CN115403502A - Ionic liquid-near infrared fluorescent probe hydrogel and preparation method and application thereof - Google Patents
Ionic liquid-near infrared fluorescent probe hydrogel and preparation method and application thereof Download PDFInfo
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- CN115403502A CN115403502A CN202210866953.0A CN202210866953A CN115403502A CN 115403502 A CN115403502 A CN 115403502A CN 202210866953 A CN202210866953 A CN 202210866953A CN 115403502 A CN115403502 A CN 115403502A
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Abstract
The invention discloses an ionic liquid-near infrared fluorescent probe hydrogel and a preparation method and application thereof.A 1- (3-aminopropyl) -3- (4-vinylbenzyl) imidazolium salt ionic liquid is polymerized and then mixed with an NIR fluorescent probe with a dicarboxyl function, the NIR fluorescent probe is grafted on the polyionic liquid through an amido bond, and meanwhile, a modified hyaluronic acid solution is added, and a 3D reticular polymer is formed through chemical and physical crosslinking. The hydrogel disclosed by the invention has good antifreezing performance, mechanical performance and biocompatibility, not only has conductivity similar to skin tissues, but also has excellent adhesion performance on the skin, and can be easily removed without causing secondary damage to the skin; the hydrogel disclosed by the invention has an obvious inhibiting effect on escherichia coli, staphylococcus aureus and bacillus, and can be used for detecting the HClO level of a wound in real time while treating the wound.
Description
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 particularly relates to a multifunctional conductive, anti-freezing and non-releasing antibacterial hydrogel combining a near infrared fluorescent probe and an 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% of the global population (nearly 4.5 billion), and the number of diabetics is increasing daily (Yang Yuan, daidi Fan, shining Shen, xiaoxuana, chemical Engineering Journal,2022, 133859. Particularly type II diabetes wounds, which are persistent inflammation, impaired angiogenesis, etc. due to the production of large amounts of Reactive Oxygen Species (ROS) in the body. Hypochlorous acid (HClO), a major highly reactive ROS, causes oxidative stress and destroys intracellular nucleic acids and proteins when present in excess concentrations in the body, which in turn triggers inflammation and delays wound healing (Xiaooli Qian, hui Yu, wenchao Zhu, xufeng Yao, wangwang Liu, shikui Yang, fangyuan Zhou, yi Liu, dyes and Pigments, volume 188,2021, 109218. 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 the level of HClO in vivo, in the past few decades, colorimetric, chromatographic, electrochemical, and fluorescent methods have been developed as detection methods. Among these methods, the fluorescence method is widely used because of its unique advantages such as easy operation and real-time analysis (Yan Zhang, haiyan Yang, mingxin Li, shuaigong, jie Song, zhonglong Wang, shifa Wang, dyes and Pigments, volume 197,2022,109861, 0143-7208). The near infrared fluorescent probe is a chemical dye which is excited by NIR fluorescence and can be used for quickly visualizing and quantifying an object to be detected after biological imaging. And the NIR fluorescence region (650-900 nm) has the advantages of high tissue penetration depth, weak photodamage to organisms, avoidance of complex background autofluorescence interference and the like (Chengg, fan J, sun W, chemical Communications,2013,50 (8): 1018-1020.). Due to its short life cycle and high activity, levels of HClO in diabetic wounds are often elusive and susceptible to interference from other ROS competitors. Therefore, the development of the hydrogel auxiliary material which can promote the healing of the diabetic wound and can quickly, sensitively and real-timely monitor the HClO level at the wound has great significance.
Disclosure of Invention
In order to improve the technical problem that the hydrogel auxiliary material can monitor the HClO level of the wound in real time while promoting the healing of the diabetic wound, the invention provides a multifunctional conductive, antifreezing and non-releasing antibacterial hydrogel with a near-infrared fluorescent probe combined with an ionic liquid, and a preparation method and application thereof. Compared with the reported polyion liquid functionalized hydrogel (Chao Zhou, chengju Sheng, linglinggao, jianguo, peng Li, bo Liu, volume 413,2021, 127429. The diabetic wound belongs to a chronic wound, the HClO concentration of the diabetic wound is abnormally high, and the excessive HClO can gradually lower or even quench the fluorescence value of the ionic liquid-near infrared fluorescent probe hydrogel. In order to rapidly quantify and visually image the HClO level of the diabetic wound, the ionic liquid-near infrared fluorescent probe hydrogel can be applied to the diabetic wound, the fluorescence change condition of the hydrogel can be seen through near infrared fluorescence living body imaging, and the HClO concentration change value of the diabetic wound can be estimated through the fluorescence value change 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, anti-freezing property, hemostatic property and non-releasing 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 the healing of the diabetic wounds.
In order to realize the 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 as shown in the formula I, which comprises the step of reacting the compound 3 with thiomorpholine to prepare the compound with the structure as 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 is 1, 1.
According to an embodiment of the present invention, the preparation process may be carried out 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 present invention, the preparation method further comprises a step of separating a solid product from the reacted mixture after the reaction is completed. For example, the reaction solution is poured into anhydrous ether, filtered, washed with anhydrous ether, the filter cake is collected, and dried to obtain the compound with the structure shown in formula I.
Preferably, the synthetic route for the compounds of formula I is as follows:
according to an embodiment of the present invention, said compound 3 is prepared by a process comprising: reacting the compound 1 with the compound 2 to obtain a compound 3;
the compound 1 has the following structure:
the compound 2 has the following structure:
according to an 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 is 1.
According to an embodiment of the present invention, in the reaction of the compound 1 with the compound 2, it is preferable to add a base.
Preferably, the molar ratio of the base to compound 2 is 1: (0.5-2), exemplary is 1.
Preferably, the base is selected from one, two or more of potassium carbonate, sodium tert-butoxide, potassium phosphate and sodium acetate.
According to an embodiment of the present invention, the preparation method further comprises a step of separating a solid product from the reacted mixture after the reaction is completed. For example, a solid product is obtained by spin-drying the solvent. Further, the preparation method also comprises the step of purifying the product. For example, the purification may be performed using a column chromatography column. Preferably, the eluent separated by the column chromatography column is dichloromethane/methanol = (40-60) 1 (v/v), exemplarily 40.
Preferably, the synthetic route of the compound 3 is as follows:
according to an embodiment of the present invention, said compound 2 is prepared by a process comprising: 2, 3-trimethyl-3H-indole and p-bromobenzoic acid are reacted to obtain the compound 2.
Preferably, the molar ratio of the 2, 3-trimethyl-3H-indole to p-bromobenzoic acid is 1: (1-2), exemplary are 1, 1.2, 1.
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 a mixture after the reaction is finished. For example, the reaction solution is poured into anhydrous ether, filtered under suction, washed with anhydrous ether, and the filter cake is collected and dried to obtain compound 2.
According to an embodiment of the present invention, the compound 1 is prepared by a method comprising: and reacting cyclohexanone with phosphorus oxychloride to obtain the compound 1.
Preferably, the molar ratio of cyclohexanone to phosphorus oxychloride is 1: (0.5-2), exemplary is 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 a step of separating a solid product from a mixture after the reaction is finished. For example, the reaction solution is poured into crushed ice, filtered with suction, washed with ethyl glacial acetate, and the filter cake is collected and dried to obtain compound 1.
Preferably, the synthetic route of the 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 from cyclohexanone and phosphorus oxychloride by using DMF as a solvent; the structural formula of the compound 1 is as follows:
(2) Synthesizing a compound 2 by using 2, 3-trimethyl-3H-indole and p-bromobenzoic acid and acetonitrile as a solvent; the structural formula of the compound 2 is as follows:
(3) Synthesizing a compound 3 from a compound 1 and a compound 2 under the action of alkali and by taking ethanol as a solvent; the structural formula of the compound 3 is as follows:
(4) Synthesizing a compound of a 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 a fluorescent probe.
The invention also provides a compound having a structure represented by 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, illustratively 40, 50, 60.
The invention also provides a preparation method of the compound with the structure shown in the formula IIThe method comprises the following steps: reacting a compound having a structure represented by formula I with poly-IL-NH 2 The ionic liquid (PIL) is subjected to amido bond reaction to obtain the near-infrared fluorescent probe grafted poly IL-NH 2 The ionic liquid and modified hyaluronic acid (HA-QA-ALD) are subjected to Schiff base reaction and amido bond reaction to prepare a compound with a structure shown in II;
the poly IL-NH 2 The structural formula of the ionic liquid (PIL) is as follows:
wherein: m and n are each a number greater than or equal to 1, 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 are exemplified by 40, 50, 60.
According to an embodiment of the invention, the compound of formula I is reacted with poly-IL-NH 2 The mass ratio of the ionic liquid is 1 (4-6), and the mass ratio is exemplarily 1.
According to an embodiment of the invention, the modified hyaluronic acid (HA-QA-ALD) is a poly-IL-NH grafted with a near-infrared fluorescent probe 2 The mass ratio of the ionic liquid is (1-4) to 5, and the ionic liquid is exemplarily 1.
According to an embodiment of the invention, the proportion of HA-QA in the modified hyaluronic acid (HA-QA-ALD) is between 46% and 53%, exemplarily 46%, 50%, 53%.
According to an embodiment of the invention, the poly IL-NH 2 The ionic liquid (PIL) is prepared by the following method comprising the following steps: reacting the compound 4 with acrylamide under the action of an initiator to obtain poly IL-NH 2 An ionic liquid (PIL);
the compound 4 has the following structure:
preferably, the reaction mass ratio of the compound 4 to acrylamide is 1: (1-2), exemplary are 1, 1.4, 1.
Preferably, the mass ratio of the compound 4 to the initiator is (10-15): 1, exemplarily 10. 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 of the ionic liquid (PIL) is as follows:
according to an embodiment of the present invention, said compound 4 is prepared by a process comprising: reaction of 1- (3-aminopropyl) -imidazole with 4-chloromethylstyrene followed by ZnCl 2 Is subjected to ZnCl reaction 3 - Coordination and Cl - Displacement of the coordination affords compound 4.
Preferably, the reaction molar ratio of the 1- (3-aminopropyl) -imidazole to the 4-chloromethylstyrene is 1: (0.5-2), exemplary is 1.
Preferably, the reaction of the 1- (3-aminopropyl) -imidazole with 4-chloromethylstyrene can 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, filtration and spin-drying of the solvent can be used to obtain the crude product. Preferably, the preparation method further comprises the step of purifying the crude product to obtain the compound 4. For example, the purification can be performed using column chromatography to give compound 4. Preferably, the eluent separated by the column chromatography column is dichloromethane and methanol = (90-110) 1 (v/v), exemplary 90.
Preferably, the synthetic route of the compound 4 is as follows:
according to an embodiment of the 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 of 1 to 100, preferably each independently selected from a number of 40 to 60, exemplary 40, 50, 60.
According to an embodiment of the invention, the modified hyaluronic acid (HA-QA-ALD) is prepared by a process comprising: first pass through NaIO 4 Oxidizing hyaluronic acid to obtain a compound with a structure shown in a formula III, and reacting with a Gillette reagent T to obtain modified hyaluronic acid (HA-QA-ALD);
the structural formula of the compound with the structure 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, exemplary being 40, 50, 60.
Preferably, the hyaluronic acid is associated with NaIO 4 The reaction mass ratio of (1): (0.4-1), exemplary is 1.
Preferably, the reaction mass ratio of the compound with the structure shown in the formula III to the Girard reagent T is 5: (1-3).
Preferably, the reaction of the compound of the structure represented by the formula iii with the gillart reagent T may be carried out in a solvent, and the solvent may be a buffer solution.
Preferably, the reaction of the compound of the structure shown in the formula III with the Giralde 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 formula iii with gillart reagent T may be carried out at room temperature.
Preferably, the preparation method of the modified hyaluronic acid (HA-QA-ALD) further comprises purifying the product after the reaction is finished. For example, the solution is dialyzed with NaCl solution at pH =7 for 2 days, and then placed in a buffer solution for dialysis for 2 days.
Preferably, the preparation method of 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:
unless otherwise specified, all the buffer solutions used in the present invention are 0.01mol/L PBS buffer solution with pH = 7.4.
The invention also provides application of the compound with the structure shown in the formula II in preparation of a fluorescent probe. Preferably in the preparation of a medicament for repairing skin wounds.
The invention also provides a fluorescent probe which comprises the compound with the structure shown in the formula II.
The invention also provides a kit which comprises 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 area animal living body 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 preparation of a medicine for promoting wound healing. Preferably in the preparation of the medicine for promoting the healing of skin wounds caused by diabetes.
The concept and principle of the present invention are illustrated as follows: the bonds formed by physical cross-linking are weaker, but the rate of bond formation and reestablishment of dynamic equilibrium is faster. The mechanical strength of the hydrogel formed by physical cross-linking is therefore weak and unsuitable for dynamic wounds. While stronger bonds can be formed by chemical cross-linking, the rate of bond formation and reestablishment of dynamic equilibrium is slower. Thus, the introduction of chemical crosslinking can improve the mechanical strength of the hydrogel. According to the invention, by utilizing the characteristics that amino and carboxyl can form physical crosslinking-amido bond, and aldehyde group and amino are easy to generate chemical crosslinking-Schiff base reaction, firstly, an NIR fluorescent probe containing carboxyl functionalization and ionic liquid containing amino functionalization are prepared, the ionic liquid is firstly mixed with an NIR probe with fluorescence quenching property in 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 polyionic liquid through amido bond, the modified hyaluronic acid is connected with the polyionic liquid through amido bond and Schiff base reaction to form 3D three-dimensional network hydrogel, and the hydrogel can visualize and quantify HClO level at a wound under a physiological state through NIR fluorescence imaging, thereby obtaining the novel multifunctional ionic liquid NIR & imaging hydrogel.
The invention has the beneficial effects that:
fluorescent probes are used for both quantitative and qualitative analysis of various analytes. Near Infrared (NIR) fluorescence probes are widely used due to their advantages of low background interference, high tissue penetration, low tissue damage, etc. 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 bioimaging are severely limited. The excitation wavelength of the near-infrared fluorescent probe SCy-7 is 680nm, the emission wavelength is 778nm, the Stokes shift is up to 98nm, and the fluorescence quantum yield is 0.18 in methanol. It is prepared byThe imaging system is used for imaging living cells and mice, has high contrast and high spatial resolution, and has satisfactory tissue imaging depth. Meanwhile, the ionic liquid is molten salt with excellent chemical and thermal stability, is classified as a green solvent, can perform polymerization reaction to form polyionic liquid, and has good ionic conductivity due to the existence of anion and cation electrolyte groups on the 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), a polymer, is a high-grade polysaccharide composed of units of D-glucuronic acid and N-acetylglucosamine, is a natural polymer which is nontoxic, colorless and tasteless, can stimulate epithelial cell migration, enhance angiogenesis and reduce inflammation, and is an ideal material for developing hydrogels. The novel multifunctional ionic liquid-NIR probe hydrogel PIL-HA is prepared by polymerizing 1- (3-aminopropyl) -3- (4-vinylbenzyl) imidazolium salt ionic liquid containing positive charge active groups, mixing the polymerized ionic liquid with an NIR fluorescent probe with a dicarboxyl function, grafting the NIR fluorescent probe on polyionic liquid through amido bonds, simultaneously adding a modified hyaluronic acid solution, and forming a 3D network polymer through chemical and physical crosslinking (Schiff base and amido bond connection). The hydrogel disclosed by the invention has good antifreezing property, mechanical property and biocompatibility, not only has conductivity similar to skin tissue, but also has excellent adhesion property on the skin, and can be easily removed without causing secondary damage to the skin; meanwhile, under the condition of not adding antibiotics, the hydrogel has obvious inhibition effect on escherichia coli, staphylococcus aureus and bacillus, and the treatment results on acute wounds and diabetic skin wounds are superior to those of commercial Tegaderm TM The film shows that the hydrogel can also detect the HClO level at the wound in real time while treating the wound through the NIR fluorescence in vivo imaging result. Specifically, the method comprises the following steps:
(1) The NIR fluorescent probe hydrogel PIL-HA can detect the HClO level at a wound in real time through in-vivo imaging so as to reduce the probability of conversion from an acute wound to a chronic wound, thereby achieving the purpose 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 is poly-PIL-NH 2 The positive charge groups contained in the ionic liquid can destroy the integrity of bacteria by generating electrostatic interaction with the anion groups of the bacterial cell walls, thereby playing a good bactericidal role.
(3) The aldehyde group and carboxyl group of the modified hyaluronic acid can generate chemical reaction with the amino group of skin tissue, and meanwhile, positive charge groups in the modified hyaluronic acid and hydrogel are combined with negative charge sialic acid groups on a mucous membrane and phospholipid of a cell membrane, so that the hydrogel has good adhesive property.
(4) The hydrogel disclosed by the invention is stable in structure, short in gelling time (within 3 min), excellent in adhesive property, capable of being firmly adhered to skin and wound parts, good in hemostatic property and capable of being easily removed without causing secondary damage to the wound.
(5) The hydrogels of the invention are associated with poly-PIL-NH 2 The increase in ionic liquid content (2 wt% to 8 wt%) gradually increased the crosslink density of the hydrogel, and increased the conductivity of the hydrogel from 0.04mS/cm to 0.67mS/cm. The hydrogel of the invention has the conductivity similar to that of skin tissue, and thus has great application potential in the process of transferring bioelectrical signals and accelerating wound healing.
(6) The hydrogel disclosed by the invention has good antifreezing capability, still has good conductivity, tensile and compressive properties under the condition that the temperature reaches-10 ℃, can be quickly recovered to the original shape within 3s after being stretched and compressed, and can protect tissues from potential damage due to excellent flexibility.
(7) The NIR fluorescent probe hydrogel PIL-HA of the invention shows far superior to commercial Tegaderm in the treatment of full-thickness skin injury models of normal and diabetic mice on the healing rate of wounds, the thickness of granulation tissues, the content of proinflammatory factors, tumor necrosis factors and interleukins TM Therapeutic effect of the film.
Drawings
FIG. 1a shows NIR fluorescence probe SCy-7 1 H NMR spectrum, FIG. 1b is infrared spectrum of NIR fluorescent probe SCy-7, FIG. 1c is mass spectrum of NIR fluorescent probe SCy-7, and FIG. 1d is 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 results of a selective experiment with NIR fluorescent probe SCy-7, FIG. 2c is a graph of fluorescence intensity variation of NIR fluorescent probe SCy-7 with HClO, and FIG. 2d is a graph of a 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 HA-QA-ALD, HA-ALD and HA of modified hyaluronic acid.
FIG. 5a is a flow chart of a preparation process for gel formation of a PIL-HA-2 hydrogel, and FIG. 5b is a diagram of a 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) hydrogel on the back of the hand; fig. 5d is a schematic of gel time for PIL-HA-x (x = 1.2.3.4) hydrogel.
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 pattern 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, and FIG. 7d is an SEM image of a PIL-HA-4 hydrogel.
Fig. 8a is a graph of tensile testing of a PIL-HA-x (x = 1.2.3.4) hydrogel, fig. 8b is a graph of compressive testing of a PIL-HA-x hydrogel, fig. 8c is a schematic of the rapid recovery of a PIL-HA-2 hydrogel within 3s after compressive testing, and fig. 8d is a graph of a splicing tower cut experiment performed on pig skin of a PIL-HA-x hydrogel.
FIG. 9a is a comparison of PIL-HA-x (x = 1.2.3.4) hydrogel before and after swelling, FIG. 9b is a bar graph of mass comparison before and after swelling of PIL-HA-x hydrogel, and FIG. 9c is a bar graph of PIL-HA-x hydrogel base area comparison before and after swelling.
Fig. 10a is a graph for evaluating the antibacterial ability of the PIL-HA-x (x = 1.2.3.4) hydrogel prepared according to the present invention by the disk method, fig. 10b is a graph for qualitatively evaluating the size of the area of inhibition generated by the hydrogel by the disk method, fig. 10c is a graph for quantitatively evaluating the antibacterial ability of the PIL-HA-x hydrogel prepared according to the present invention, and fig. 10d is a graph for evaluating the antibacterial rate of the hydrogel by the colony counting method.
FIG. 11a is a graph showing the test results of protein adsorption performance of the PIL-HA-x hydrogel prepared according to the present invention (x = 1.2.3.4), FIG. 11b is a histogram showing the water content of the PIL-HA-x hydrogel prepared according to the present invention, and FIG. 11c is a graph showing the maximum tensile set of the PIL-HA-x hydrogel prepared according to the present invention. FIG. 11d is a histogram of statistical PIL-HA-x hydrogel elongation.
FIG. 12a is a staining pattern of viable and dead cells after 1 day and 2 days of co-culture of NIR fluorescence probe SCy-7 directly prepared by the present invention and fibroblast cells L929, FIG. 12b is a staining pattern of viable and dead cells after 1 day and 2 days of co-culture of NIR fluorescence probe SCy-7 directly prepared by the present invention and embryonic fibroblast cells NIH-3T3, FIG. 12c is a staining pattern of viable and dead cells after 1 day and 2 days of co-culture of PIL-HA-x (x = 1.2.3.4) hydrogel prepared by the present invention and fibroblast cells L929, and FIG. 12d is a staining pattern of viable and dead cells after 1 day and 2 days of co-culture of PIL-HA-x hydrogel prepared by the present invention and embryonic fibroblast cells NIH-3T 3.
FIG. 13a is a statistical graph of cell survival numbers of NIR fluorescent probe SCy-7 prepared by the present invention after 1, 2 and 3 days of co-culture with fibroblast cell L929 directly, FIG. 13b is a statistical graph of cell survival numbers of NIR fluorescent probe SCy-7 prepared by the present invention after 1, 2 and 3 days of co-culture with embryonic fibroblast cell NIH-3T3 directly, FIG. 13c is a statistical graph of cell survival numbers of PIL-HA-x (x = 1.2.3.4) hydrogel prepared by the present invention after equilibrium swelling and fibroblast cell L929 after 1, 2 and 3 days of co-culture, and FIG. 13d is a statistical graph of cell survival numbers of PIL-HA-x hydrogel prepared by the present invention after equilibrium swelling and embryonic fibroblast cell NIH-3T3 after 1, 2 and 3 days of co-culture.
FIG. 14a is a graph showing the hemolytic activity of a PIL-HA-x (x = 1.2.3.4) hydrogel prepared according to the present invention; FIG. 14b is a test chart of the cell adhesion test of the PIL-HA-x hydrogel prepared by the present invention to blood.
Fig. 15a is a graph showing the conductivity measurement of the PIL-HA-x (x = 1.2.3.4) hydrogel prepared according to the present invention, and fig. 15b is a schematic diagram showing the conductivity of the PIL-HA-2 hydrogel.
Fig. 16a is a low-temperature conductive pattern of the PIL-HA-2 hydrogel prepared according to the present invention, fig. 16b is a stretch-recovery pattern of the PIL-HA-2 hydrogel at a low temperature, and fig. 16c is a compression-recovery pattern of the PIL-HA-2 hydrogel at a low temperature.
FIG. 17a is a comparison of the hemostatic capacities of the blank control group and the PIL-HA-2 hydrogel prepared according to the present invention in vitro, FIG. 17b is a comparison of the hemostatic capacities of the blank control group and the PIL-HA-2 hydrogel prepared according to the present invention in vivo, and FIG. 17c is a statistical chart of the hemostatic capacities of the blank control group and the PIL-HA-2 hydrogel prepared according to the present invention.
FIG. 18a is a graph showing the adhesion of the PIL-HA-2 hydrogel prepared by the present invention to pigskin, FIG. 18b shows that the PIL-HA-2 hydrogel can be easily peeled off from skin, and FIG. 18c shows a schematic graph showing the adhesion of the PIL-HA-2 hydrogel to various articles and the adhesion diagram of the PIL-HA-2 hydrogel to various articles.
Fig. 19a is a micrograph of in vitro L929 cell scratch distances before and after 6, 12 and 18h of blank and PIL-HA-x (x = 1.2.3.4) hydrogel treatment made according to the present invention, fig. 19b is a quantitative statistical plot of in vitro L929 cell scratch distances before and after 6, 12 and 18h of blank and PIL-HA-x hydrogel treatment made according to the present invention, fig. 19c is a micrograph of in vitro NIH-3T3 cell scratch distances before and after 6, 12 and 18h of blank and PIL-HA-x hydrogel treatment made according to the present invention, and fig. 19d is a quantitative statistical plot of in vitro NIH-3T3 cell scratch distances before and after 6, 12 and 18h of blank and PIL-HA-x hydrogel treatment made according to the present invention.
FIG. 20a is an inverted fluorescence imaging microscope image of the prepared NIR fluorescence probe SCy-7 and L929 cell, FIG. 20b is an inverted fluorescence imaging microscope image of the prepared NIR fluorescence probe SCy-7 and NIH-3T3 cell, FIG. 20c is an inverted fluorescence imaging microscope image of the prepared NIR fluorescence probe hydrogel PIL-HA-2 and L929 cell, and FIG. 20d is an inverted fluorescence imaging microscope image of the prepared NIR fluorescence probe hydrogel PIL-HA-2 and NIH-3T3 cell.
FIG. 21a is an NIR fluorescence in vivo imaging graph of the NIR fluorescence probe hydrogel PIL-HA-2 prepared by the invention taken on wounds of diabetic mice with different blood sugar values, FIG. 21b is a graph obtained by quantifying and plotting the fluorescence value of the hydrogel adhered to the diabetic mice with different blood sugar values, FIG. 21c is an NIR fluorescence in vivo imaging graph of the NIR fluorescence probe hydrogel PIL-HA-2 prepared by the invention taken on wounds of diabetic mice with different blood sugar values, and FIG. 21d is a graph obtained by quantifying and plotting the fluorescence value of the wounds of diabetic mice with different blood sugar values.
FIG. 22a is a NIR fluorescence live imaging graph of the NIR fluorescence probe hydrogel PIL-HA-2 prepared by the invention on wounds of normal blood glucose mice with the increase of PMA content at the wounds; FIG. 22b is a graph of fluorescence of an adherent hydrogel as a function of PMA content at a wound site in mice; FIG. 22c is a chart of NIR fluorescence in vivo imaging of the NIR fluorescence probe hydrogel PIL-HA-2 prepared by the invention on wounds of normal blood glucose mice with increasing HClO content at the wounds; FIG. 22d is a graph of fluorescence of an adherent hydrogel in mice as a function of HClO content at the wound site.
FIG. 23a is a graph of fluorescence in vivo images of acute wounds on days 0, 3, 7, 10 and 12 using different protocols for treatment of skin, FIG. 23b is a graph of the degree of healing of acute wounds on days 0, 3, 7, 10 and 12 using different protocols for treatment of skin, FIG. 23c is a graph of fluorescence in vivo images of chronic wounds on days 0, 3, 7, 10 and 14 using different protocols for treatment of diabetic skin, and FIG. 23b is a graph of the degree of healing of chronic wounds on days 0, 3, 7, 10 and 14 using different protocols for treatment of diabetic skin.
Fig. 24 is a hematoxylin and eosin (H & E) staining image of the organs of diabetic mice after completion of wound healing.
FIG. 25a is a graph of H & E staining of a PIL-HA-2 hydrogel prepared according to the invention after 12 days of acute wound treatment; FIG. 25b is a graph of Masson staining after 12 days treatment of acute wounds with a PIL-HA-2 hydrogel prepared according to the present invention; FIG. 25c is a graph of the quantification of the number of blood vessels in an acute wound; FIG. 25d is a graph of the quantification of the collagen index of acute wounds; FIG. 25e is a graph of the quantitative analysis of epidermal thickness at acute wounds.
FIG. 26a is a 3M Tegaderm film passed over a commercial film TM And PIL-HA-x (x = 1.2.3.4) hydrogel treated H of skin wounds of diabetic mice&E, staining pattern; FIG. 26b is a 3M Tegaderm film passed over a commercial film TM And Masson staining of PIL-HA-x (x = 1.2.3.4) hydrogel treated diabetic mouse skin wounds; FIG. 26c is a graph showing the quantitative analysis of the number of blood vessels at the skin wound of diabetic mice; FIG. 26d is a graph showing the quantitative analysis of collagen index at a skin wound of a diabetic mouse, and FIG. 25e is a graph showing the quantitative analysis of the epidermal thickness at a skin wound of a diabetic mouse.
FIG. 27a is a PIL-HA-2 hydrogel and a commercial film of 3M Tegaderm prepared by the present invention TM Immunofluorescent staining patterns for IL-6 12 days after treatment of acute wounds; FIG. 27b is a PIL-HA-2 hydrogel and a commercial film of 3M Tegaderm prepared by the present invention TM Immunofluorescent staining pattern for TNF-alpha 12 days after treatment of acute wounds; FIG. 27c is a 3M Tegaderm commercial film pass through TM And a plot of immunofluorescence staining of IL-6 following treatment of skin wounds in diabetic mice with PIL-HA-x (x = 1.2.3.4) hydrogel; FIG. 27d is a 3M Tegaderm film passed over a commercial film TM And a plot of immunofluorescent staining for TNF- α after treatment of skin wounds in diabetic mice with PIL-HA-x (x = 1.2.3.4) hydrogel.
FIG. 28 is a schematic diagram of the NIR fluorescent probe PIL-HA hydrogel prepared by the invention for promoting healing of diabetic wounds and rapidly and visually monitoring HClO levels of the diabetic wounds.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the techniques realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.
Example 1
The preparation method of the multifunctional ionic liquid & NIR imaging hydrogel PIL-HA comprises the following steps:
(1) Preparation of NIR fluorescent Probe SCy-7:
10mL of DMF is placed in a 50mL single-neck flask, and under the ice bath condition, the mixture is stirred and slowly added with a phosphorus oxychloride solution (6.8mL, 7.5 mmol), stirred for 30min, warmed to room temperature, added with 2mL of cyclohexanone, and heated to 50 ℃ to continue the reaction for 6h. After the reaction is finished, pouring the reaction liquid into 100g of crushed ice in batches, stirring for 5h, separating out yellow precipitate, performing suction filtration, washing with ethyl glacial acetate for 3 times, and refrigerating the obtained yellow powder (compound 1) for later use.
Under a nitrogen atmosphere, 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, 100mL acetonitrile was added, the mixture was heated to 81 ℃ and reacted for 10 hours, the mixture was cooled to room temperature, the reaction solution was poured into 100mL of anhydrous ether in portions, precipitation was immediately generated, suction filtration was performed, washing was performed 3 times with anhydrous ether, the filter cake was collected, and vacuum drying was performed to obtain 4.8630g of a product (Compound 2).
Compound 1 (3.0000 g, 17.4 mmol), compound 2 (10.7613 g,36.6 mmol), anhydrous sodium acetate (3.0023g, 36.6 mmol) were placed in a 150mL flask under a nitrogen atmosphere, 120mL of ethanol was added, heated to 70 ℃ for reaction for 2h, cooled to room temperature, extracted with dichloromethane (4 × 100 mL), the upper layer liquid was collected, the solvent was rotary evaporated, column chromatography separation and purification (eluent dichloromethane: methanol =50, 1, v/v), the product was collected, the solvent was rotary evaporated, vacuum drying was carried out, and 2.7487g of a dark green product (compound 3) with metallic luster was obtained.
Under the nitrogen atmosphere, placing compound 3 (1.0000 g and 1.25 mmol) and thiomorpholine (388 mg and 3.76 mmol) in a 50mL flask, adding 15mL of DMF, stirring at room temperature for reaction for 2h, pouring the reaction liquid in batches into a large amount of anhydrous ether, generating precipitate immediately, performing suction filtration, washing for 3 times by using the anhydrous ether, collecting a filter cake, and performing vacuum drying to obtain 0.8152g of product, 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 dissolved in 80mL of methanol sufficiently, 10.68g of 4-chloromethylstyrene (0.07 mol) was added. After stirring vigorously at room temperature for 12h, saturated ZnCl is used 2 Is stirred at 30 ℃ for 24h to carry out ZnCl 3 - Coordination and Cl - Replacement of the coordination. Filtering the solution after stirring, spin-drying the filtered solution by a vacuum rotary evaporator, purifying the residual product by silica gel column chromatography with dichloromethane and methanol (100/1, v/v), drying the obtained yellow product in a vacuum drying oven at 60 ℃ to obtain the yellow product 1- (3-aminopropyl) -3- (4-vinylbenzyl) imidazolium salt ionic liquid, and recording the ionic liquid as IL-NH 2 An ionic liquid.
(b) Poly IL-NH 2 Preparation of ionic liquid:
to a 10mL round bottom flask was added 1mL of 0.01mol/L PBS (pH = 7.4) buffer solution, 0.7g of acrylamide and 0.04g of ammonium persulfate were added thereto, and 0.5g of IL-NH obtained in step (a) was further added 2 Stirring the ionic liquid for 5min to obtain poly IL-NH 2 Ionic liquid, abbreviated as PIL.
(3) Preparation of modified hyaluronic acid:
(a) Preparation of modified hyaluronic acid HA-QA: 1g hyaluronic acid (MW =340 000Da) powder was well dissolved in a beaker containing 100mL 0.01mol/L PBS buffer (pH = 7.4) and the beaker was wrapped with tinfoil paper, 0.6g sodium periodate was added after obtaining a transparent solution, and stirred at room temperature for 12h. 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 000Da). And finally, freeze-drying the dialyzed product in a vacuum freeze dryer to obtain a white cotton-shaped 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) HA-ALD aqueous solution, and adding 0.6g Girard ‘s Reagent T, adjusting the pH value of the solution to 4.5-5, stirring the reaction at room temperature for 26h, dialyzing with NaCl solution with pH =7 for 2 days, then dialyzing in deionized water for 2 days, and freeze-drying the dialyzed product by a vacuum freeze-dryer to obtain a white cotton-shaped product HA-QA-ALD.
(4) Preparation of a novel multifunctional ionic liquid-NIR imaging hydrogel PIL-HA:
after solubilizing the NIR fluorescence probe SCy-7 prepared in step (1) with 100. Mu.L of methanol, the probe was concentrated by adding 0.01mol/L PBS (pH = 7.4)Degree of arrangement is 10 -5 g/mL to obtain an NIR fluorescent probe solution;
subjecting the poly IL-NH obtained in the step (2) 2 The ionic liquid is dissolved in 0.01mol/L PBS (pH = 7.4) to prepare 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 the concentration of 5 wt%;
taking 200 μ L of 10 -5 g/mL NIR fluorescent probe solutions were mixed with 1mL of poly IL-NH dissolved in 0.01mol/L PBS (pH = 7.4) at 2wt%,4wt%,6wt%,8wt%, respectively 2 Mixing the ionic liquid solutions, respectively adding 5wt% of modified hyaluronic acid solution with the same volume, uniformly mixing, and then mutually crosslinking at 60 ℃ for 1-3min to respectively obtain the multifunctional ionic liquid&NIR probe hydrogels PIL-HA-1, PIL-HA-2, PIL-HA-3, PIL-HA-4, abbreviated PIL-HA-x (wherein wt% is the addition polymerized IL-NH) 2 Mass percent of ionic liquid solution).
The synthetic route of the NIR fluorescence probe SCy-7 prepared in the example is as follows:
poly IL-NH was prepared in this example 2 The synthetic route of the ionic liquid is as follows:
the synthetic route for HA-QA-ALD obtained in this example is as follows:
the synthetic route for preparing the PIL-HA hydrogel in this example is as follows:
wherein: m, n, x, y and z are each a number of 40 to 60.
The schematic diagram of the PIL-HA hydrogel combined with the NIR fluorescent probe for realizing the promotion of the wound healing of the diabetic patient and simultaneously monitoring the HClO level of the wound in real time is shown in fig. 28.
The PIL-HA hydrogel prepared in this example was subjected to characterization and performance tests, and the results are shown in FIGS. 1 to 27:
FIG. 1a is a nuclear magnetic resonance hydrogen spectrogram of an NIR fluorescent probe SCy-7, which can be obtained by analyzing chemical shifts and attribution of corresponding hydrogen atoms in a molecular structure.
FIG. 1b is a Fourier infrared spectrum of an NIR fluorescence probe SCy-7, wherein 3460cm -1 The presence of carboxyl groups was confirmed by the absorption peak at 2925cm -1 Is CH 2 ＝CH 2 Stretching vibration peak of middle C-H, 1534cm -1 、1378cm -1 And 1165cm -1 Is the vibration peak of the skeleton of the benzene ring, 925cm -1 、798cm -1 And 752cm -1 Respectively comprises a C-N stretching vibration peak, a C-S stretching vibration peak and a C-C stretching vibration peak. From the above results, the present invention has successfully prepared the SCy-7 near infrared fluorescent probe.
FIG. 1c is a mass spectrometric characterization of the NIR fluorescent probe SCy-7, and the results in the figure show that M/z =791.4787 ([ M + H ] +) of SCy-7 is consistent with the formula weight of the target compound, thereby demonstrating that the NIR fluorescent probe SCy-7 is successfully prepared according to the invention.
FIG. 1d is a graph of the ultraviolet fluorescence spectrum of an NIR fluorescence probe SCy-7, the characteristic absorption peak at 778nm indicating that the probe belongs to the near infrared fluorescence region (650 nm to 900 nm).
FIG. 2a is a pH stability plot of NIR fluorescent probe SCy-7, investigating the effect of different pH on HClO response. In the case of no HClO addition, the fluorescence spectrum of the NIR fluorescent probe SCy-7 solution hardly changes in the pH range of 4.0 to 8.0; 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 figure show that: the probe can sensitively detect HClO in any wound environment.
FIG. 2b is a graph of the results of a selectivity experiment for the NIR fluorescent probe SCy-7, testing the selective specificity of the NIR fluorescent probe SCy-7 of the invention for HClO to detect the potential for use of the probe in complex biochemical environments. The experimental results show that: when HClO (80. Mu.M) was added, the fluorescence intensity of the NIR fluorescent probe SCy-7 was significantly reduced; and 80. Mu.M of an inorganic salt (Ca) was added 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 - ) The fluorescence intensity of the NIR fluorescent probe SCy-7 hardly changed. From this result, it can be seen that: the NIR fluorescent probe SCy-7 can realize specific detection on HClO.
FIG. 2c is a graph of the change in fluorescence intensity of NIR fluorescent probe SCy-7 versus HClO. Fluorescence intensity of the NIR fluorescent probe SCy-7 at a wavelength of 778nm was measured when SCy-7 interacted with different concentrations of HClO (0-100. Mu.M). The results in the figure show that: as the concentration of HClO increases, the fluorescence intensity of the 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. Fluorescence intensity values of the NIR fluorescent probe SCy-7 were determined when the NIR fluorescent probe SCy-7 was interacted with different concentrations of HClO (0-100. Mu.M). The fluorescence intensity values were plotted on the ordinate and the concentration of HClO added on the abscissa. The results in the figure show that: the HClO concentration can be quantitatively detected through the change of the fluorescence intensity of the probe.
FIG. 3 is IL-NH 2 The nuclear magnetic resonance hydrogen spectrogram can be obtained by analyzing the chemical shift and the attribution of the corresponding hydrogen atoms in the molecular structure, and the IL-NH is successfully synthesized by the method 2 An ionic liquid.
FIG. 4 is a Fourier Infrared (FT-IR) spectrum of HA-QA-ALD, HA-ALD and HA, the results of which show: the FT-IR spectrum of HA-ALD was 1650cm compared to the starting HA -1 A new characteristic peak is shown, which is attributed to the hydrazide group. For HA-QA-ALD, we can easily observe aldehyde group at 1747cm -1 Characteristic peak and quaternary ammonium group of (1) at 1470cm -1 And 1491cm -1 Thus proving 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 gel-forming PIL-HA hydrogel. First, 1g of modified HA-QA-ALD dissolved in 5mL of 0.01mol/L PBS buffer (pH = 7.4) was recorded as solution 1; 0.2g of acrylamide, 0.4g of ammonium persulfate, 200. Mu.L of an NIR fluorescent probe SCy-7 and 0.5mL of IL-NH 2 Dissolve in 3mL of 0.01mol/L PBS (pH = 7.4) buffer solution and mix well as record as solution 2; mixing 1mL of solution 1 and 1mL of solution 2, placing the mixture in a water bath kettle at 60 ℃, and obtaining the PIL-HA hydrogel within 3 min.
FIG. 5b is a peptized view of a PIL-HA-2 hydrogel. After 1mL of hydroxylamine (1 mol/L) is added into the 0.5g of PIL-HA hydrogel, the hydrogel is placed at 37 ℃, and the conversion process from gel to sol can be realized within 30min. This is due to the competition of PIL-NH by hydroxylamine 2 To break the schiff base and amide bond of the hydrogel.
Fig. 5c is a schematic illustration of the adhesion of PIL-HA-x (x = 1.2.3.4) hydrogel on the back of the hand. The results in the figure indicate that the PIL-HA hydrogel HAs good adhesion on skin.
Fig. 5d is a schematic diagram of gel time of the PIL-HA-x (x = 1.2.3.4) hydrogel, which was placed in a water bath at 60 ℃, and the gel was observed and recorded by photographing at intervals of 1min. The results in the figure show that the gel time of PIL-HA-1 is 3min at the slowest and that the gel time of PIL-HA-4 is 1min at the fastest. Thus, it was demonstrated that the PIL-HA hydrogel of the present invention can form a gel within 3min at 60 ℃.
FIG. 6a is a Fourier Infrared Spectroscopy plot of a PIL-HA-x (x = 1.2.3.4) hydrogel, which can be seen at 3440cm -1 A wide and strong peak is caused by the stretching vibration of the hydroxyl; 2926cm -1 The characteristic absorption peak at (a) is due to carbon-hydrogen bond stretching vibrations between the schiff base and the amide bond. Due to IL-NH 2 Can be introduced intoImidazole ring was seen at 1627cm -1 And the intensity of the absorption peak is dependent on IL-NH 2 Increased content, which demonstrates IL-NH 2 Was successfully incorporated into the PIL-HA hydrogel.
Fig. 6b is an XRD pattern of the PIL-HA-x (x = 1.2.3.4) hydrogel, in which one ton peak appears at 2 θ =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 a PIL-HA-x (x = 1.2.3.4) hydrogel, and a direct comparison shows that as poly IL-NH is incorporated into the hydrogel 2 The increase in ionic liquid concentration increased the thermal stability constants of the four PIL-HA hydrogels from 106.9 ℃ to 289.7 ℃, 327.6 ℃ and 330.6 ℃, probably because the increase in crosslink density resulted in an increase in hydrogel thermal stability constants.
FIG. 6d is a Zeta potential diagram 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 side-indicates that the PIL-HA-2 hydrogel HAs antibacterial properties.
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 observed from the field emission scanning electron microscope image, all PIL-HA hydrogels showed interconnected 3D porous structure and were accompanied by poly IL-NH 2 Increasing the amount of ionic liquid, poly-IL-NH 2 The chance that the amino group of the ionic liquid reacts with the aldehyde group and the carboxyl group in the modified hyaluronic acid HA-QA-ALD is increased, and the aperture area of the hydrogel prepared by the method is from (5000 +/-1500) mu m 2 (PIL-HA-1 hydrogel) 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 the breathability of the wound and the growth and survival of cells, as well as for the transport of nutrients and the absorption of metabolic waste.
Fig. 8a is a graph of a tensile test of a PIL-HA-x (x = 1.2.3.4) hydrogel. The test method comprises the following steps: the PIL-HA-x (x = 1.2.3.4) hydrogel was cut into a rectangular hydrogel (10 mm. Times.20 mm. Times.10 mm) as a tensile sample, and a film tensile machine (instron 5) was used943, usa) the tensile properties of the four hydrogels were tested, with the tensile rate kept constant at 10mm/min, giving a linear relationship between tensile and stress. It can be observed from the figure that with poly IL-NH 2 The tensile stress of the hydrogel is increased from 0.18MPa to 0.43MPa, 0.44MPa and 0.82MPa by increasing the dosage of the ionic liquid. Therefore, 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 shows the result of the compressive property test of the PIL-HA-x (x = 1.2.3.4) hydrogel, which was made into a cylindrical shape (radius =10mm, height =40 mm) and tested for compressive property by an electronic universal material testing machine (AI-7000M, china) at a compressive testing rate of 5mm/min, giving a linear relationship between compression and stress. It can be observed from the figure that with poly IL-NH 2 The stress of the hydrogel when the hydrogel is compressed by 80 percent is increased from 0.21MPa to 0.42MPa, 0.55MPa and 0.74MPa by increasing the dosage of the ionic liquid.
FIG. 8c shows that the PIL-HA-2 hydrogel can recover its original shape rapidly within 3s after being compressed to 80%. Therefore, the PIL-HA hydrogel disclosed by the invention HAs better mechanical properties with human skin, and can better resist external force, so that potential damage to tissues is avoided.
Fig. 8d is a splicing tower cut experiment using pig skin to evaluate the ability of PIL-HA-x (x = 1.2.3.4) hydrogel to adhere to skin tissue. The specific experimental method is as follows: fresh pigskins were purchased from the market, cut into a rectangle 5cm long and 2cm wide after removal of excess fat, and re-soaked in 10ml of 0.01mol/L PBS (pH = 7.4) solution to ensure that the pigskins were always in a moist environment before the experiment in order to prevent the pigskins from drying out. To test the adhesion of the hydrogel on the pigskin, two rectangular pigskins were removed and the surface residual 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 pigskins at room temperature, and the two pigskins were sandwiched by a film tension machine, respectively, and a 100N weighing cell was used for 10mm min -1 The strain rate of (2) was measured and the results are shown in the figure8d are shown. As can be seen from the figure, the tensile stress of all the hydrogels showed adhesive strengths of (3.21. + -. 0.45) KPa to (24.36. + -. 0.84) KPa. The PIL-HA-x (x = 2.3.4) hydrogel of the invention had 3-8 times its adhesive strength compared to the clinically established fibringluce adhesive strength (about 3 KPa).
A graph comparing the change from left to right of the PIL-HA-x (x = 1.2.3.4) hydrogel after reaching the swelling limit in 3ml of 0.01mol/L PBS (pH = 7.4) solution is shown in fig. 9 a. As can be seen from the figure: the volume of the swelled hydrogel is obviously increased, thereby showing that the PIL-HA hydrogel HAs better water absorption.
Fig. 9b is a graph comparing the mass of the PIL-HA-x (x = 1.2.3.4) hydrogel before and after swelling, as can be seen: the PIL-HA-x (x = 1.2.3.4) hydrogel of the present invention can absorb water 6-11 times more than its own weight.
Fig. 9c is a plot of the base area of the PIL-HA-x (x = 1.2.3.4) hydrogel before and after swelling, as seen by: the floor area of the PIL-HA-x (x = 1.2.3.4) hydrogel of the invention increased 6-11 times after swelling compared to before swelling.
Fig. 10a is a qualitative assessment of the antimicrobial capacity of PIL-HA-x (x = 1.2.3.4) hydrogel using the disk method as follows: 500. Mu.L of each of E.coli, S.aureus and Bacillus subtilis suspensions (1X 10) 8 CFU/mL) was uniformly spread on an LB medium (30 mL) by a roll ball method, and then the sterilized PIL-HA-x (x = 1.2.3.4) hydrogel (1 g) was uniformly spread on the surface of the medium, respectively, and incubated at 37 ℃ for 24 hours to observe the growth of bacteria around the hydrogel. As can be seen from the figure, with poly IL-NH 2 The area of a bacteriostatic zone around the hydrogel is increased along with the increase of the dosage of the ionic liquid, and no colony grows on the surface of the hydrogel, so that the non-release antibacterial performance of the PIL-HA hydrogel is laterally verified.
Fig. 10b is a graph representing the evaluation of the antimicrobial capacity of the hydrogels using the disc method to evaluate the size of the zone of inhibition of the four hydrogels. It can be seen from the figure that: different poly IL-NH 2 The PIL-HA hydrogel with the ionic liquid content generates bacteriostasis along with the increase of the dosage of the ionic liquidThe area of the loop increases. This is due to the quaternary ammonium cation and poly-IL-NH on HA-QA-ALD in the PIL-HA hydrogel 2 The imidazole group with positive charge can generate electrostatic interaction with phosphate group with anion on the bacterial cell wall, and then the lipophilic part is inserted into the phospholipid bilayer of the bacteria to destroy the cell membrane, so that the bacteria are cracked and killed.
Fig. 10c quantitatively assesses the antibacterial ability of PIL-HA-x (x = 1.2.3.4) hydrogels using colony counting method as follows: the PIL-HA-x (x = 1.2.3.4) hydrogel was subjected to ultraviolet sterilization in advance, and then the concentration was adjusted to 1X 10 8 CFU/mL of 200. Mu.L suspension of Escherichia coli, staphylococcus aureus and Bacillus subtilis was transferred to sterilized LB medium (10 mL), respectively, and then sterilized PIL-HA-x (x = 1.2.3.4) hydrogel (1 g) was spread uniformly on the surface of the medium, respectively, and incubated at 37 ℃ at 120rpm on a shaker, and after 24h the bacterial suspension was diluted with LB medium to the original concentration of 10 -6 20 μ L of the diluted bacterial suspension was uniformly dispersed on LB solid medium by the ball method and incubated at 37 ℃ for 24 hours. As can be seen from the figure, with poly IL-NH 2 The increase of the dosage of the ionic liquid improves the antibacterial capability of the hydrogel, which indicates that the PIL-HA hydrogel HAs the characteristic of non-release antibacterial property.
FIG. 10d is a graph in which the number of Colony Forming Units (CFU) was counted, respectively, and the test was repeated for more than three independent times for each colony, and a bacterial suspension to which no hydrogel was added was used as a control group of the experiment (control, whose antibacterial ability was regarded as 0, on the basis of which the antibacterial ratio of the other groups was calculated). Poly IL-NH compared to PIL-HA-1 hydrogel 2 The antibacterial activity of the PIL-HA-2, PIL-HA-3 and PIL-HA-4 hydrogel prepared after the dosage of the ionic liquid is increased is obviously improved, and especially the antibacterial activity to staphylococcus aureus and bacillus subtilis is obviously improved. In addition, the PIL-HA-2, PIL-HA-3 and PIL-HA-4 hydrogels can inhibit about 90% of Escherichia coli and more than 95% of Staphylococcus aureus and Bacillus subtilis. The above results further demonstrate that the PIL-HA hydrogels of the present invention are effective against e.coli, s.aureus and b.subtilisBacterial activity, also demonstrates its great potential in the treatment of bacterial infections.
Fig. 11a is a result of testing the adsorption capacity of the PIL-HA-x (x = 1.2.3.4) hydrogel for 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 rectangular shapes (about 1g in weight), and sterilized under an ultraviolet lamp while being sterilized with 75% medical alcohol for 1 hour. After soaking in PBS (pH =7.4, 0.01mol/L) to reach a swelling equilibrium, the treated PIL-HA-x (x = 1.2.3.4) hydrogel samples were added to 5mL of 10mg/mL Bovine Serum Albumin (BSA) solution, respectively, and incubated in a shaker at 37 ℃ for 24h, each sample was repeated three times, the ultraviolet light intensity of the solution was measured at 595nm using Shimadzu UV-2550 to calculate the BSA remaining in the solution, the Bovine Serum Albumin (BSA) solution without the hydrogel sample added was set as a control group, and the adsorption capacity of the hydrogel material was evaluated based on the BSA concentration measured by absorptiometry. The calculation formula is as follows:
wherein Co and Cx are the concentration (mg/mL) of BSA after adsorption of the control and the hydrogel with added PIL-HA-x (x = 1.2.3.4), W is the weight of the PIL-HA hydrogel, and V is the volume of BSA added, respectively. 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 also the electrostatic interaction between the positively charged PIL and BSA can contribute to the improvement of 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) hydrogel. At a temperature of less than-90 ℃, the vacuum degree: in the case of < 5Pa, the PIL-HA-x (x = 1.2.3.4) hydrogel was vacuum freeze-dried for 24h and then weighed again and recorded, and the water content of the four hydrogels was estimated by calculating the water loss after freeze-drying. The calculation formula is as follows:
wherein W 0 And W x The quality of the hydrogel before and after lyophilization respectively. As can be seen from the figure: the water content of the PIL-HA-x (x = 1.2.3.4) hydrogel of the present invention is about (80 ± 3)%, so that it can provide a moist environment for the wound to relieve the pain of the wound and promote the wound healing.
Fig. 11c is a drawing of a PIL-HA-x (x = 1.2.3.4) hydrogel cut into cuboids of equal volume (12 mm x 5mm x 3 mm) and stretched with two hands to record the maximum tensile deformation rate of the four hydrogels of PIL-HA-x (x = 1.2.3.4). As can be seen from the figure, with poly IL-NH 2 The use amount of the ionic liquid is increased, and the tensile deformation rate of the hydrogel is increased, which indicates that the hydrogel has excellent mechanical properties.
FIG. 11d is a graph which shows the maximum tensile set of four hydrogels, and it can be seen that with the poly PIL-NH in the hydrogel 2 The maximum tensile set of the hydrogel increased gradually from 215% to 248%, 426% and 837% with increasing ionic liquid content. Therefore, the hydrogel disclosed by the invention has good tensile deformation performance, so that skin tissues can be better protected.
FIGS. 12a and 12b show the comparison of the NIR fluorescence probe SCy-7 of the present invention with mouse fibroblast cells L929 (1X 10) 6 Cell/culture dish) and embryonic fibroblasts NIH-3T3 (1X 10) 6 Cell/culture dish) were co-cultured for 24h (1 day) and 48h (2 days) for staining of live and dead cells, while staining of live and dead cells was performed using calcein double fluorescence staining and observed. As can be seen from the figure, few dead cells appeared in red, indicating that the NIR fluorescent probe SCy-7 has good biocompatibility.
FIGS. 12c and 12d show that the PIL-HA-x (x = 1.2.3.4) hydrogel (0.1 g) prepared by the present invention is fully swollen and equilibrated and sterilized with mouse fibroblast L929 (1X 10) 6 Cell/culture dish) and embryonic fibroblasts NIH-3T3 (1X 10) 6 Cell/petri dish) staining pattern of live and dead cells for 24h (1 day) and 48h (2 days), the mass of PIL-HA-x (x = 1.2.3.4) hydrogel was 0.5g. Live and dead cells were also stained with calcein and observed. As can be seen from the figure, all the green viable cells were present, while few red dead cells were present, thus indicating that the PIL-HA-x (x = 1.2.3.4) hydrogel of the present invention HAsHas good biocompatibility.
FIGS. 13a and 13b show mouse fibroblast cells L929 (1X 10) 5 Cells/well) and embryonic fibroblasts NIH-3T3 (1X 10) 5 Cell/well) with the NIR fluorescent probe of the invention SCy-7 for 1 day, 2 days and 3 days. From the figure, it can be seen that the cell activities of both L929 and NIH-3T3 cells were above 90%, and no statistically significant difference was observed between the control group and the probe-treated group, further illustrating that the NIR fluorescent probe SCy-7 of the present invention has good biocompatibility.
FIGS. 13c and 13d are statistical plots of the proportion of cell survival for mouse fibroblast L929 and embryonic fibroblast NIH-3T3, respectively, after direct contact with PIL-HA-x (x = 1.2.3.4) hydrogels made in accordance with the present invention for 1, 2 and 3 days after sufficient swelling equilibration and sterilization. The experimental method comprises the following steps: respectively mixing mouse fibroblast L929 and embryo fibroblast NIH-3T3 at 1 × 10 5 After overnight incubation in 96-well plates, 10mg PIL-HA-x (x = 1.2.3.4) hydrogel, which reached swelling equilibrium in modified DMEM medium (10% fetal bovine serum, 1% diabody (penicillin-streptomycin)), and different amounts of NIR fluorescent probe SCy-7 (concentration 5 μ M, 10 μ M, 15 μ M, 20 μ M) dissolved in modified DMEM medium were each cultured in direct contact with the cells, with the concentration of NIR fluorescent probe SCy-7 being 0 μ M as a negative control group, and 200 μ L of 0.01mol/L PBS (pH = 7.4)/well as a blank group. Adding cck-8 solution (10. Mu.L/well) to 96-well plate on day 1, day 2 and day 3, respectively, and keeping the plate in the dark, and then keeping the plate at 37 deg.C and 5% CO 2 Was cultured for 1 hour, and then the Optical Density (OD) value of each well was measured by a microplate reader at a wavelength of 450 nm. The cell viability is calculated as:
in the formula: OD T 、OD blank 、OD neg The optical densities of the experimental group, blank group and negative control group were measured. The results in the figure show that: poly-PIL-NH in hydrogel with SCy-7 Probe concentration 2 Increase in Ionic liquid concentration despite slight decrease in cellular ActivityBut still maintain a survival rate of over 80%. Therefore, the SCy-7 probe and the PIL-HA hydrogel have good cell compatibility.
Figure 14a is an assessment of the hemocompatibility of PIL-HA-x (x = 1.2.3.4) hydrogel by in vitro hemolysis assay. In a hemolytic test, fresh blood of a mouse is obtained by picking eyeballs and taking blood, the fresh blood of the mouse is diluted by 10 times by using normal saline, the blood is evenly mixed and divided into five parts, red blood cells collected from whole blood are washed by the normal saline for multiple times, 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 placed into prepared red blood cell stock solution, and a negative control and a positive control are respectively added with the normal saline and a buffer solution. After incubation for 6h at 37 ℃ the hydrogel was removed and the red blood cell suspension was centrifuged (1200 rpm) for 12min and after recording by photography, the supernatant was aspirated and the absorbance was measured at 545nm using a UV-visible spectrophotometer. After co-culturing the four hydrogels with erythrocytes for 3h at 37 ℃ on a shaker, the hydrogels were taken out and the color contrast of the four hydrogel groups and the positive control group (buffer solution) was photographed. As can be seen from the figure: the supernatants of the experimental group to which PIL-HA-x (x = 1.2.3.4) hydrogel was added were all pale yellow, while the positive control group was bright red. Quantitative analysis revealed that the hemolysis rates of the experimental groups with the PIL-HA-x (x = 1.2.3.4) hydrogel were (0.96. + -. 0.13)%, (1.20. + -. 2.6)%, (2.11. + -. 0.09)% and (2.90. + -. 0.04)%, respectively. It can be seen that the hemolysis rate of the hydrogel slightly increases with the ionic liquid content, but all showed good blood compatibility.
FIG. 14b is a blood cell adhesion assay to evaluate the compatibility of the PIL-HA-x (x = 1.2.3.4) hydrogel with blood cells by first co-culturing 5mL of fresh whole mouse blood with four 10mm diameter samples of the PIL-HA-x (x = 1.2.3.4) hydrogel, followed by 30min incubation at 37 ℃ with a shaker, carefully removing the hydrogel and gently rinsing it twice with saline and recording it by photography. As can be seen from the figure: the hydrogel hardly adhered to dead blood cells except for slight yellowing, and thus it was found that the hydrogel prepared according to the present invention was almost not damaged to blood cells, thereby exhibiting good hemocytocompatibility.
FIG. 15a is a graph showing the electrical conductivity of a PIL-HA-x (x = 1.2.3.4) hydrogel prepared according to the present invention, which was evaluated by a four-probe method, as seen from the graph, as poly-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 that: the hydrogel samples of the present invention all have conductivity values similar to those of skin tissue, and thus have great potential in transferring bioelectric signals and accelerating wound healing.
FIG. 15b is a schematic of the electrical conductivity of the 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 an LED bulb in a series circuit, and the hydrogel prepared by the method is more intuitively proved to have good electric conductivity by observing the connection, cutting and splicing modes.
FIG. 16a shows the low temperature electrical conductivity mapping method of the PIL-HA-2 hydrogel prepared by the present invention as follows: the PIL-HA-2 hydrogel and the temperature sensor were simultaneously frozen at-20 ℃ for 24h and connected to a series circuit, and the PIL-HA-2 hydrogel was used as an electrical conductor to connect the LED bulb and the battery. It can be seen from the figure that: the brightness of the LED bulb at around-10 ℃ was the same as the brightness at room temperature of 25 ℃, thus indicating that the hydrogel of the present invention has good conductivity at low temperatures.
FIG. 16b is a graph of the stretch-recovery of the PIL-HA-2 hydrogel at low temperature. The method comprises the following steps: the PIL-HA-2 hydrogel is stretched to 3 times of the length of the hydrogel at a low temperature of about-10 ℃. As can be seen from the figure: the hydrogel of the present invention can rapidly recover the initial state within 3 seconds.
FIG. 16c is a graph of compression-recovery of a PIL-HA-2 hydrogel at low temperature. The method comprises the following steps: the PIL-HA-2 hydrogel is extruded at a low temperature of about-10 ℃ and then is stopped. As can be seen from the figure: the hydrogel of the present invention can be rapidly restored to its original shape within 3 seconds under the condition of complete compression. Therefore, the PIL-HA-2 hydrogel HAs excellent anti-freezing performance and still HAs good mechanical performance at low temperature.
FIG. 17a is a graph established on a mouse tailgating hemorrhage model to confirm the hemostatic capacity of the PIL-HA-2 hydrogel in vitro. The experimental method is as follows: the anesthetized mice were fixed on a foam surgical board with pins, plastic film pads with the same size were cut on the bottom to prevent bleeding and run off, filter paper was placed between the tail of the mouse and the plastic to absorb the blood flowing out of the tail, finally the tail of one mouse was cut with scissors and the foam board was tilted to induce tail bleeding, and after the other tail was cut, the tail was immediately stopped with PIL-HA-2 hydrogel at the tail cut of the mouse, and the foam board was similarly tilted to induce tail bleeding. After 3min the filter paper was weighed and the experiments were performed in at least three replicates. As can be seen from the figure, there was almost no blood stain on the filter paper in the case of the hydrogel group mice as compared with the blank group mice, thereby indicating that the PIL-HA hydrogel of the present invention HAs good hemostatic ability in vitro.
FIG. 17b is a graph established in a mouse model of liver bleeding to confirm the hemostatic ability of the PIL-HA-2 hydrogel in vivo. The experimental method is as follows: the anesthetized mice were also fixed, and the mice were thoracically cut with a scalpel and the livers were carefully removed. And similarly, a plastic film is firstly placed, then the filter paper is placed between the liver and the plastic film, the needle of a 1mL sterile syringe is used for inducing liver bleeding, one group uses the PIL-HA-2 hydrogel for liver hemostasis, the other group does not carry out any treatment, and the foam plate is inclined to enable the blood to better flow out of the liver and flow to the filter paper. The filter paper was weighed after 3min and the experiments were also run in at least triplicate. As can be seen from the figure, the blood flow on the filter paper of the hydrogel group mice is much smaller than that of the mice without hemostasis treatment, thereby indicating that the PIL-HA hydrogel provided by the invention HAs good in vivo hemostasis capability.
FIG. 17c depicts a statistical plot of the hemostatic capabilities of the PIL-HA-2 hydrogel, with the control mice in the tailed group exhibiting blood loss of (130.04. + -. 10.37) mg, while the PIL-HA-2 hydrogel group exhibited blood loss of only (10.24. + -. 0.13) mg. Also in the model of liver bleeding of mice guided by a needle, the amount of blood lost from the liver of the mice in the control group was (237. + -. 14.84) mg, while that of the PIL-HA-2 hydrogel group was only (50.3. + -. 9.79) mg. The control group had obvious blood flow traces on the filter paper, while the hydrogel group had no blood flow traces on the filter paper. Therefore, the hydrogel prepared by the invention has good hemostatic capability.
FIG. 18a is a drawing showing the adhesion of the PIL-HA-2 hydrogel prepared by the present invention to the pigskin, and by adhering the PIL-HA-2 hydrogel to the pigskin, it can be seen that the hydrogel and the pigskin are adhered tightly at different angles with almost no gaps as the pigskin is twisted and folded.
Fig. 18b shows the process from adhesion to gradual peeling of the PIL-HA-2 hydrogel on the pigskin, and little fluctuation of the pigskin can be seen, thereby demonstrating that the PIL-HA-2 hydrogel can be easily removed without causing secondary damage to the wound.
FIG. 18c shows a schematic diagram of the adhesion of PIL-HA-2 hydrogel to various articles and the adhesion diagram on various articles. As can be seen from the figure: the PIL-HA hydrogel of the present invention can be tightly adhered not only to the tissue surface but also to the surface of various other materials (including rubber, paper, polyethylene (PE), polypropylene (PP), glass, and metal). The hydroxyl groups on the surface of the hydrogel according to the invention have a strong adhesion to various substrates and can interact with different substrates by covalent or non-covalent bonds. Covalent bonds can be formed, for example, by schiff bases on certain specific substrates containing amine or thiol groups; also non-covalent bonds (e.g., hydrogen bonding, π - π stacking, etc.) may exist between the hydrogel and the surface of other objects.
FIG. 19a, FIG. 19b, FIG. 19c, FIG. 19d are graphs evaluating the magnitude of the PIL-HA-x (x = 1.2.3.4) hydrogel's ability to promote proliferation of fibroblast L929 and embryonic fibroblast NIH-3T3 cells by cell scratch assay. The experimental method is as follows: fibroblast cells L929 and embryonic fibroblast cells NIH-3T3 were separately added at 1.2X 10 per well 6 The density of individual cells was seeded in a 6-well plate, after the cells were attached and fully grown, three scratches were vertically scribed in each well of the 6-well plate with a 200 μ L sterile tip, and four sterilized PIL-HA-x (x = 1.2.3.4) hydrogels were placed, and the distances between the scratches were recorded by taking pictures with an inverted fluorescence microscope (IX 73, OLYMPUS) at 0h, 8h, and 16h, respectively. And wells without hydrogel samples were used as controls (control). As is evident from the figures: compared with the control group, the PIL-HA hydrogel is helpful for improving the proliferation capacity of the fibroblast L929, so that the water condensation is carried out at a time point 8 hours after the hydrogel is addedThe scratch distance of the gel group was (800. + -.50) μm smaller than that of the control group (1100. + -.50). And the above phenomenon was more pronounced at the time point 16h after the addition of the hydrogel, i.e., the scratch distance of the hydrogel group was only (380. + -.30) μm, whereas the scratch distance of the control group was as high as (780. + -.20) μm. Also, the PIL-HA hydrogel significantly contributes to the proliferation of embryonic fibroblast 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 hydrogel addition for 8h, whereas the scratch distance of the hydrogel group was only (200. + -.25) μm and that of the control group was (510. + -.20) μm at the time point of hydrogel addition for 16 h. Therefore, the hydrogel provided by the invention has a remarkable promoting effect on migration and proliferation of fibroblast L929 and embryonic fibroblast NIH-3T 3.
FIGS. 20a and 20b show the results of experiments on the response of the NIR fluorescent probe SCy-7 to HClO in living cells and fluorescence imaging of fibroblasts L929 and embryonic fibroblasts NIH-3T3 cells. The experimental method is as follows: l929 cells and NIH-3T3 cells were imaged after incubation with 10. Mu.M of a 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. Whereas 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 a hydro-maleic anhydride (PMA) activator, which triggers an increase in mitochondrial ROS levels by activating the phosphoinositide signaling Pathway (PKC), thereby producing endogenous HClO. Fluorescence in L929 cells and NIH-3T3 cells was significantly quenched (NIR probe + PMA) after stimulation with PMA (1 ng/mL). Carbaryl (NAC) can inhibit and eliminate active oxygen in cells such as HClO. To investigate the effect of NAC on the levels of HClO endogenous to the 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 had 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 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. The NIR fluorescent probe hydrogel PIL-HA-2 is subjected to the response of HClO in living cells and the fluorescence imaging experiment on fibroblast L929 and embryonic fibroblast NIH-3T3 cells according to the imaging experiment method of the probe SCy-7 in the same way as the fluorescence imaging experiment of the probe SCy-7. Fluorescence (fluorescent hydrogel) can be observed after the NIR fluorescent probe hydrogel PIL-HA-2 is added from a confocal inverted fluorescence microscope; whereas intracellular fluorescence quenching was observed after addition of 10 μ M NIR fluorescent probe hydrogel PIL-HA-2 and 10 μ M HClO (fluorescent hydrogel + HClO); fluorescence in L929 cells and NIH-3T3 cells was significantly quenched after stimulation with PMA (fluorescent hydrogel + PMA); while L929 cells and NIH-3T3 cells were treated with PMA for 20min and then incubated with NAC (5 ng/mL) for 30min. Cells incubated with 10 μ M NIR fluorescent probe hydrogel PIL-HA-2 in the presence of NAC still showed 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 visualize HClO in living animals. Because the near infrared light penetrates the tissue deeply, the interference of autofluorescence is small, and the damage to the biological sample is small. Kunming (KM) mice were therefore selected. By continuously taking 5 days at 50mg kg -1 The diabetic mouse model was induced by intraperitoneal injection of Streptozotocin (STZ) into healthy KM mice with fasting plasma and blood glucose between 3.0-6.0 mmol/L. Randomly selecting a diabetes mouse successfully modeled to realize general anesthesia by injecting 5% chloral hydrate into the abdominal cavity, shaving most of hairs on the back of the mouse by using a pet hair shaver, and cleaning the hairs by using nonirritating depilatory cream. After thoroughly disinfecting it with alcohol cotton, a 10mm by 10mm square full thickness skin defect (wound depth up to the sarcolemma) was created at the epilation site using scissors and forceps. Four kinds of NIR fluorescent probe hydrogel (PIL-HA-2) containing the same amount of SCy-7 probe, which is prepared by the invention, are closely attached to the wound of the mouse for 30min, and then the wound of the mouse is subjected to in vivo fluorescence imaging through a living body imaging system of the mouse. As can be seen in fig. 21 b: the fluorescence intensity of the NIR fluorescent probe hydrogel PIL-HA-2 is increased along with the increase of blood sugar of diabetic mice, and the HClO level at wounds is increased along with the increase of blood sugar of diabetic mice, so that the NIR fluorescent probe hydrogel PIL-HA-2 is further increased, and the fluorescence intensity of the NIR fluorescent probe hydrogel PIL-HA-2 is further increased along with the increase of the blood sugar of diabetic mice, so that the HClO level at the wounds is further increasedThe fluorescence values of the NIR fluorescent probe hydrogels gradually decreased, while the fluorescence of the hydrogels was gradually quenched from the stronger fluorescence as can also be seen in the fluorescence imaging of fig. 21 a.
Similarly, fig. 21c and 21d evaluate the applicability of the NIR fluorescent probe hydrogel to visualization of HClO on wounds of living animals, and further explore the fluorescence imaging of the NIR fluorescent probe hydrogel on cells at wounds of diabetic mice. FIG. 21d shows that the fluorescence at the wound site of the mouse decreases with increasing blood glucose level of the mouse. Therefore, the NIR fluorescent probe hydrogel prepared by the invention can detect the HClO level at a wound of a living body without being interfered by a background signal. More importantly, all images in fig. 21c were obtained from KM mice that were not completely dehaired due to the near infrared absorption and emission characteristics of the probe.
Fig. 22a, 22d evaluate the reactivity of NIR fluorescent probe hydrogels to visualize the increase in HClO levels in real time in living animals. Further, PMA solution (1 ng/mL) was used to simulate an increase in endogenous HClO levels in the cells at the wound site, HClO solution (1X 10) -5 ) Mimicking the elevation of exogenous HClO levels in cells at the wound. Randomly selecting two non-diabetic KM mice, anesthetizing the mice, completely removing hairs on the backs of the mice, establishing a full-thickness skin defect model, covering with NIR fluorescent probe hydrogel, and imaging the mice by a small animal in-vivo imaging system. Subsequently, the hydrogel adjuvant was removed from this group of mice, 100 μ L of PMA solution was added to the wound, the hydrogel adjuvant was reapplied, and 3min later, the mice were imaged again, and the procedure was repeated four times. Along with the increase of the endogenous HClO level of the mouse wound, the fluorescence value of the supplementary material PIL-HA-2 of the NIR fluorescent probe hydrogel of the mouse wound is reduced.
Similarly, taking off the NIR fluorescent probe hydrogel PIL-HA-2 hydrogel auxiliary material of another group of mice, adding 100 mu L HClO solution to the wound, coating the hydrogel auxiliary material for 3min again, and then performing living body imaging on the mice again. Repeat four times to get FIG. 22c. As can be seen from the characterization of FIG. 22d, the fluorescence of the adjuvant at the wound of the mouse decreased with the increase of the exogenous HClO level at the wound of the mouse. Therefore, the NIR fluorescent probe hydrogel prepared by the invention can be used for rapidly, sensitively and real-timely monitoring the increase of HClO level in a mouse wound model, and plays a great potential in the aspect of preventing and treating diabetic wounds.
To evaluate the efficacy of PIL-HA-2 hydrogel in the treatment of acute wounds in normal mice, a commercial Tegaderm was used TM The film served as Control (Control). The experimental method is as follows: normal blood sugar mice were randomly selected, anesthetized and alcohol sterilized, and a square full thickness skin defect (10 mm. Times.10 mm) reaching the depth of the sarcolemma was created with scissors and forceps, using NIR fluorescent probe hydrogel PIL-HA-2 hydrogel as adjuvant and Tegaderm, respectively TM The film treats the disease. The wound area of the mice gradually decreased with increasing post-operative time, and the wounds were substantially closed after treatment with the PIL-HA-2 hydrogel group 12 days later. Figure 23a is a graph of fluorescence in vivo imaging of acute wounds treated with different regimens on days 0, 3, 7, 10, and 12, and figure 23b is a histogram of the extent of healing after acute wounds treated with different regimens on days 0, 3, 7, 10, and 12. Throughout the repair (12 days), in the quantitative analysis of the wound area: the wound healing rate (98.74%) using the PIL-HA-2 hydrogel group was much greater than that of commercial Tegaderm TM Film group (76.32%).
To evaluate the efficacy of the PIL-HA-x (x = 1.2.3.4) hydrogel on wound healing in diabetic mice, PBS was used as a blank group (Control), commercial Tegaderm TM The film served as a control. Similarly, a square full thickness skin defect (10 mm. Times.10 mm) was created to a depth of the sarcolemma after anesthetizing and disinfecting the mice and treated with different excipients. The wound area of all groups decreased gradually with increasing post-operative time. After 14 days of treatment, the wounds were substantially closed after the PIL-HA hydrogel treatment. FIG. 23c is a graph of fluorescence live images of diabetic skin wounds treated with different regimens on days 0, 3, 7, 10 and 14, and FIG. 23d is a bar graph of the degree of healing after treatment of diabetic skin wounds with different regimens on days 0, 3, 7, 10 and 14. After the wound is healed for 14 days, the area of the wound can be quantitatively analyzed to obtain: the wound healing rate of the PIL-HA-1 hydrogel group is 97.79 percent, and the wound healing rate of the PIL-HA-2 hydrogel group is99.31%, the wound healing rate of the PIL-HA-3 hydrogel group was 98.56%, and the wound healing rate of the PIL-HA-4 hydrogel group was 99.76%, which were both much greater than the wound healing rate of the blank group (71.61%) and commercial Tegaderm TM Wound healing rate of the thin film group (78.61%).
To evaluate the biocompatibility of the hydrogel in mice. After completion of the above wound healing experiment, the mice were sacrificed and their major organs (heart, liver, spleen, lung and kidney) were collected, fixed with 4% paraformaldehyde solution, paraffin-embedded, sectioned, stained with hematoxylin and eosin (H & E), and then examined by pathology photographing using an optical microscope, and the results are shown in fig. 24. A blank mouse organ section without hydrogel was used as a control group (control). It can be seen from the figure that the wounds of the mice after the treatment with the PIL-HA hydrogel can maintain normal tissue structure without any significant organ damage or inflammatory lesions. Thus, 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 evaluated 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 (with Tegaderm) TM Film treatment), whereas the inflammatory cells of the skin tissue of mice treated with the PIL-HA-2 hydrogel of the invention were significantly reduced due to the excellent antibacterial and anti-infective properties of the hydrogel. The hydrogel group also showed more newly formed blood vessels and hair follicles and was more favorable for the formation of granulation tissue than the control group. The above results indicate that the PIL-HA hydrogel of the invention promotes cell proliferation and migration.
FIG. 25b is PIL-HA hydrogel and control group (using Tegaderm) TM Film treatment) Masson staining pattern 12 days after treatment of acute wounds. As can be seen from the figure, the wounds treated with the PIL-HA hydrogel of the invention showed more collagen deposition and collagen alignment more orderly, closer to normal skin tissue, than the control group.
For acute injuryThe mouth is respectively passed through NIR fluorescent probe hydrogel PIL-HA-2 auxiliary material and Tegaderm TM The number of regenerated blood vessels in the newborn skin 12 days after the film (control group) treatment was quantitatively analyzed by the Roldreview software, and the results are shown in FIG. 25 c. As can be seen from the figure, the number of blood vessels in the regenerated skin of the mice treated with the hydrogel PIL-HA-2 adjuvant group is much greater than that of the commercial adjuvant group, which indicates that the PIL-HA hydrogel can promote wound healing more effectively.
Subjecting acute wound to NIR fluorescent probe hydrogel PIL-HA-2 adjuvant and Tegaderm TM The collagen index of the newly formed skin of the wound tissue after 12 days of film (control) treatment was quantitatively analyzed by the Roldreview software, and the result is shown in FIG. 25 d. As can be seen from the figure, the collagen index of the regenerated skin of the mice treated by the hydrogel PIL-HA-2 adjuvant group is superior to that of the commercial adjuvant group, which indicates that the PIL-HA hydrogel can promote wound healing more effectively.
FIG. 25e shows the results of quantitative analysis of the epidermal thickness of the acute wounds on the Roldreview software 12 days after the acute wounds were treated with the hydrogel group and the control group, as shown in FIG. 25 e. As can be seen from the figure, the thickness of the epidermis of the regenerated skin of the mouse treated by the hydrogel PIL-HA-2 adjuvant group is larger than that of the commercial adjuvant group, which indicates that the PIL-HA hydrogel can promote wound healing more effectively.
In order to further monitor the promotion effect of the PIL-HA hydrogel prepared by the invention on wound healing of diabetic mice, a commercial film 3M Tegaderm was respectively adopted TM And PIL-HA-x (x = 1.2.3.4) hydrogel dressing for healing wounds of diabetic mice for 14 days, and then H is performed on skin tissues at the wounds&E staining, results are shown in FIG. 26 a. As can be seen from the results in the figure, poly IL-NH was included in the hydrogel according to the present invention as compared to the blank (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 the epidermis and relieves the inflammation, and further shows that the PIL-HA hydrogel HAs a remarkable promoting effect on the healing of the skin wound of the diabetic mouse.
FIG. 26b is a graph of Masson staining of PIL-HA hydrogel prepared according to the present invention, blank and control (commercial 3M Tegader film) after 14 days of treatment of skin wounds in diabetic mice. As can be seen from the figure, the skin wounds of diabetic mice treated with the PIL-HA hydrogel of the invention showed more collagen deposition and collagen alignment more orderly and closer to normal skin tissue than the other groups through the entire healing process.
For commercial film 3M Tegaderm TM And PIL-HA-x (x = 1.2.3.4) hydrogel treatment the number of regenerated blood vessels of skin wounds of diabetic mice 14 days after treatment was quantitatively analyzed using the rollreview software, and the results are shown in fig. 26 c. As can be seen from the figure, the number of regenerated blood vessels of the mouse regenerated skin treated with the PIL-HA-x (x = 1.2.3.4) hydrogel group was much greater than those of the commercial adjuvant group and the blank group, indicating that the PIL-HA hydrogel could more effectively promote the healing of diabetic wounds.
For commercial film 3M Tegaderm TM And PIL-HA-x (x = 1.2.3.4) hydrogel treatment of skin wounds of diabetic mice 14 days later, collagen indices were quantitatively analyzed using the rollreview software, and the results are shown in fig. 26 d. As can be seen from the figure, the collagen index of the regenerated skin of mice treated with the PIL-HA-x (x = 1.2.3.4) hydrogel group is far superior to that of the commercial adjuvant group and the blank group, indicating that the PIL-HA hydrogel can more effectively promote the healing of diabetic wounds.
For commercial film 3M Tegaderm TM And PIL-HA-x (x = 1.2.3.4) hydrogel treatment 14 days after diabetic mouse skin wounds were quantified for skin thickness using the rollreview software, the results are shown in fig. 26 e. As can be seen from the figure, the regenerated skin thickness of the mice treated with the PIL-HA-x (x = 1.2.3.4) hydrogel group was greater than that of the commercial adjuvant group and the blank group, indicating that the PIL-HA hydrogel can more effectively promote the healing of diabetic wounds.
To further explore the content of a series of inflammatory factors (interleukin (IL-6) and tumor necrosis factor (TNF-alpha)) in acute wounds, the commercial film 3M Tegaderm was used TM Immunofluorescence with IL-6 and TNF-alpha at skin wound tissue of diabetic mice 12 days after PIL-HA-2 hydrogel treatmentThe results of the photostaining are shown in fig. 27a and 27b, respectively. As can be seen from the figure, the levels of pro-inflammatory chemokines (IL-6 and TNF-. Alpha.) at the skin wounds of diabetic mice were significantly reduced following treatment of acute wounds with the PIL-HA-2 hydrogel compared to the control group, thereby indicating that the PIL-HA hydrogel may be effective in promoting the conversion of macrophages from the inflammatory cell M1 phenotype to the reparative phenotype M2 at the early inflammatory stage.
To further explore the content of a series of inflammatory factors (interleukin (IL-6) and tumor necrosis factor (TNF-alpha)) in skin wounds of diabetic mice, a commercial film of 3M Tegaderm was used TM And PIL-HA-x (x = 1.2.3.4) were immunofluorescent stained for IL-6 and TNF-a at the skin wound tissue of diabetic mice 14 days after treatment, and the results are shown in fig. 27c, fig. 27d, respectively. As can be seen from the figure, the levels of proinflammatory chemokines (IL-6 and TNF-alpha) at the skin lesion of diabetic mice were significantly reduced after the PIL-HA hydrogel treatment compared to the control group, and as poly IL-NH was polymerized in the PIL-HA hydrogel 2 The amount of the PIL-HA hydrogel increased, the number of hair follicles in the tissue increased and the arrangement gradually tended to be regular, thereby indicating that the PIL-HA hydrogel could effectively resist inflammation and promote wound healing in the 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, or improvement made without departing from the spirit and principle of the present invention shall fall within the protection scope of the present invention.
Claims (10)
1. A compound having the structure of formula I:
2. the method for preparing the compound with the structure shown in the formula I in the claim 1 is characterized by comprising the steps of reacting a compound 3 with thiomorpholine to prepare the compound with the structure shown in the formula I;
the compound 3 has the following structure:
3. the method of claim 2, wherein the molar ratio of thiomorpholine to compound 3 is 1: (1-5).
Preferably, the compound 3 is prepared by a method comprising the following steps: reacting the compound 1 with the compound 2 to obtain a compound 3;
the compound 1 has the following structure:
the compound 2 has the following structure:
preferably, the molar ratio of compound 1 to compound 2 is 1: (1-5).
Preferably, the compound 2 is prepared by a method comprising the following steps: 2, 3-trimethyl-3H-indole and p-bromobenzoic acid are reacted to obtain the compound 2.
Preferably, the molar ratio of the 2, 3-trimethyl-3H-indole to p-bromobenzoic acid is 1: (1-2).
Preferably, the compound 1 is prepared by a method comprising the following steps: and reacting cyclohexanone with phosphorus oxychloride to obtain the compound 1.
Preferably, the molar ratio of cyclohexanone to phosphorus oxychloride is 1: (0.5-2).
4. Use of a compound of formula I according to claim 1 and/or a compound of formula I prepared by a method according to any one of claims 2 to 3 for the preparation of a fluorescent probe.
5. A compound having the structure of 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.
6. A process for preparing a compound of formula ii as claimed in claim 5, comprising: reacting a compound of formula I as defined in claim 1 with poly-IL-NH 2 The ionic liquid (PIL) is subjected to amido bond reaction to obtain the near-infrared fluorescent probe grafted poly IL-NH 2 The ionic liquid and modified hyaluronic acid (HA-QA-ALD) are subjected to Schiff base reaction and amido bond reaction to prepare a compound with a structure shown in II;
the poly IL-NH 2 The structural formula of the ionic liquid (PIL) is as follows:
preferably, the compound with the structure shown in the formula I and poly IL-NH 2 The mass ratio of the ionic liquid is 1 (4-6).
Preferably, the modified hyaluronic acid (HA-QA-ALD) is poly-IL-NH grafted with a near-infrared fluorescent probe 2 The mass ratio of the ionic liquid is (1-4) to 5.
Preferably, the poly IL-NH 2 The ionic liquid (PIL) is prepared by the following method comprising the following steps: reacting the compound 4 with acrylamide under the action of an initiator to obtain poly IL-NH 2 An ionic liquid (PIL);
the compound 4 has the following structure:
preferably, the reaction mass ratio of the compound 4 to acrylamide is 1: (1-2).
Preferably, the mass ratio of the compound 4 to the initiator is (10-15): 1.
preferably, the compound 4 is prepared by a method comprising: reaction of 1- (3-aminopropyl) -imidazole with 4-chloromethylstyrene followed by ZnCl 2 Is subjected to ZnCl reaction 1- Coordination and Cl 2- Displacement of the coordination affords compound 4.
Preferably, the reaction molar ratio of the 1- (3-aminopropyl) -imidazole to the 4-chloromethylstyrene is 1: (0.5-2).
Preferably, 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 of 1 to 100, preferably each independently selected from a number of 40 to 60.
7. Use of a compound of formula ii according to claim 5 for the preparation of a fluorescent probe. Preferably in the preparation of a medicament for repairing skin wounds.
8. A fluorescent probe comprising a compound of formula ii according to claim 5.
9. A kit comprising the fluorescent probe of claim 8.
10. A biosensor comprising the fluorescent probe of claim 8.
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