ The increase of the amount of ionic liquid increases the tensile deformation rate of the hydrogel, which indicates that the hydrogel has excellent mechanical properties.

FIG. 11d is a graph depicting the maximum tensile deformation of four hydrogels, as can be seen from the graph, as PIL-NH is polymerized in the hydrogels ₂ The maximum tensile deformation of the hydrogel was also gradually increased from 215% to 248%, 426% and 837% by increasing the ionic liquid content. Therefore, the hydrogel has good tensile deformation performance, so that skin tissues can be better protected.

FIGS. 12a and 12b show the comparison of the NIR fluorescent probe SCy-7 of the present invention with mouse fibroblast L929 (1×10) ⁶ Cell/culture dish) and embryonic fibroblast NIH-3T3 (1X 10) ⁶ Cell/petri dish) staining patterns of live and dead cells for 24h (1 day) and 48h (2 days) were co-cultured, while staining and observation of live and dead cells was performed using calcein double fluorescent staining. From the figure, it can be seen that almost no dead cells appeared in red, indicating that the NIR fluorescent probe SCy-7 has good biocompatibility.

FIGS. 12c and 12d show the PIL-HA-x (x=1.2.3.4) hydrogel (0.1 g) prepared according to the present invention after sufficient swelling balance and disinfection with mouse fibroblast L929 (1X 10) ⁶ Cell/culture dish) and embryonic fibroblast NIH-3T3 (1X 10) ⁶ Cell/petri dish) staining pattern of live and dead cells for 24h (1 day) and 48h (2 days), mass of PIL-HA-x (x=1.2.3.4) hydrogel was 0.5g. Viable and dead cells were also stained with calcein and observed. From the figure, it can be seen that the whole green living cells, but almost no red dead cells appear, thus indicating that the PIL-HA-x (x=1.2.3.4) hydrogels of the present invention have good biocompatibility.

FIGS. 13a and 13b are, respectively, mouse fibroblast L929 (1X 10) ⁵ Cells/well) and embryonic fibroblast NIH-3T3 (1X 10) ⁵ Cell/well) with the NIR fluorescent probe SCy-7 of the present invention for 1 day, 2 days and 3 days. From the figure, the cell activities of L929 and NIH-3T3 cells are above 90%, no statistically significant difference is observed between the control group and the probe treatment group, and the good biocompatibility of the NIR fluorescent probe SCy-7 is further demonstrated.

FIG. 13c and FIG. 13dThe cell survival ratio statistical graphs of the mouse fibroblast L929 and embryo fibroblast NIH-3T3 and PIL-HA-x (x=1.2.3.4) hydrogel prepared by the invention after full swelling balance and disinfection are respectively in direct contact for 1 day, 2 days and 3 days. The experimental method comprises the following steps: mouse fibroblast L929 and embryonic fibroblast NIH-3T3 were isolated at 1X 10 ⁵ After overnight incubation in 96-well plates, 10mg of PIL-HA-x (x=1.2.3.4) hydrogel in modified DMEM medium (10% fetal bovine serum, 1% diantigen (penicillin-streptomycin)) to reach swelling equilibrium and varying amounts of NIR fluorescent probe SCy-7 (5 μm, 10 μm, 15 μm, 20 μm) dissolved in modified DMEM medium were incubated in direct contact with cells, respectively, with NIR fluorescent probe SCy-7 at a concentration of 0 μm as negative control, and 200 μl of 0.01mol/L PBS (ph=7.4)/well as blank. Cck-8 solution (10. Mu.L/well) was added to 96-well plates at 1 day, 2 days, and 3 days, respectively, and the plates were kept at 37℃and 5% CO after being protected from light ₂ Is incubated for 1h and then the Optical Density (OD) value of each well is measured by an enzyme-labeled instrument at a wavelength of 450 nm. The cell viability was calculated as:wherein: OD (optical density) _T 、OD _blank 、OD _neg Optical densities of experimental, blank, and negative control groups, respectively. The results in the figures show that: with SCy-7 probe concentration and poly-PIL-NH in hydrogel ₂ The increase in ionic liquid concentration, although slightly decreasing the activity of the cells, can maintain a viability of more than 80%. Therefore, the SCy-7 probe and the PIL-HA hydrogel have good cell compatibility.

Fig. 14a is an evaluation of blood compatibility of PIL-HA-x (x=1.2.3.4) hydrogels by an in vitro hemolysis test. In the hemolysis test, fresh blood of a mouse is obtained by taking blood from an eyeball, the fresh blood of the mouse is diluted 10 times by using normal saline, the mixture is uniformly mixed and divided into five parts, red blood cells collected from whole blood are washed for a plurality of times by using the normal saline, finally, the red blood cells are diluted to 3mL by using the normal saline, rectangular PIL-HA-x (x=1.2.3.4) hydrogel (15 mm multiplied by 3mm multiplied by 2 mm) is respectively put into configured red blood cell stock solutions, and normal saline and buffer solution are respectively added into negative control and positive control. After incubation for 6h at 37℃the hydrogel was removed and the red blood cell suspension was centrifuged (1200 rpm) for 12min, photographed and recorded, and the supernatant was aspirated and absorbance was measured at 545nm using an ultraviolet-visible spectrophotometer. After co-culturing the four hydrogels with red blood cells on a shaker at 37 ℃ for 3 hours, the hydrogels were removed and the color comparison of the four hydrogel groups and the positive control group (buffer solution) was photographed. As can be seen from the figures: the supernatants of the experiments with the addition of PIL-HA-x (x=1.2.3.4) hydrogels were all pale yellow, while the positive control group was bright red. The quantitative analysis showed that the hemolysis rates of the experimental group to which PIL-HA-x (x=1.2.3.4) was added were (0.96.+ -. 0.13)%, (1.20.+ -. 2.6)%, (2.11.+ -. 0.09)% and (2.90.+ -. 0.04)%, respectively. Thus, it was found that the hemolysis rate of the hydrogel increased slightly with increasing ionic liquid content, but all showed good blood compatibility.

Fig. 14b is a graph of the compatibility of PIL-HA-x (x=1.2.3.4) hydrogels to blood cells evaluated by a blood cell adhesion test, 5mL of fresh mouse whole blood was first co-cultured with four samples of PIL-HA-x (x=1.2.3.4) hydrogels each having a diameter of 10mm, and after incubation in a shaker at 37 ℃ for 30min, the hydrogels were carefully removed and gently rinsed twice with normal saline and photographed for recording. As can be seen from the figures: the hydrogel has little adhesion of dead blood cells except slight yellowing, so that the hydrogel prepared by the invention has little damage to the blood cells, thereby showing good blood cell compatibility.

FIG. 15a is a graph showing the conductivity of PIL-HA-x (x=1.2.3.4) hydrogels prepared according to the present invention, which was evaluated by a four-probe method, as seen with PIL-NH ₂ The content of the ionic liquid in the hydrogel is increased, and the conductivity of the hydrogel is increased from 0.04mS/cm to 0.67mS/cm. It can be seen from this: the hydrogel samples of the present invention all have conductivity values similar to skin tissue and thus have great potential in transferring bioelectric signals and accelerating wound healing.

FIG. 15b is a schematic diagram of the conductivity of PIL-HA-2 hydrogel. The method comprises the following steps: the PIL-HA-2 hydrogel is used as an electric conductor to replace an electric wire to be connected to the LED bulbs of the series circuit, and the connection, cutting and re-splicing modes are observed to more intuitively prove that the prepared hydrogel HAs good electric conductivity.

FIG. 16a is a low temperature conductivity diagram of PIL-HA-2 hydrogels prepared according to the present invention as follows: the PIL-HA-2 hydrogel and the temperature sensor were connected to a series circuit after being frozen at-20℃for 24 hours at the same time, and the LED bulb and the battery were connected using the PIL-HA-2 hydrogel as an electrical conductor. From the figures it can be seen that: the brightness of the LED bulb is the same as the brightness at the room temperature of 25 ℃ at about-10 ℃, thereby showing that the hydrogel has good conductivity at low temperature.

FIG. 16b is a stretch-recovery plot of PIL-HA-2 hydrogel at low temperature. The method comprises the following steps: the PIL-HA-2 hydrogel was stretched at a low temperature of about-10℃and stopped when it was stretched to 3 times its own length. As can be seen from the figures: the hydrogel of the invention can quickly recover the initial state within 3 seconds.

FIG. 16c is a compression-recovery plot of PIL-HA-2 hydrogel at low temperature. The method comprises the following steps: the PIL-HA-2 hydrogel is stopped after being completely extruded at the low temperature of about-10 ℃. As can be seen from the figures: the hydrogels of the present invention quickly recovered their original shape within 3s under full extrusion. Therefore, the PIL-HA-2 hydrogel HAs excellent freezing resistance and good mechanical properties at low temperature.

FIG. 17a is a model of bleeding from a tail-end of a mouse to confirm the in vitro hemostatic capacity of PIL-HA-2 hydrogels. The experimental method is as follows: fixing anesthetized mice on a foam operation plate by using a pin, cutting a plastic film pad with consistent size at the lowest surface to prevent blood from exuding and losing, placing filter paper between the tail of the mice and the plastic to absorb blood flowing out of the tail, cutting the tail of one mouse by using scissors and tilting the foam plate to induce tail bleeding, immediately stopping bleeding at the tail breaking position of the mice by using PIL-HA-2 hydrogel after cutting the tail, and tilting the foam plate to induce tail bleeding. After 3min the filters were weighed and at least three experiments were performed in parallel. From the figure, it can be seen that the mice using the hydrogel group have little blood stain on the filter paper compared with the mice of the blank group, thereby showing that the PIL-HA hydrogel of the present invention HAs good in vitro hemostatic ability.

FIG. 17b is a model of liver hemorrhage in mice to confirm the hemostatic capacity of PIL-HA-2 hydrogel in vivo. The experimental method is as follows: the anesthetized and mice were also fixed, the mouse chest was dissected with a scalpel and the liver carefully removed. A plastic film is put on the filter paper, the filter paper is put between the liver and the plastic film, the needle of a 1mL sterile syringe is used for inducing liver bleeding, one group of the filter paper uses PIL-HA-2 hydrogel for liver hemostasis, the other group of the filter paper does not perform any treatment, and the foam plate is inclined to enable blood to better flow out of the liver and flow out of the filter paper. After 3min the filter paper was weighed and the experiment was also performed at least three times in parallel. From the graph, the blood flow on the filter paper of the hydrogel group mice is far smaller than that of the mice without the hemostasis treatment, so that the PIL-HA hydrogel HAs good in-vivo hemostasis capability.

FIG. 17c is a statistical plot of hemostatic capacity of PIL-HA-2 hydrogel, showing that mice in the control group of the tail-biting group had a blood loss of (130.04.+ -. 10.37) mg, while the PIL-HA-2 hydrogel group had only a blood loss of (10.24.+ -. 0.13) mg. Also in the needle-guided mouse liver hemorrhage model, the control group had a blood loss of (237.+ -. 14.84) mg in the mouse liver, while the PIL-HA-2 hydrogel group had a blood loss of only (50.3.+ -. 9.79) mg. The control group has obvious blood flow marks on the filter paper, while the hydrogel group has no obvious blood flow marks on the filter paper. Therefore, the hydrogel prepared by the invention has good hemostatic capability.

FIG. 18a is a drawing showing the adhesion of PIL-HA-2 hydrogel prepared by the present invention to pigskin, wherein the adhesion of the PIL-HA-2 hydrogel to pigskin is tight and almost without gaps due to the twisting and folding of the pigskin.

FIG. 18b shows the gradual peeling of the PIL-HA-2 hydrogel from the skin, and little fluctuation in the skin is seen, thus demonstrating that the PIL-HA-2 hydrogel can be easily removed without secondary damage to the wound.

FIG. 18c shows a schematic of the adhesion of PIL-HA-2 hydrogel to different articles. As can be seen from the figures: the PIL-HA hydrogel disclosed by the invention can be tightly adhered to the surface of tissues, and can be tightly adhered to the surfaces of other various materials (including rubber, paper, polyethylene (PE), polypropylene (PP), glass and metal). The hydroxyl groups of the hydrogel surface of the present invention have strong adhesion to various substrates, which can interact with different substrates through covalent or non-covalent bonds. Covalent bonds may be formed, for example, by Schiff bases on certain specific substrates containing amine or thiol groups; non-covalent bonds (e.g., hydrogen bonds, pi-pi stacking, etc.) may also exist between the hydrogel and other object surfaces.

Fig. 19a, 19b, 19c, 19d are the evaluation of the size of PIL-HA-x (x=1.2.3.4) hydrogels to promote fibroblast L929 and embryonic fibroblast NIH-3T3 cell proliferation capacity by cell scratch test. The experimental method is as follows: fibroblast L929 and embryonic fibroblast NIH-3T3 were isolated at 1.2X10 per well ⁶ Density of individual cells was seeded in 6-well plates, after the cells had adhered to and grown up, three scratches were vertically made in each well of the 6-well plates with a 200 μl sterile gun head, and four sterilized PIL-HA-x (x=1.2.3.4) hydrogels were placed, respectively, and the distances between the cell scratches were recorded by photographing with an inverted fluorescence microscope (IX 73, OLYMPUS) at 0h, 8h, and 16h, respectively. And wells to which no hydrogel sample was added were used as control groups. As is evident from the figures: compared with the control group, the PIL-HA hydrogel is beneficial to improving the proliferation capacity of the fibroblast L929, so that the scratch distance (800+/-50) of the hydrogel group is smaller than the scratch distance (1100+/-50) of the control group at the time point after the hydrogel is added for 8 hours. And the above phenomenon is more remarkable at the time point after 16h of adding the hydrogel, namely, the scratch distance of the hydrogel group is only (380+ -30) μm, and the scratch distance of the control group is as high as (780+ -20) μm. Also the PIL-HA hydrogel significantly contributed to proliferation of embryonic fibroblasts NIH-3T3 cells. The scratch distance of the hydrogel group was smaller (100.+ -.10) μm than that of the control group at the time point of 8h of addition of the hydrogel, whereas the scratch distance of the hydrogel group was only (200.+ -.25) μm at the time point of 16h of addition of the hydrogel, and the scratch distance of the control group was (510.+ -.20) μm. From this, it can be seen that the present invention Has remarkable promoting effect on migration and proliferation of fibroblast L929 and embryonic fibroblast NIH-3T 3.

FIGS. 20a and 20b show the results of fluorescence imaging experiments of NIR fluorescent probe SCy-7 on HClO in living cells and on fibroblasts L929 and embryonic fibroblasts NIH-3T3 cells. The experimental method is as follows: l929 cells and NIH-3T3 cells were incubated with 10. Mu.M NIR fluorescent probe SCy-7, respectively, for 30min at 37℃in a cell incubator, and fluorescence (NIR probe) was observed from a confocal inverted fluorescence microscope. While intracellular fluorescence quenching was observed after addition of 10. Mu.M SCy-7 and 10. Mu.M HClO (NIR probe+HClO). Subsequently, L929 cells and NIH-3T3 cells were stimulated with hydrolytic polymaleic anhydride (PMA) activators, which triggered an increase in mitochondrial ROS levels by activating the phosphatidylinositol signaling Pathway (PKC), thereby producing endogenous HClO. After stimulation with PMA (1 ng/mL), fluorescence in L929 cells and NIH-3T3 cells was significantly quenched (NIR probe+PMA). Carbaryl (NAC) can inhibit and eliminate intracellular active oxygen such as HClO. To investigate the effect of NAC on endogenous HClO levels in cells, L929 cells and NIH-3T3 cells were first treated with PMA for 20min, respectively, and then incubated with NAC (5 ng/mL) for 30min. Cells incubated with probe SCy-7 in the presence of NAC still have a fluorescent response, further demonstrating the ability of NAC to scavenge ROS (PMA+NAC+NIR probe). Cell fluorescence imaging experiments prove that the probe SCy-7 can successfully realize real-time visual detection of the HClO level in living cells.

FIGS. 20c and 20d further explore fluorescence imaging experiments of NIR fluorescent probe hydrogels on HClO in L929 cells and NIH-3T3 cells. And performing fluorescence imaging experiments on the response of HClO in living cells and the fibroblasts L929 and embryonic fibroblasts NIH-3T3 cells by using the NIR fluorescence probe hydrogel PIL-HA-2 according to the imaging experimental method of the probe SCy-7 in the same manner as the fluorescence imaging experiment of the probe SCy-7. Fluorescence (fluorescent hydrogel) was observed after addition of the NIR fluorescent probe hydrogel PIL-HA-2 from a confocal inverted fluorescence microscope; while intracellular fluorescence quenching was observed after addition of 10. Mu.M NIR fluorescent probe hydrogel PIL-HA-2 and 10. Mu.M HClO (fluorescent hydrogel+HClO); fluorescence in L929 cells and NIH-3T3 cells quenched significantly after stimulation with PMA (fluorohydrogel+pma); whereas L929 cells and NIH-3T3 cells were treated with PMA for 20min, respectively, followed by incubation with NAC (5 ng/mL) for 30min. Cells incubated with 10. Mu.M NIR fluorescent probe hydrogel PIL-HA-2 in the presence of NAC still had a fluorescent response, further demonstrating the ability of NAC to scavenge ROS (PMA+NAC+fluorescent hydrogel).

Fig. 21a, 21b evaluate the applicability of NIR fluorescent probe hydrogels to visualise HClO in living animals. Because near infrared light penetrates deeper into tissues, the autofluorescence interference is small, and the damage to biological samples is small. Thus, kunming (KM) mice were selected. 50mg kg by continuous 5 days ^-1 Is administered intraperitoneally with Streptozotocin (STZ) to healthy KM mice with fasting blood glucose between 3.0 and 6.0mmol/L to induce a diabetic mouse model. Randomly selecting successfully modeled diabetic mice, performing general anesthesia by injecting 5% chloral hydrate into the abdominal cavity, then shaving most of the hair on the back of the mice by using a pet shaver, and then cleaning the hair by using a non-irritating depilatory cream. After thoroughly sterilizing it with alcohol cotton, a 10mm x 10mm square full thickness skin defect (wound depth up to myomembrane) was created at the dehairing site using scissors and forceps. After four NIR fluorescent probe hydrogels (PIL-HA-2) containing equivalent amounts of SCy-7 probes prepared by the invention are tightly attached to the wounds of mice for 30min, the wounds of the mice are subjected to in-vivo fluorescent imaging through a small animal in-vivo imaging system. As can be seen from fig. 21 b: the fluorescence intensity of the NIR fluorescent probe hydrogel PIL-HA-2 increases with the increase of blood glucose in diabetic mice, and the HClO level at the wound increases accordingly, so that the fluorescence value of the NIR fluorescent probe hydrogel gradually decreases, and the fluorescence of the hydrogel is gradually quenched from stronger fluorescence as can also be seen in the fluorescence imaging of FIG. 21 a.

Similarly, fig. 21c and 21d evaluate the applicability of NIR fluorescent probe hydrogels to visualized HClO of living animal wounds, further exploring the fluorescent imaging of NIR fluorescent probe hydrogels to cells at wounds of diabetic mice. Fig. 21d shows that the fluorescence value at the wound of the mice decreases with increasing blood glucose in the mice. It was thus demonstrated that the NIR fluorescent probe hydrogels prepared according to the present invention can detect HClO levels at a living wound without interference from background signals. More importantly, all images in fig. 21c were obtained from KM mice that did not completely dehaire due to the near infrared absorption and emission properties of the probe.

FIGS. 22a, 22d evaluate the ability of NIR fluorescent probe hydrogels to visualize the reaction of detecting increased HClO levels in real time in living animals. Further, the increase in endogenous HClO levels of cells at the wound site was simulated with PMA solution (1 ng/mL), and the wound site was treated with HClO solution (1X 10) ^-5 ) Mimicking the increase in exogenous HClO levels in cells at the wound. Two non-diabetic KM mice are randomly selected, after the mice are anesthetized, the hairs on the back of the mice are totally removed, a full-thickness skin defect model is built, and after the NIR fluorescent probe hydrogel is covered, the mice are imaged through a small animal living body imaging system. Subsequently, the hydrogel adjuvant was removed from the group, 100 μl of PMA solution was added to the wound site, the hydrogel adjuvant was reapplied, and after 3min, the mice were imaged again and repeated four times. As the endogenous HClO level at the wound of the mouse increases, the fluorescence value of the NIR fluorescent probe hydrogel PIL-HA-2 auxiliary material at the wound of the mouse also decreases.

Similarly, another group of mice is taken out of the NIR fluorescent probe hydrogel PIL-HA-2 hydrogel auxiliary material, 100 mu L of HClO solution is added at the wound, the hydrogel auxiliary material is coated again for 3min, and then living body imaging is carried out on the mice again. Four repetitions may result in fig. 22c. From the characterization of fig. 22d, it is seen that as the exogenous HClO level increases in the wound site of the mice, the fluorescence value of the adjuvant in the wound site of the mice also decreases. Therefore, the NIR fluorescent probe hydrogel prepared by the invention can rapidly and sensitively monitor the increase of HClO level in a mouse wound model in real time, thereby playing a great potential in the aspects of preventing and treating diabetes wounds.

To evaluate the efficacy of the PIL-HA-2 hydrogel in treating acute wounds in normal mice, commercial Tegaderm was used ^TM The film served as a Control. The experimental method is as follows: normoglycemic mice were randomly selected, anesthetized and sterilized with alcohol, and a square full thickness skin defect (10 mm. Times.10 mm) was created with scissors and forceps to a depth of myomembrane, and NIR fluorescent probe hydrogel PIL-HA-2 hydrogel adjuvants were used, respectively、Tegaderm ^TM The film treats it. With increasing time after surgery, the wound area of the mice gradually decreased, and after 12 days of treatment, the wound treated with the PIL-HA-2 hydrogel group was substantially closed. Fig. 23a is a fluorescent in vivo image of acute wounds treated with different protocols on days 0, 3, 7, 10 and 12, and fig. 23b is a bar graph of the extent of healing after acute wounds treated with different protocols on days 0, 3, 7, 10 and 12. Throughout the repair (12 days), quantitative analysis of wound area: the wound healing rate (98.74%) using the PIL-HA-2 hydrogel group was far greater than that of commercial Tegaderm ^TM Film set (76.32%).

To evaluate the efficacy of PIL-HA-x (x=1.2.3.4) hydrogels on wound healing in diabetic mice, PBS was used as a Control, commercial Tegaderm ^TM The film served as a control. Similarly, mice were anesthetized and sterilized to create a square full thickness skin defect (10 mm x 10 mm) with depth up to the myomembrane, and treated with different adjuvants. As the post-operative time increases, the wound area of all groups also gradually decreases. After 14 days of treatment, the wounds were substantially closed after treatment with the PIL-HA hydrogel. FIG. 23c is a fluorescent in vivo image of diabetic skin wounds treated with different protocols on days 0, 3, 7, 10 and 14, and FIG. 23d is a bar graph of the extent of healing after diabetic skin wounds treated with different protocols on days 0, 3, 7, 10 and 14. After 14 days of healing, the wound area can be obtained after quantitative analysis: the wound healing rate of the PIL-HA-1 hydrogel group is 97.79%, the wound healing rate of the PIL-HA-2 hydrogel group is 99.31%, the wound healing rate of the PIL-HA-3 hydrogel group is 98.56%, the wound healing rate of the PIL-HA-4 hydrogel group is 99.76%, which are far greater than the wound healing rate of the blank group (71.61%) and the commercial Tegaderm ^TM Wound healing rate of the film group (78.61%).

To assess the biocompatibility of the hydrogels in mice. After completion of the above wound healing experiments, mice were sacrificed and their major organs (heart, liver, spleen, lung and kidney) were collected, fixed with 4% paraformaldehyde solution, and after paraffin-embedded sections, stained with hematoxylin eosin (H & E), and then observed using an optical microscope for examination of pathology photographs, the results of which are shown in fig. 24. Control was obtained by slicing organs of mice from a blank group without hydrogel. From the figure, it can be seen that the mice wound can maintain normal tissue structure without any obvious organ injury or inflammatory lesions after treatment with the PIL-HA hydrogel. The hydrogel prepared by the invention has good in vivo biocompatibility.

The therapeutic effect of the PIL-HA hydrogel on acute wound healing was further assessed by histological analysis and the results are shown in fig. 25 a. As can be seen from the figure, the skin tissue H is compared with the blank group&E staining results, 12 days after treatment, control group (Tegaderm used ^TM Thin film treatment) still had significant inflammatory cell infiltration, whereas the inflammatory cells of the skin tissue of mice treated with the PIL-HA-2 hydrogels of the invention were significantly reduced due to the excellent antibacterial and anti-infective capabilities of the hydrogels. The hydrogel group also showed more newly formed blood vessels and hair follicles and was more favorable for granulation tissue formation than the control group. The above results indicate that the PIL-HA hydrogels of the present invention promote cell proliferation and migration.

FIG. 25b is a schematic illustration of PIL-HA hydrogel and control (using Tegaderm) ^TM Membrane treatment) map of Masson staining after 12 days of acute wound treatment. As can be seen from the figure, wounds treated with the PIL-HA hydrogels of the invention showed more collagen deposition and more ordered collagen alignment, closer to normal skin tissue, than the control group.

The acute wound is treated by NIR fluorescent probe hydrogel PIL-HA-2 auxiliary material and Tegaderm respectively ^TM The regenerated skin after 12 days of film (control) treatment was quantitatively analyzed for the number of regenerated blood vessels by the Roldreview software, and the results are shown in FIG. 25 c. From the figure, the blood vessel number of the regenerated skin of the mice treated by the hydrogel PIL-HA-2 auxiliary material group is far more than that of the mice treated by the commercial auxiliary material group, which proves that the PIL-HA hydrogel can promote wound healing more effectively.

The acute wound is treated by NIR fluorescent probe hydrogel PIL-HA-2 auxiliary material and Tegaderm respectively ^TM 12 days after the treatment of the film (control group), the wound tissue is regeneratedThe collagen index of skin was quantitatively analyzed by Roldreview software and the results are shown in FIG. 25 d. From the figure, the collagen index of the regenerated skin of the mice treated by the hydrogel PIL-HA-2 auxiliary material group is better than that of the mice treated by the commercial auxiliary material group, which proves that the PIL-HA hydrogel can promote wound healing more effectively.

Fig. 25e shows the results of quantitative analysis of the epidermis thickness at the wound site of the acute wound 12 days after the hydrogel and control treatments using the roltview software, as shown in fig. 25 e. From the figure, the regenerated skin of the mice treated by the hydrogel PIL-HA-2 auxiliary material group HAs a larger epidermis thickness than that of the mice treated by the commercial auxiliary material group, which proves that the PIL-HA hydrogel can promote wound healing more effectively.

To further monitor the promotion effect of PIL-HA hydrogels prepared according to the present invention on wound healing in diabetic mice, commercial film 3M Tegaderm was used, respectively ^TM And PIL-HA-x (x=1.2.3.4) hydrogel dressing H was performed on skin tissue at wound site 14 days after healing treatment of diabetic mouse wound&E staining, the results are shown in FIG. 26 a. As can be seen from the results in the figures, the poly IL-NH was incorporated into the hydrogels of the present invention as compared to the control and control commercial 3M Tegader films ₂ The increase of the content of the ionic liquid increases the number of new blood vessels at the wound of the diabetic mouse, increases the thickness of epidermis and reduces the inflammation condition, and further shows that the PIL-HA hydrogel HAs remarkable promotion effect on the healing of the skin wound of the diabetic mouse.

FIG. 26b is a map of Masson staining of diabetic mice 14 days after skin wounds treated with PIL-HA hydrogels prepared according to the present invention, blank and control (commercial 3M Tegader film). From the figure, it can be seen that the skin wound of diabetic mice treated with the PIL-HA hydrogel of the present invention showed more collagen deposition and collagen arrangement was more ordered and closer to normal skin tissue than other groups through the entire healing process.

For 3M Tegaderm passing through commercial film ^TM And PIL-HA-x (x=1.2.3.4) hydrogel the number of regenerated blood vessels from skin wounds in diabetic mice after 14 days of treatment was quantified using the rolreview softwareThe results of the analysis are shown in FIG. 26 c. From the figure, the regenerated blood vessel number of the regenerated skin of the mice treated by the PIL-HA-x (x=1.2.3.4) hydrogel group is far more than that of the commercial auxiliary material group and the blank group, which indicates that the PIL-HA hydrogel can more effectively promote the healing of the diabetes wound.

For 3M Tegaderm passing through commercial film ^TM And PIL-HA-x (x=1.2.3.4) hydrogel skin wounds of diabetic mice after 14 days of treatment were quantitatively analyzed for collagen index using the rolreview software, and the results are shown in fig. 26 d. From the figure, the collagen index of regenerated skin of mice treated by the PIL-HA-x (x=1.2.3.4) hydrogel group is far better than that of the commercial auxiliary material group and the blank group, which shows that the PIL-HA hydrogel can more effectively promote the healing of diabetes wounds.

For 3M Tegaderm passing through commercial film ^TM And PIL-HA-x (x=1.2.3.4) hydrogel skin wounds of diabetic mice after 14 days of treatment were quantified for skin thickness using the rolreview software, and the results are shown in fig. 26 e. From the figure, it can be seen that the regenerated skin of mice treated with the PIL-HA-x (x=1.2.3.4) hydrogel group had a greater epidermis thickness than the commercial adjuvant group and the blank group, demonstrating that the PIL-HA hydrogel can more effectively promote healing of diabetic wounds.

To further explore the content of a range of inflammatory factors (interleukin (IL-6) and tumor necrosis factor (TNF-. Alpha.)) at acute wounds, 3M Tegaderm was passed through a commercial film ^TM And IL-6 and TNF-alpha at skin wound tissue of diabetic mice after 12 days of PIL-HA-2 hydrogel treatment were immunofluorescent stained, and the results are shown in FIG. 27a and FIG. 27b, respectively. As can be seen from the figure, the levels of pro-inflammatory chemokines (IL-6 and TNF- α) at skin wounds of diabetic mice were significantly reduced after acute wounds were treated with PIL-HA-2 hydrogels compared to the control group, thus indicating that PIL-HA hydrogels may be effective in promoting macrophage transformation from inflammatory cell M1 phenotype to reparative phenotype M2 during early inflammatory phases.

To further explore the content of a range of inflammatory factors (interleukin (IL-6) and tumor necrosis factor (TNF-. Alpha.)) in skin wounds of diabetic mice, a commercial film 3M Tegade was usedrm ^TM And PIL-HA-x (x=1.2.3.4) IL-6 and TNF- α at skin wound tissue of diabetic mice were immunofluorescent stained after 14 days of treatment, and the results are shown in fig. 27c, 27d, respectively. As can be seen from the figure, the content of pro-inflammatory chemokines (IL-6 and TNF-alpha) at skin wounds of diabetic mice was significantly reduced after PIL-HA hydrogel treatment compared to control group, and as the PIL-HA hydrogel was polymerized with IL-NH ₂ The increase in content, the number of hair follicles at the tissue also increases and the arrangement gradually tends to be regular, thus indicating that the PIL-HA hydrogel can effectively resist inflammation and promote wound healing at an early inflammatory stage.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A compound of the structure of formula ii:

wherein: m, n, x, y and z are each a number greater than or equal to 1.

2. A compound of the structure of formula ii according to claim 1 wherein m, n, x, y and z are each independently selected from the group consisting of numbers 1 to 100.

3. A compound of the structure of formula ii according to claim 2 wherein m, n, x, y and z are each independently selected from numbers 40 to 60.

4. A process for the preparation of a compound of the structure of formula ii according to claim 1, which comprises: the compound with the structure shown in the formula I and the poly IL-NH ₂ The ionic liquid (PIL) generates amide bond reaction to obtain near infrared fluorescent probe grafting Poly IL-NH ₂ The ionic liquid and modified hyaluronic acid (HA-QA-ALD) undergo Schiff base reaction and amide bond reaction to prepare a compound with a structure shown as II;

a compound of the structure shown in formula I:

the poly IL-NH ₂ The ionic liquid (PIL) has the structural formula:

5. the method of claim 4, wherein the compound of formula I is a compound of formula I and a poly-IL-NH ₂ The mass ratio of the ionic liquid is 1 (4-6).

6. The method of claim 4, wherein the modified hyaluronic acid (HA-QA-ALD) is grafted with a near infrared fluorescent probe to form a poly-IL-NH ₂ The mass ratio of the ionic liquid is (1-4): 5.

7. The method of claim 4, wherein the poly IL-NH is ₂ An ionic liquid (PIL) is prepared by a process comprising: reacting the compound 4 with acrylamide under the action of an initiator to obtain the poly-IL-NH ₂ Ionic Liquids (PILs);

the compound 4 has the following structure:

8. the preparation method according to claim 7, wherein the mass ratio of the compound 4 to the acrylamide is 1: (1-2);

the reaction mass ratio of the compound 4 to the initiator is (10-15): 1.

9. the method of claim 7, wherein the compound 4 is prepared by a process comprising: 1- (3-aminopropyl) -imidazole was reacted with 4-chloromethylstyrene, followed by ZnCl ₂ ZnCl in ethanol solution ^1- Coordination and Cl ^2- Replacement of coordination to give compound 4;

the reaction mole ratio of the 1- (3-aminopropyl) -imidazole to the 4-chloromethyl styrene is 1: (0.5-2).

10. The method of claim 4, wherein the modified hyaluronic acid (HA-QA-ALD) HAs the structural formula:

wherein: x, y and z are each a number greater than or equal to 1.

11. The method of claim 10, wherein x, y and z are each independently selected from the group consisting of numbers from 1 to 100.

12. The method of claim 11, wherein x, y and z are each independently selected from the group consisting of numbers from 40 to 60.

13. The process according to claim 4, wherein the process for producing a compound having a structure represented by formula I comprises reacting compound 3 with thiomorpholine to produce the above compound having a structure represented by formula I;

the compound 3 has the following structure:

the molar ratio of thiomorpholine to compound 3 is 1: (1-5);

the compound 3 is prepared by a method comprising the following steps: reacting the compound 1 with the compound 2 to obtain the compound 3;

the compound 1 has the following structure:

the compound 2 has the following structure:

14. Use of a compound of the structure of formula ii according to any one of claims 1 to 3 for the preparation of a fluorescent probe.

15. Use of a compound of the structure of formula ii according to any one of claims 1 to 3 for the preparation of a medicament for the repair of skin wounds.

16. A fluorescent probe comprising a compound of the structure of formula ii according to any one of claims 1 to 3.

17. A kit comprising the fluorescent probe of claim 16.

18. A biosensor comprising the fluorescent probe of claim 16.