Functionalized adhesive hydrogel as well as preparation method and application thereof

Abstract

The invention discloses functionalized adhesive hydrogel as well as a preparation method and application thereof, and belongs to the technical field of biomedical materials. The functional adhesive hydrogel is prepared from the following raw materials: recombinant humanized I-type collagen subjected to methacrylation, recombinant humanized I-type collagen modified by N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy) butyramide, a photoinitiator and water, and the functional adhesive hydrogel is prepared from the following raw materials: recombinant humanized I-type collagen subjected to methacrylation, recombinant humanized I-type collagen modified by N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy) butyramide, a photoinitiator and water; each milliliter of the functionalized adhesive hydrogel also comprises 80 to 120 U of deoxyribonuclease I, 400 to 600 ng of a clomastine free drug and 5 to 15 [mu] g of a clomastine liposome. The invention also discloses a preparation method and application of the functionalized adhesive hydrogel. The functionalized adhesive hydrogel provided by the invention can reduce secondary injury, inflammatory response and myelin sheath injury of a cerebral hemorrhage part, inhibit formation of neutrophil extracellular traps, and realize multi-level intervention after cerebral hemorrhage.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and in particular relates to a functionalized adhesive hydrogel and a preparation method and application thereof. Background Art

[0002] Intracerebral hemorrhage (ICH) is a serious disease with high mortality and disability rates, often leading to severe neurological deficits. The formation of neutrophil extracellular traps (NETs) has been widely documented as an important inflammatory response after ICH, which further aggravates brain damage. NETs are composed of DNA and proteins excreted by neutrophils, with an overall reticular structure, and play a vital role in the inflammatory cascade. Recent studies have shown that NETs have harmful effects on multiple neural cell types, but the specific interaction between NETs and oligodendrocytes responsible for myelination in the central nervous system remains poorly studied.

[0003] At present, the treatment methods for inflammatory response and neural regeneration after cerebral hemorrhage are mostly focused on drug intervention and physical therapy. Research in related fields mainly focuses on the use of anti-inflammatory drugs and antioxidants, as well as early intervention methods for neural repair after hematoma removal. These methods mainly target a single pathological process and cannot fully cope with the complex pathological changes after cerebral hemorrhage. At the same time, research specifically targeting the formation of NETs, ​​myelin repair, and comprehensive treatment strategies to reduce brain damage is relatively limited. In addition, although existing hydrogel materials have certain applications in hemostasis and drug sustained release, they lack targeted adhesion and multifunctional synergistic therapeutic effects. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a functionalized adhesive hydrogel and a preparation method and application thereof, which can perform multi-level intervention on the inflammatory response after cerebral hemorrhage, the formation of neutrophil extracellular traps and myelin damage, and has broad application prospects.

[0005] The technical solution adopted to solve the technical problem is to provide a functionalized adhesive hydrogel, comprising the following raw materials in parts by weight: 5 to 10 parts of methacrylylated recombinant humanized type I collagen, 1 to 5 parts of recombinant humanized type I collagen modified by N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide, 0.01 to 0.1 parts of a photoinitiator and 90 to 120 parts of water.

[0006] The beneficial effects of the technical scheme of the present invention are as follows: recombinant humanized type I collagen protein is used as the matrix material of the hydrogel, and after being modified and grafted with methacryloyl and N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide, it has the characteristics of photo-crosslinking; the photoinitiator decomposes under ultraviolet light to generate free radicals and initiate a cross-linking reaction, and the methacryloyl groups on the methacryloylated recombinant humanized type I collagen are rapidly self-crosslinked to form the first layer network structure of the hydrogel; at the same time, photo-initiation causes the amino groups on N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide to undergo a chemical reaction to become aldehyde groups, and N-(2-aminoethyl)-4-(4-(hydroxymethyl) The aldehyde groups on the recombinant humanized type I collagen modified with N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide react with the amino groups on the recombinant humanized type I collagen acylated with methacryloyl to form a second layer of cross-linked network inside the hydrogel; at the interface between the hydrogel and the tissue, the aldehyde groups on the recombinant humanized type I collagen modified with N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide also react with the amino groups in the tissue protein to form a Schiff base, thereby enhancing adhesion; as the reaction proceeds, the remaining aldehyde groups continue to combine with the amino groups in the tissue and the methacryloyl recombinant humanized type I collagen, further strengthening the tissue adhesion and the degree of internal cross-linking. The synergistic effect of the two gives the hydrogel good mechanical properties and biocompatibility.

[0007] Preferably, the functionalized adhesive hydrogel comprises the following raw materials in parts by weight: 10 parts of methacrylylated recombinant humanized type I collagen, 1 part of recombinant humanized type I collagen modified with N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide, 0.05 parts of a photoinitiator and 100 parts of water.

[0008] Preferably, each milliliter of the functionalized adhesive hydrogel further comprises 80-120 U of deoxyribonuclease I, 400-600 ng of clemastine free drug, and 5-15 μg of clemastine liposomes.

[0009] The beneficial effects of the above technical scheme adopted in the present invention are as follows: deoxyribonuclease I is embedded in the hydrogel, which can quickly release and degrade neutrophil extracellular traps, reducing their damage to neural tissue; the free drug of clemastine achieves the effect of reducing oligodendrocyte damage at an early stage, and inhibits the therapeutic effect of neutrophil extracellular trap release; liposome-encapsulated clemastine achieves sustained release, promotes myelin repair and inhibits the formation of neutrophil extracellular traps for a long time.

[0010] More preferably, each milliliter of the functionalized adhesive hydrogel further comprises 100 U of deoxyribonuclease I, 500 ng of clemastine free drug, and 8 μg of clemastine liposomes.

[0011] Preferably, the photoinitiator is a LAP photoinitiator.

[0012] Preferably, the clemastine liposomes are prepared by the following steps: (1) Dissolve phospholipids, cholesterol, octadecylamine and clemastine in anhydrous ethanol to obtain a stock solution; (2) injecting the stock solution into the buffer solution by ethanol injection to obtain liposome vesicles; (3) The liposome vesicles are subjected to rotary evaporation, ultrasonication, centrifugation and freeze-drying.

[0013] Preferably, the mass ratio of phospholipid, cholesterol, octadecylamine and clemastine is (3-5):1:(0.6-1):(0.2-0.6); the injection speed in step (2) is 8-12 μL / s; the rotary evaporation temperature in step (3) is 35-38°C, and the time is 0.5-1.5 h; the ultrasonic power is 180-220 W, and the ultrasonic process is performed at intervals of 3-5 s for a total of 2-4 min.

[0014] More preferably, the mass ratio of phospholipid, cholesterol, octadecylamine and clemastine is 4:1:0.8:0.4; the injection speed in step (2) is 10 μL / s; the rotary evaporation temperature in step (3) is 37°C and the time is 1 h; the ultrasonic power is 200 W, and the ultrasonic process is performed at intervals of 5 s for a total of 3 min.

[0015] More preferably, in step (3), the centrifugal speed is 3000 rpm and the time is 40 min.

[0016] More preferably, the particle size of the clemastine liposome is 100-200 nm; and the mass concentration of clemastine in the clemastine liposome is 200-600 μg / mL.

[0017] Preferably, the methacrylylated recombinant humanized type I collagen is prepared by the following steps: The recombinant humanized type I collagen is dissolved in a buffer solution, and methacrylic anhydride is added under stirring conditions to carry out acylation reaction at 35-45°C for 3-5 hours and dialyzed to obtain the product.

[0018] More preferably, the solid-liquid ratio of recombinant humanized type I collagen to methacrylic anhydride is 1 g:0.1 mL; the acylation reaction temperature is 40° C., and the reaction time is 4 h.

[0019] Preferably, the recombinant humanized type I collagen modified with N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide is prepared by the following steps: N-hydroxysuccinimide and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride are dissolved in a recombinant humanized type I collagen solution, the pH is adjusted to 5.5-6.5, N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide is added for stirring reaction, and then dialyzed, centrifuged and freeze-dried to obtain the product.

[0020] More preferably, the stirring reaction temperature is 35-38°C, the time is 5-7 h; the solid-liquid ratio of the recombinant humanized type I collagen solution to N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide is (40-60) mL:327.33 mg.

[0021] More preferably, the stirring reaction temperature is 37° C. and the time is 6 h; the solid-liquid ratio of the recombinant humanized type I collagen solution to N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide is 50 mL:327.33 mg.

[0022] More preferably, the mass concentration of the recombinant humanized type I collagen solution is 1%.

[0023] The present invention also provides a method for preparing the functionalized adhesive hydrogel, comprising the following steps: The materials except the photoinitiator are mixed evenly, and then the photoinitiator is added to perform cross-linking under ultraviolet light to obtain the functionalized adhesive hydrogel.

[0024] Preferably, the method for preparing the functionalized adhesive hydrogel comprises the following steps: Methacrylylated recombinant humanized type I collagen and recombinant humanized type I collagen modified by N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide are dissolved in water, a photoinitiator is added, and then cross-linking is performed under ultraviolet light to obtain a functionalized adhesive hydrogel; or methacrylylated recombinant humanized type I collagen and recombinant humanized type I collagen modified by N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide are dissolved in water, deoxyribonuclease I, clemastine free drug, clemastine liposomes and a photoinitiator are added, and then cross-linking is performed under ultraviolet light to obtain a functionalized adhesive hydrogel.

[0025] Preferably, the wavelength of the ultraviolet light is 365 nm; and the cross-linking time is 40 s.

[0026] The present invention also provides application of the functionalized adhesive hydrogel in the preparation of materials for treating cerebral hemorrhage.

[0027] The present invention has the following beneficial effects: (1) The functionalized adhesive hydrogel of the present invention integrates multiple functional components to perform multi-level intervention on inflammatory response, neutrophil extracellular trap formation and myelin damage after cerebral hemorrhage, thereby achieving a synergistic therapeutic effect; the hydrogel matrix is ​​composed of methacrylylated recombinant humanized type I collagen and recombinant humanized type I collagen modified with N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide, and the adhesive component of the recombinant humanized type I collagen modified with N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide is The adhesion property enables the hydrogel to firmly adhere to the bleeding site, playing a role in physical hemostasis; at the same time, the biocompatibility and adhesion of the hydrogel help reduce secondary damage and inflammatory response at the bleeding site; the incorporated deoxyribonuclease I can be quickly released after reaching the site of cerebral hemorrhage, degrading neutrophil extracellular traps, reducing the inflammatory cascade caused by neutrophil extracellular traps and direct damage to neural tissue; clemastine can promote the survival and myelination of oligodendrocytes, inhibit the formation of neutrophil extracellular traps, and achieve sustained release of clemastine through liposome encapsulation, providing long-term therapeutic effects; (2) The present invention achieves rapid release of the drug by incorporating clemastine and deoxyribonuclease I into the hydrogel in free form, promoting the survival of oligodendrocytes and rapidly degrading neutrophil extracellular traps; at the same time, liposomes are used to encapsulate clemastine to achieve slow release of the drug and continuously promote myelin repair; this dual release strategy takes into account both immediate and long-term treatment needs and significantly improves the treatment effect; (3) In actual application, the functionalized adhesive hydrogel of the present invention is implanted by a combination of semi-liquid hydrogel injection and in situ ultraviolet light-induced gelation. Ultraviolet light is guided to the area around the hematoma through a thin ultraviolet optical fiber to induce the gelation of the hydrogel at the target site, thereby ensuring the precise positioning of the hydrogel and avoiding damage to the surrounding normal tissues. The adhesion of recombinant humanized type I collagen modified with N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide is used to enhance the hemostatic and adhesion effects of the hydrogel. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1The figures are rheological results of functionalized adhesive hydrogels; wherein (a) is a rheological result of the functionalized adhesive hydrogel prepared in Comparative Example 1; (b) is a rheological result of the functionalized adhesive hydrogel prepared in Example 1; (c) is a rheological result of the functionalized adhesive hydrogel prepared in Example 2; (d) is a rheological result of the functionalized adhesive hydrogel prepared in Comparative Example 2; Figure 2 The figure is the adhesion performance result of the functionalized adhesion hydrogel; Figure 3 The brain tissue compatibility results of the functionalized adhesive hydrogel are shown in FIG. 1 ; (a) is the staining result diagram; (b) is the GFAP-positive area ratio diagram; (c) is the IBA-1-positive area ratio diagram; Figure 4 The figures are the characterization diagrams of blank liposomes and clemastine liposomes prepared in Example 5; wherein, (a) is a SEM image, and the scale bars in the figures are all 100 nm; (b) is a liposome particle size diagram; and (c) is a liposome particle size statistical diagram; Figure 5 Characterization diagrams of the functionalized adhesive hydrogels prepared in Example 1 and Example 5; wherein (a) is a SEM diagram of the functionalized adhesive hydrogel prepared in Example 1, and the scale bars in the diagrams are all 20 μm; (b) is a SEM diagram of the functionalized adhesive hydrogel prepared in Example 5, and the scale bars in the diagrams are 20 μm and 5 μm from left to right, respectively; Figure 6 The results of blood coagulation and blood compatibility of functionalized adhesive hydrogels; (a) is the result of hemostasis in the liver bleeding model of functionalized adhesive hydrogels; (b) is the statistical graph of blood loss; (c) is the result of plasma recalcification experiment; (d) is the statistical graph of plasma recalcification time; Figure 7 Schematic diagram of the brain implantation and in vitro gelation of functionalized adhesive hydrogels in animal experiments; (a) is a schematic diagram of the implantation of functionalized adhesive hydrogels in mice with cerebral hemorrhage; (b) is a schematic diagram of the in vitro gelation of functionalized adhesive hydrogel droplets; Figure 8 The results of the functionalized adhesive hydrogel on the neutrophil extracellular traps in mice with cerebral hemorrhage are shown in the figure. The scale bars in the figure are all 20 μm. Fig. 9 This is the result diagram of the effect of functionalized adhesive hydrogel on the inflammatory response of mice with cerebral hemorrhage. The scale bars in the figure are all 50μm. Fig.10 This is a diagram showing the effect of functionalized adhesive hydrogel on promoting myelin repair in mice with cerebral hemorrhage. In the figure, the scales of the sham operation control group, cerebral hemorrhage group, Example 1 and Example 5 from left to right are 50 μm, 20 μm, 20 μm, 20 μm and 20 μm, respectively. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiment.It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention, that is, the embodiments described are only part of the embodiments of the present invention, rather than all embodiments.If the specific conditions are not indicated in the embodiments, they are carried out according to the conditions recommended by the normal conditions or the manufacturers.If the manufacturer is not indicated in the reagents or instruments used, they are all conventional products that can be obtained by commercial purchase.

[0030] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention.

[0031] The features and performance of the present invention are further described in detail below in conjunction with embodiments.

[0032] Example 1 A functionalized adhesive hydrogel comprises the following raw materials in parts by weight: 10 parts of methacrylylated recombinant humanized type I collagen, 1 part of recombinant humanized type I collagen modified by N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide, 0.05 parts of LAP photoinitiator and 100 parts of water; The methacrylylated recombinant humanized type I collagen is prepared by the following steps: 2 g of recombinant humanized type I collagen was dissolved in 15 mL of carbonate buffer solution with a pH of 8.5-9.5, the temperature was maintained at 40°C, and 0.2 mL of methacrylic anhydride was added dropwise at 40°C with stirring at 600 rpm for 4 h. After the reaction was completed, it was dialyzed in ultrapure water for 3 days and freeze-dried to obtain methacrylylated recombinant humanized type I collagen. Wherein, the recombinant humanized type I collagen modified with N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide is prepared by the following steps: 1 mmol of N-hydroxysuccinimide and 1 mmol of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride were dissolved in 50 mL of 1% recombinant humanized type I collagen solution, the pH was adjusted to 6.0, and then 327.33 mg of N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide was added. The mixture was stirred magnetically at 37°C and 600 rpm for 6 h, and then dialyzed in ultrapure water for 3 d. After being taken out, it was centrifuged at 4000 rpm for 5 min, and the supernatant was freeze-dried to obtain recombinant humanized type I collagen modified with N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide.

[0033] This embodiment also provides a method for preparing the functionalized adhesive hydrogel, comprising the following steps: Methacrylylated recombinant humanized type I collagen and recombinant humanized type I collagen modified by N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide were dissolved in water, stirred evenly, and then LAP photoinitiator was added. The mixture was then irradiated under 365 nm ultraviolet light for cross-linking for 40 s to obtain functionalized adhesive hydrogel.

[0034] Example 2 A functionalized adhesive hydrogel comprises the following raw materials in parts by weight: 10 parts of methacrylylated recombinant humanized type I collagen, 5 parts of recombinant humanized type I collagen modified by N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide, 0.05 parts of LAP photoinitiator and 100 parts of water; The preparation methods of the methacryloylated recombinant humanized type I collagen and the recombinant humanized type I collagen modified by N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide are the same as those in Example 1.

[0035] This embodiment also provides a method for preparing the functionalized adhesive hydrogel, comprising the following steps: Methacrylylated recombinant humanized type I collagen and recombinant humanized type I collagen modified by N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide were dissolved in water, stirred evenly, and then LAP photoinitiator was added. The mixture was then irradiated under 365 nm ultraviolet light for cross-linking for 40 s to obtain functionalized adhesive hydrogel.

[0036] Example 3 A functionalized adhesive hydrogel comprises the following raw materials in parts by weight: 10 parts of methacrylylated recombinant humanized type I collagen, 1 part of N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide-modified recombinant humanized type I collagen, 0.05 parts of LAP photoinitiator, and 100 parts of water; wherein each milliliter of the functionalized adhesive hydrogel further comprises 100 U of deoxyribonuclease I, 500 ng of clemastine free drug, and 13 μg of clemastine liposomes; The particle size of the clemastine liposomes is 100-200 nm; the mass concentration of clemastine in the clemastine liposomes is 200 μg / mL; Wherein, the clemastine liposomes are prepared by the following steps: (1) Dissolve 3 mg of phospholipid, 1 mg of cholesterol, 600 μg of octadecylamine, and 200 μg of clemastine in 1 mL of anhydrous ethanol to obtain a stock solution; (2) Add 4 mL of PBS buffer to a 25 mL glass beaker containing a magnetic stirrer, heat to 45°C and stir at 700 rpm to obtain a buffer solution; (3) Transfer the stock solution to a 1 mL syringe equipped with a 26 G needle, tap the syringe and push the plunger to expel air bubbles; (4) injecting the stock solution in step (3) into the buffer solution at a rate of 8 μL / s using an ethanol injection method to obtain liposome vesicles; (5) The liposome vesicles were transferred to a 25 mL round-bottom flask and the residual ethanol was evaporated using a rotary evaporator at 35 °C for 1.5 h to obtain a suspension; (6) The suspension was transferred to a 10 mL centrifuge tube and ultrasonicated using a probe ultrasonic processor at a power of 180 W. During the ultrasonication process, the suspension was ultrasonicated for 3 s and then left to stand for 3 s. The total ultrasonication time was 2 min. After ultrasonication, the suspension was transferred to a 10 kDa ultrafiltration tube and centrifuged at 3000 rpm for 40 min. This was repeated 3 times and then freeze-dried to obtain the product. The methacrylylated recombinant humanized type I collagen is prepared by the following steps: 2 g of recombinant humanized type I collagen was dissolved in 15 mL of carbonate buffer solution with a pH of 8.5-9.5, and the temperature was maintained at 35°C. 0.2 mL of methacrylic anhydride was added dropwise at 35°C with stirring at 600 rpm for acylation reaction for 3 h. After the reaction was completed, it was dialyzed in ultrapure water for 3 days and freeze-dried to obtain methacrylylated recombinant humanized type I collagen. Wherein, the recombinant humanized type I collagen modified with N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide is prepared by the following steps: 1 mmol of N-hydroxysuccinimide and 1 mmol of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride were dissolved in 40 mL of 1% recombinant humanized type I collagen solution, the pH was adjusted to 5.5, and then 327.33 mg of N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide was added. The mixture was stirred magnetically at 35°C and 600 rpm for 5 h, and then dialyzed in ultrapure water for 3 d. After being taken out, it was centrifuged at 4000 rpm for 5 min, and the supernatant was freeze-dried to obtain recombinant humanized type I collagen modified with N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide.

[0037] This embodiment also provides a method for preparing the functionalized adhesive hydrogel, comprising the following steps: Methacrylylated recombinant humanized type I collagen and recombinant humanized type I collagen modified by N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide were dissolved in water, and deoxyribonuclease I, clemastine free drug and clemastine liposomes were added. After stirring evenly, LAP photoinitiator was added and then irradiated under 365nm ultraviolet light for cross-linking for 40 s to obtain functionalized adhesive hydrogel.

[0038] Example 4 A functionalized adhesive hydrogel comprises the following raw materials in parts by weight: 10 parts of methacrylylated recombinant humanized type I collagen, 1 part of N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide-modified recombinant humanized type I collagen, 0.05 parts of LAP photoinitiator, and 100 parts of water; wherein each milliliter of the functionalized adhesive hydrogel further comprises 100 U of deoxyribonuclease I, 500 ng of clemastine free drug, and 7 μg of clemastine liposomes; The particle size of the clemastine liposomes is 100-200 nm; the mass concentration of clemastine in the clemastine liposomes is 600 μg / mL; Wherein, the clemastine liposomes are prepared by the following steps: (1) Dissolve 5 mg of phospholipid, 1 mg of cholesterol, 1 mg of octadecylamine, and 600 μg of clemastine in 1 mL of anhydrous ethanol to obtain a stock solution; (2) Add 4 mL of PBS buffer to a 25 mL glass beaker containing a magnetic stirrer, heat to 45°C and stir at 700 rpm to obtain a buffer solution; (3) Transfer the stock solution to a 1 mL syringe equipped with a 26 G needle, tap the syringe and push the plunger to expel air bubbles; (4) injecting the stock solution in step (3) into the buffer solution at a rate of 12 μL / s using an ethanol injection method to obtain liposome vesicles; (5) The liposome vesicles were transferred to a 25 mL round-bottom flask and the residual ethanol was evaporated using a rotary evaporator at 38 °C for 0.5 h to obtain a suspension; (6) The suspension was transferred to a 10 mL centrifuge tube and ultrasonicated at a power of 220 W using a probe ultrasonic processor. During the ultrasonication process, the suspension was ultrasonicated for 5 s and then left to stand for 5 s. The total ultrasonication time was 4 min. After ultrasonication, the suspension was transferred to a 10 kDa ultrafiltration tube and centrifuged at 3000 rpm for 40 min. This was repeated 3 times and then freeze-dried to obtain the product. The methacrylylated recombinant humanized type I collagen is prepared by the following steps: 2 g of recombinant humanized type I collagen was dissolved in 15 mL of carbonate buffer solution with a pH of 8.5-9.5, the temperature was maintained at 45°C, and 0.2 mL of methacrylic anhydride was added dropwise at 45°C with stirring at 600 rpm for acylation reaction for 5 h. After the reaction was completed, it was dialyzed in ultrapure water for 3 days and freeze-dried to obtain methacrylylated recombinant humanized type I collagen. Wherein, the recombinant humanized type I collagen modified with N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide is prepared by the following steps: 1 mmol of N-hydroxysuccinimide and 1 mmol of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride were dissolved in 60 mL of 1% recombinant humanized type I collagen solution, the pH was adjusted to 6.5, and then 327.33 mg of N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide was added. The mixture was stirred magnetically at 38°C and 600 rpm for 7 h, and then dialyzed in ultrapure water for 3 d. After being taken out, it was centrifuged at 4000 rpm for 5 min, and the supernatant was freeze-dried to obtain recombinant humanized type I collagen modified with N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide.

[0039] This embodiment also provides a method for preparing the functionalized adhesive hydrogel, comprising the following steps: Methacrylylated recombinant humanized type I collagen and recombinant humanized type I collagen modified by N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide were dissolved in water, and deoxyribonuclease I, clemastine free drug and clemastine liposomes were added. After stirring evenly, LAP photoinitiator was added and then irradiated under 365nm ultraviolet light for cross-linking for 40 s to obtain functionalized adhesive hydrogel.

[0040] Example 5 A functionalized adhesive hydrogel comprises the following raw materials in parts by weight: 10 parts of methacrylylated recombinant humanized type I collagen, 1 part of N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide-modified recombinant humanized type I collagen, 0.05 parts of LAP photoinitiator, and 100 parts of water; wherein each milliliter of the functionalized adhesive hydrogel further comprises 100 U of deoxyribonuclease I, 500 ng of clemastine free drug, and 8 μg of clemastine liposomes; The particle size of the clemastine liposomes is 100-200 nm; the mass concentration of clemastine in the clemastine liposomes is 400 μg / mL; Wherein, the clemastine liposomes are prepared by the following steps: (1) Dissolve 4 mg of phospholipid, 1 mg of cholesterol, 800 μg of octadecylamine, and 400 μg of clemastine in 1 mL of anhydrous ethanol to obtain a stock solution; (2) Add 4 mL of PBS buffer to a 25 mL glass beaker containing a magnetic stirrer, heat to 45°C and stir at 700 rpm to obtain a buffer solution; (3) Transfer the stock solution to a 1 mL syringe equipped with a 26 G needle, tap the syringe and push the plunger to expel air bubbles; (4) injecting the stock solution in step (3) into the buffer solution at a rate of 10 μL / s using an ethanol injection method to obtain liposome vesicles; (5) The liposome vesicles were transferred to a 25 mL round-bottom flask and the residual ethanol was evaporated using a rotary evaporator at 37 °C for 1 h to obtain a suspension; (6) The suspension was transferred to a 10 mL centrifuge tube and ultrasonicated at a power of 200 W using a probe ultrasonic processor. During the ultrasonication process, the suspension was ultrasonicated for 5 s and then left to stand for 5 s. The total ultrasonication time was 3 min. After ultrasonication, the suspension was transferred to a 10 kDa ultrafiltration tube and centrifuged at 3000 rpm for 40 min. This was repeated 3 times and then freeze-dried to obtain the product. The preparation methods of the methacryloylated recombinant humanized type I collagen and the recombinant humanized type I collagen modified by N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide are the same as those in Example 1.

[0041] This embodiment also provides a method for preparing the functionalized adhesive hydrogel, comprising the following steps: Methacrylylated recombinant humanized type I collagen and recombinant humanized type I collagen modified by N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide were dissolved in water, and deoxyribonuclease I, clemastine free drug and clemastine liposomes were added. After stirring evenly, LAP photoinitiator was added and then irradiated under 365nm ultraviolet light for cross-linking for 40 s to obtain functionalized adhesive hydrogel.

[0042] Example 6 A functionalized adhesive hydrogel comprises the following raw materials in parts by weight: 10 parts of methacrylylated recombinant humanized type I collagen, 5 parts of N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide-modified recombinant humanized type I collagen, 0.05 parts of LAP photoinitiator, and 100 parts of water; wherein each milliliter of the functionalized adhesive hydrogel further comprises 100 U of deoxyribonuclease I, 500 ng of clemastine free drug, and 8 μg of clemastine liposomes; The particle size of the clemastine liposomes is 100-200 nm; the mass concentration of clemastine in the clemastine liposomes is 400 μg / mL; Among them, the preparation method of clemastine liposomes is the same as that in Example 5, and the preparation method of methacrylylated recombinant humanized type I collagen and N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide-modified recombinant humanized type I collagen is the same as that in Example 1.

[0043] This embodiment also provides a method for preparing the functionalized adhesive hydrogel, comprising the following steps: Methacrylylated recombinant humanized type I collagen and recombinant humanized type I collagen modified by N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide were dissolved in water, and deoxyribonuclease I, clemastine free drug and clemastine liposomes were added. After stirring evenly, LAP photoinitiator was added and then irradiated under 365nm ultraviolet light for cross-linking for 40 s to obtain functionalized adhesive hydrogel.

[0044] Comparative Example 1 A functionalized adhesive hydrogel comprises the following raw materials in parts by weight: 10 parts of methacrylylated recombinant humanized type I collagen, 0.05 parts of LAP photoinitiator, and 100 parts of water; The preparation method of methacrylylated recombinant humanized type I collagen is the same as that in Example 1.

[0045] This comparative example also provides a method for preparing the functionalized adhesive hydrogel, comprising the following steps: Methacrylylated recombinant humanized type I collagen was dissolved in water, stirred evenly, and then LAP photoinitiator was added. The mixture was then irradiated under 365 nm ultraviolet light for cross-linking for 40 s to obtain a functionalized adhesive hydrogel.

[0046] Comparative Example 2 A functionalized adhesive hydrogel comprises the following raw materials in parts by weight: 15 parts of methacrylylated recombinant humanized type I collagen, 1 part of recombinant humanized type I collagen modified by N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide, 0.05 parts of LAP photoinitiator, and 100 parts of water; The preparation methods of the methacryloylated recombinant humanized type I collagen and the recombinant humanized type I collagen modified by N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide are the same as those in Example 1.

[0047] This comparative example also provides a method for preparing the functionalized adhesive hydrogel, comprising the following steps: Methacrylylated recombinant humanized type I collagen and recombinant humanized type I collagen modified by N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide were dissolved in water, stirred evenly, and then LAP photoinitiator was added. The mixture was then irradiated under 365 nm ultraviolet light for cross-linking for 40 s to obtain functionalized adhesive hydrogel.

[0048] Experimental example 1. The functionalized adhesive hydrogels prepared in Examples 1 and 2 and Comparative Examples 1 and 2 were subjected to rheological tests. The specific test method was as follows: at 37°C, the storage modulus (G′) and loss modulus (G″) were measured using a 25 mm plate-to-plate rheometer at a constant strain of 1% and a frequency of 10 rad / s. The results are shown in Table 1. Figure 1 shown.

[0049] Hydrogels with a loss modulus of less than 1000 Pa can match the modulus of brain tissue. Figure 1It can be seen that when the ratio of methacrylylated recombinant humanized type I collagen to recombinant humanized type I collagen modified with N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide is 10:0 (Comparative Example 1), the G′ of the hydrogel is 552 Pa; when the ratio of methacrylylated recombinant humanized type I collagen to recombinant humanized type I collagen modified with N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide is 10:1 (Example 1), the G′ of the hydrogel is 710 Pa; when the ratio of methacrylylated recombinant humanized type I collagen to recombinant humanized type I collagen modified with N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide is 10:5 (Example 2), the G′ of the hydrogel is 996 Pa; when the ratio of methacryloylated recombinant humanized type I collagen to recombinant humanized type I collagen modified with N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide is 15:1, the hydrogel G′ is 3585 Pa; it can be seen that when the ratio of methacryloylated recombinant humanized type I collagen is higher (Comparative Example 2), the obtained functionalized adhesion hydrogel has a low degree of matching with the brain tissue modulus and is not suitable for the preparation of cerebral hemorrhage treatment materials.

[0050] 2. The functionalized adhesive hydrogels prepared in Examples 1 and 2 and Comparative Examples 1 and 2 were subjected to an adhesion test. The specific test method was as follows: the functionalized adhesive hydrogels were subjected to an adhesion test using a universal calorimeter. The hydrogels were bonded to the surface of pig skin with an adhesion area of ​​1 cm 2 Then, a tensile force was gradually applied by a universal mechanical instrument to measure the adhesion strength between the hydrogel and the pig skin surface; the results are shown in Figure 2 shown.

[0051] from Figure 2 It can be seen that the functionalized adhesive hydrogel prepared in Comparative Example 1 has the lowest adhesion. The adhesion performance of the hydrogel can be significantly enhanced by increasing the weight of recombinant humanized type I collagen modified with N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide. There is no obvious difference between the functionalized adhesive hydrogels prepared in Example 1 and Example 2, and the functionalized adhesive hydrogel prepared in Comparative Example 2 has the strongest adhesion performance.

[0052] 3. The functionalized adhesive hydrogels prepared in Examples 1-2 and Comparative Example 2 were subjected to brain tissue compatibility tests. The specific test method was as follows: the functionalized adhesive hydrogels were implanted into mouse brain tissues, and the tissues were collected for histological analysis 28 days after implantation; GFAP was used to mark astrocytes and IBA-1 was used to mark microglia; the results were as follows: Figure 3 shown.

[0053] from Figure 3 It can be seen that the excellence degree of the proportion of GFAP-positive areas of the functionalized adhesive hydrogels is PBS>Example 1>Example 2>Comparative Example 2; the excellence degree of the proportion of IBA-1-positive areas is PBS>Example 1>Comparative Example 2>Example 2, among which the functionalized adhesive hydrogel prepared in Example 1 has the best tissue compatibility and causes the smallest astrocyte and microglial reactions.

[0054] 4. Characterize the clemastine liposomes and the functionalized adhesive hydrogel. First, measure the size of the clemastine liposomes: dilute the liposomes 10 times with PBS buffer, and then use Nano ZS® Zetasizer to perform dynamic laser scattering to measure the size of the liposomes. Prepare blank liposomes. The difference between the preparation method and the preparation method of the clemastine liposomes in Example 5 is that no clemastine is added in step (1), and the remaining steps and parameters are the same as the preparation method of the clemastine liposomes in Example 5; SEM characterization and particle size statistics are performed on the blank liposomes and the clemastine liposomes prepared in Example 5, and SEM characterization is performed on the functionalized adhesive hydrogels prepared in Example 1 and Example 5. The results are as follows: Figures 4 and 5 shown.

[0055] from Figures 4 and 5 It can be seen that the particle size of the clemastine liposomes is slightly larger than that of the blank liposomes, but the difference is not significant. It can be seen from the microstructure of the functionalized adhesive hydrogel prepared in Example 5 of the present invention that the clemastine liposomes are successfully encapsulated in the functionalized adhesive hydrogel.

[0056] 5. Perform coagulation and blood compatibility tests on the functionalized adhesive hydrogel. The specific test methods are as follows: (1) Using a mouse liver bleeding model, liver bleeding was induced by applying standardized trauma to the liver incision; after bleeding, the functionalized adhesive hydrogel was applied to the bleeding site, and the amount of blood loss was measured using filter paper; the amount of blood loss was statistically calculated by recording the weight change of the filter paper before and after the operation; (2) 6 mL of peripheral blood was collected from a healthy volunteer, anticoagulated with 3.2% sodium citrate and centrifuged at 1200 × g for 10 min to obtain platelet-poor plasma (PPP). 0.2 g of the functionalized adhesive hydrogel was taken and 1 mL of PPP was added to completely cover the platelet. The PPP without the functionalized adhesive hydrogel was used as the blank control, and the PPP containing the glass slide was used as the positive control. All samples were incubated at 37°C and 100 rpm for 60 min. After incubation, 100 µL of PPP was added to a 96-well plate, and 100 µL of 25 mM calcium chloride solution was added. The absorbance at 405 nm was immediately monitored at 37°C using an ELISA reader at 1-min intervals for 1 h, and the plasma recalcification kinetic curve was plotted. The results are shown in Table 2. Figure 6 shown.

[0057] from Figure 6 It can be seen that the functionalized adhesive hydrogel prepared by the present invention has good in vivo and in vitro hemostatic ability. When it is used as a therapeutic material for cerebral hemorrhage, it can have a good prognostic effect on the inflammatory response of cerebral hemorrhage.

[0058] 6. The functionalized adhesive hydrogel is gelled and implanted into the brain in animal experiments. The schematic diagram of implantation and in vitro gelling is shown in the figure. Figure 7 As shown in the figure, the effect of functionalized adhesive hydrogel on neutrophil extracellular traps (NETs) in mice with intracerebral hemorrhage was studied. NETs were labeled with Ly6g and Cit-H3 cells. The specific test method was as follows: first, a semi-liquid hydrogel was injected into the ICH site of the mouse brain, and ultraviolet light was guided to the area around the hematoma using an ultraviolet optical fiber to induce the gelation of the hydrogel at the target site; then, the mouse brain tissue was extracted and fixed, and Ly6g and Cit-H3 were labeled by immunofluorescence staining to detect the formation of NETs; the labeling results were observed under a microscope, and the number of NETs-positive cells was counted; the results are shown in the figure. Figure 8 shown.

[0059] from Figure 7 It can be seen that the functionalized adhesive hydrogel in the present invention is implanted by a combination of semi-liquid hydrogel injection and in situ ultraviolet light-induced gelation. The ultraviolet light is guided to the area around the hematoma through a thin ultraviolet optical fiber to induce the hydrogel to gel at the target site, thereby ensuring the precise positioning of the hydrogel and avoiding damage to the surrounding normal tissues. Figure 8 It can be seen that the functionalized adhesive hydrogel prepared by the present invention can significantly reduce the production of NETs in mice with cerebral hemorrhage.

[0060] 7. The effect of functionalized adhesive hydrogel on the inflammatory response of mice with cerebral hemorrhage was studied. Microglia are the main inflammatory response cells in brain tissue. IBA-1 was used to label total microglia. Fig. 9 shown.

[0061] from Fig. 9 It can be seen that the functionalized adhesive hydrogel prepared by the present invention can reduce the positive area of ​​IBA-1 in mice with cerebral hemorrhage and reduce the inflammatory activation of microglia.

[0062] 8. The effect of functionalized adhesive hydrogel on promoting myelin repair in mice with cerebral hemorrhage was studied. MOG was used to label myelin and SOX10 was used to label oligodendrocyte precursor cells. The results were as follows: Fig.10 shown.

[0063] from Fig.10 It can be seen that the functionalized adhesive hydrogel prepared by the present invention can enhance the myelin repair in the hydrogel implantation area of ​​cerebral hemorrhage mice and promote the proliferation of oligodendrocyte precursor cells.

[0064] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A functionalized adhesive hydrogel, characterized in that: The invention comprises the following raw materials in parts by weight: 5 to 10 parts of methacrylylated recombinant humanized type I collagen, 1 to 5 parts of recombinant humanized type I collagen modified by N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide, 0.01 to 0.1 parts of a photoinitiator and 90 to 120 parts of water.

2. The functionalized adhesive hydrogel according to claim 1, characterized in that The invention comprises the following raw materials in parts by weight: 10 parts of methacrylylated recombinant humanized type I collagen, 1 part of recombinant humanized type I collagen modified by N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide, 0.05 parts of a photoinitiator and 100 parts of water.

3. The functionalized adhesive hydrogel according to claim 1 or 2, characterized in that: Each milliliter of the functionalized adhesive hydrogel also includes 80-120 U of deoxyribonuclease I, 400-600 ng of clemastine free drug and 5-15 μg of clemastine liposome.

4. The functionalized adhesive hydrogel according to claim 3, characterized in that The clemastine liposomes are prepared by the following steps: (1) Dissolve phospholipids, cholesterol, octadecylamine and clemastine in anhydrous ethanol to obtain a stock solution; (2) injecting the stock solution into the buffer solution by ethanol injection to obtain liposome vesicles; (3) The liposome vesicles are subjected to rotary evaporation, ultrasonication, centrifugation and freeze-drying.

5. The functionalized adhesive hydrogel according to claim 4, characterized in that The mass ratio of the phospholipid, cholesterol, octadecylamine and clemastine is (3-5):1:(0.6-1):(0.2-0.6); the injection speed in the step (2) is 8-12 μL / s; the rotary evaporation temperature in the step (3) is 35-38°C, and the time is 0.5-1.5 h; the ultrasonic power is 180-220 W, and the ultrasonic process is performed at intervals of 3-5 s for a total of 2-4 min.

6. The functionalized adhesive hydrogel according to claim 1 or 2, characterized in that: The methacrylylated recombinant humanized type I collagen is prepared by the following steps: The recombinant humanized type I collagen is dissolved in a buffer solution, and methacrylic anhydride is added under stirring conditions to carry out acylation reaction at 35-45°C for 3-5 hours and dialyzed to obtain the product.

7. The functionalized adhesive hydrogel according to claim 1 or 2, characterized in that: The recombinant humanized type I collagen modified by N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide is prepared by the following steps: N-hydroxysuccinimide and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride are dissolved in a recombinant humanized type I collagen solution, the pH is adjusted to 5.5-6.5, N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide is added for stirring reaction, and then dialyzed, centrifuged and freeze-dried to obtain the product.

8. The functionalized adhesive hydrogel according to claim 7, characterized in that The stirring reaction temperature is 35-38° C., and the reaction time is 5-7 h. The solid-liquid ratio of the recombinant humanized type I collagen solution to N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide is (40-60) mL:327.33 mg.

9. The method for preparing the functionalized adhesive hydrogel according to any one of claims 1 to 8, characterized in that: The following steps are involved: The materials except the photoinitiator are mixed evenly, and then the photoinitiator is added to perform cross-linking under ultraviolet light to obtain the functionalized adhesive hydrogel.

10. Use of the functionalized adhesive hydrogel according to any one of claims 1 to 8 in the preparation of therapeutic materials for cerebral hemorrhage.

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