A functionalized adhesive hydrogel, its preparation method and application

Abstract

The present invention discloses a functionalized adhesive hydrogel and its preparation method and application, belonging to the technical field of biomedical materials. The functionalized adhesive hydrogel comprises the following raw materials: methacrylated recombinant humanized type I collagen, recombinant humanized type I collagen modified with N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy) butyramide, a photoinitiator and water; per milliliter of the functionalized adhesive hydrogel also comprises 80-120 U of deoxyribonuclease I, 400-600 ng of clemastine free drug and 5-15 μg of clemastine liposome. The present invention also discloses the preparation method and application of the functionalized adhesive hydrogel. The functionalized adhesive hydrogel of the present invention can reduce the occurrence of secondary injury, inflammatory response and myelin injury at the intracerebral hemorrhage site, inhibit the formation of neutrophil extracellular traps, and can achieve multi-level intervention after intracerebral hemorrhage.

Description

Technical Field

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

[0002] Intracerebral hemorrhage (ICH) is a severe disease with high mortality and high 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 intracerebral hemorrhage, which further exacerbates brain injury. NETs are composed of DNA and proteins discharged by neutrophils, presenting an overall reticular structure and playing a crucial role in the inflammatory cascade reaction. Recent studies have shown that NETs have harmful effects on various types of nerve cells, but the specific interaction between NETs and oligodendrocytes responsible for myelin formation in the central nervous system still lacks in-depth research.

[0003] Currently, the treatment methods for the inflammatory response and nerve regeneration after intracerebral hemorrhage mainly focus on drug intervention and physical therapy. The research in related fields mainly focuses on the use of anti-inflammatory drugs and antioxidants, as well as the early intervention methods for nerve repair after hematoma removal. These methods mainly target a single pathological process and cannot comprehensively address the complex pathological changes after intracerebral hemorrhage. At the same time, the research on comprehensive treatment strategies specifically targeting the formation of NETs, myelin repair, and reduction of brain injury is relatively limited. In addition, although existing hydrogel materials have certain applications in hemostasis and drug sustained release, they lack targeted adhesiveness and multi-functional synergistic treatment effects. Summary of the Invention

[0004] Aiming at the above deficiencies in the prior art, the present invention provides a functionalized adhesive hydrogel, a preparation method thereof, and an application thereof, which can perform multi-level interventions on the inflammatory response, the formation of neutrophil extracellular traps, and myelin damage after intracerebral hemorrhage, and has a wide application prospect.

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

[0006] The beneficial effects of the present invention adopting the above technical solutions are as follows: Recombinant humanized type I collagen protein is used as the matrix material of the hydrogel. After being modified by grafting methacryloyl group and N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy) butyramide, it has the characteristics of photocrosslinking; The photoinitiator decomposes under ultraviolet light irradiation to generate free radicals and initiate the crosslinking reaction. The methacryloyl groups on the methacrylated recombinant humanized type I collagen rapidly self-crosslink to form the first network structure of the hydrogel; At the same time, photocatalysis causes the amino group on N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy) butyramide to undergo a chemical reaction to become an aldehyde group. The aldehyde group on the N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy) butyramide-modified recombinant humanized type I collagen reacts with the amino group on the methacrylated recombinant humanized type I collagen to form the second crosslinking network inside the hydrogel; At the interface between the hydrogel and the tissue, the aldehyde group on the N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy) butyramide-modified recombinant humanized type I collagen also reacts with the amino group in the tissue protein to form a Schiff base, enhancing adhesion; As the reaction proceeds, the remaining aldehyde groups continue to combine with the amino groups in the tissue and the methacrylated recombinant humanized type I collagen, further strengthening the tissue adhesion and the degree of internal crosslinking. 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 methacrylated 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 part of 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 liposome.

[0009] The beneficial effects of the present invention adopting the above technical solutions are as follows: Deoxyribonuclease I is embedded in the hydrogel, which can be rapidly released and degrade neutrophil extracellular traps, reducing their damage to nerve tissue; The clemastine free drug achieves the effect of reducing oligodendrocyte damage at an early stage and inhibits the therapeutic effect of neutrophil extracellular trap release; Liposome-encapsulated clemastine realizes sustained release, promoting myelin repair and inhibiting the formation of neutrophil extracellular traps in the long term.

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

[0011] Preferably, the photoinitiator is LAP photoinitiator.

[0012] Preferably, the clemastine liposome is prepared through the following steps:

[0013] (1) Dissolve phospholipids, cholesterol, octadecylamine, and clemastine in absolute ethanol to obtain a stock solution;

[0014] (2) Inject the stock solution into a buffer solution by the ethanol injection method to obtain liposome vesicles;

[0015] (3) Rotavaporize the liposome vesicles, then ultrasonicate, and then centrifuge and freeze-dry to obtain the product.

[0016] Preferably, the mass ratio of phospholipids, cholesterol, octadecylamine, and clemastine is (3 - 5):1:(0.6 - 1):(0.2 - 0.6); the injection rate in step (2) is 8 - 12 μL / s; the rotavaporization 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 carried out with an interval of 3 - 5 s for a total of 2 - 4 min.

[0017] More preferably, the mass ratio of phospholipids, cholesterol, octadecylamine, and clemastine is 4:1:0.8:0.4; the injection rate in step (2) is 10 μL / s; the rotavaporization temperature in step (3) is 37 °C, and the time is 1 h; the ultrasonic power is 200 W, and the ultrasonic process is carried out with an interval of 5 s for a total of 3 min.

[0018] More preferably, the centrifugation speed in step (3) is 3000 rpm, and the time is 40 min.

[0019] More preferably, the particle size of the clemastine liposome is 100 - 200 nm; the mass concentration of clemastine in the clemastine liposome is 200 - 600 μg / mL.

[0020] Preferably, the methacrylated recombinant humanized type I collagen is prepared through the following steps:

[0021] Dissolve the recombinant humanized type I collagen in a buffer solution, and add methacrylic anhydride under stirring conditions to carry out an acylation reaction at 35 - 45 °C for 3 - 5 h and then dialyze to obtain the product.

[0022] More preferably, the material 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 time is 4 h.

[0023] Preferably, the recombinant humanized type I collagen modified with N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy) butyramide is prepared through the following steps:

[0024] Dissolve N-hydroxysuccinimide and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride in the recombinant humanized type I collagen solution, adjust the pH to 5.5 - 6.5, add N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy) butyramide and carry out a stirring reaction, and then obtain the product after dialysis, centrifugation and freeze-drying.

[0025] More preferably, the stirring reaction temperature is 35 - 38 °C and the time is 5 - 7 h; the material 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.

[0026] More preferably, the stirring reaction temperature is 37 °C and the time is 6 h; the material 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.

[0027] More preferably, the mass concentration of the recombinant humanized type I collagen solution is 1%.

[0028] The present invention also provides a preparation method of the above functionalized adhesive hydrogel, including the following steps:

[0029] Mix the substances except the photoinitiator, then add the photoinitiator, and carry out crosslinking under ultraviolet light to obtain the functionalized adhesive hydrogel.

[0030] Preferably, the preparation method of the functionalized adhesive hydrogel includes the following steps:

[0031] Dissolve methacrylated recombinant humanized type I collagen and N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide-modified recombinant humanized type I collagen in water, add a photoinitiator, and then crosslink under ultraviolet light to obtain a functionalized adhesive hydrogel; or dissolve methacrylated recombinant humanized type I collagen and N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide-modified recombinant humanized type I collagen in water, add deoxyribonuclease I, clemastine free drug, clemastine liposome and a photoinitiator, and then crosslink under ultraviolet light to obtain a functionalized adhesive hydrogel.

[0032] Preferably, the wavelength of the ultraviolet light is 365 nm; the crosslinking time is 40 s.

[0033] The present invention also provides the application of the above functionalized adhesive hydrogel in the preparation of a treatment material for cerebral hemorrhage.

[0034] The present invention has the following beneficial effects:

[0035] (1) The functionalized adhesive hydrogel of the present invention integrates multiple functional components to perform multi-level intervention on the inflammatory response, neutrophil extracellular trap formation and myelin injury after cerebral hemorrhage, achieving a synergistic treatment effect; the hydrogel matrix is composed of methacrylated recombinant humanized type I collagen and N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide-modified recombinant humanized type I collagen. The adhesion characteristics of N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide-modified recombinant humanized type I collagen can make the hydrogel firmly adhere to the bleeding site, playing a role in physical hemostasis; at the same time, the biocompatibility and adhesiveness of the hydrogel help to reduce secondary injury and inflammatory response at the bleeding site; for the incorporated deoxyribonuclease I, it can be rapidly released after reaching the cerebral hemorrhage site, degrade neutrophil extracellular traps, and reduce the inflammatory cascade reaction and direct damage to nerve tissue caused by neutrophil extracellular traps; clemastine can promote the survival and myelin formation of oligodendrocytes, inhibit the formation of neutrophil extracellular traps, and achieve the sustained slow release of clemastine through liposome encapsulation, providing a long-term treatment effect;

[0036] (2) By incorporating clemastine and deoxyribonuclease I into the hydrogel in a free form, the present invention realizes the rapid release of drugs, promotes the survival of oligodendrocytes and rapidly degrades neutrophil extracellular traps; at the same time, clemastine is encapsulated in liposomes to achieve the slow release of drugs and continuously promote myelin repair; this dual release strategy takes into account the immediate and long-term treatment needs and significantly improves the treatment effect;

[0037] (3) In the actual application process of the functionalized adhesive hydrogel of the present invention, semi-liquid hydrogel injection combined with in-situ ultraviolet light-induced gelation is used for implantation. 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, ensuring the precise positioning of the hydrogel, avoiding damage to the surrounding normal tissues, and enhancing the hemostatic and adhesion effects of the hydrogel by using the adhesiveness of recombinant humanized type I collagen modified with N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy) butyramide. Description of the Drawings

[0038] Figure 1 It is a rheological result diagram of the functionalized adhesive hydrogel; among them, (a) is the rheological result diagram of the functionalized adhesive hydrogel prepared in Comparative Example 1; (b) is the rheological result diagram of the functionalized adhesive hydrogel prepared in Example 1; (c) is the rheological result diagram of the functionalized adhesive hydrogel prepared in Example 2; (d) is the rheological result diagram of the functionalized adhesive hydrogel prepared in Comparative Example 2;

[0039] Figure 2 It is an adhesion performance result diagram of the functionalized adhesive hydrogel;

[0040] Figure 3 It is a brain tissue compatibility result diagram of the functionalized adhesive hydrogel; among them, (a) is the staining result diagram; (b) is the proportion diagram of the GFAP-positive area; (c) is the proportion diagram of the IBA-1-positive area;

[0041] Figure 4 It is a characterization diagram of blank liposomes and clemastine liposomes prepared in Example 5; among them, (a) is the SEM diagram, and the scale bars in the figures are all 100 nm; (b) is the liposome particle size diagram; (c) is the liposome particle size statistical diagram;

[0042] Figure 5 It is a characterization diagram of the functionalized adhesive hydrogels prepared in Example 1 and Example 5; among them, (a) is the SEM diagram of the functionalized adhesive hydrogel prepared in Example 1, and the scale bar in the figure is 20 μm; (b) is the SEM diagram of the functionalized adhesive hydrogel prepared in Example 5, and the scale bars from left to right are 20 μm and 5 μm in turn;

[0043] Figure 6 It is the coagulation and blood compatibility results of the functionalized adhesive hydrogel; among them, (a) is the hemostatic ability result diagram of the functionalized adhesive hydrogel in the liver bleeding model; (b) is the blood loss statistical diagram; (c) is the plasma recalcification experiment result diagram; (d) is the plasma recalcification time statistical diagram;

[0044] Figure 7Schematic diagram of brain implantation of functionalized adhesive hydrogel in animal experiments and gelation in vitro; among them, (a) is the schematic diagram of implanting functionalized adhesive hydrogel in a mouse with cerebral hemorrhage; (b) is the schematic diagram of gelation of functionalized adhesive hydrogel droplets in vitro;

[0045] Figure 8 Results of the effect of functionalized adhesive hydrogel on neutrophil extracellular traps in mice with cerebral hemorrhage; the scale bars in the figures are all 20 μm;

[0046] Figure 9 Results of the effect of functionalized adhesive hydrogel on the inflammatory response in mice with cerebral hemorrhage; the scale bars in the figures are all 50 μm;

[0047] Figure 10 Effect diagram of functionalized adhesive hydrogel promoting myelin repair in mice with cerebral hemorrhage; in the figure, the scale bars of the sham operation control group, cerebral hemorrhage group, Example 1 and Example 5 are 50 μm, 20 μm, 20 μm, 20 μm and 20 μm from left to right. Detailed implementation mode

[0048] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention, that is, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Those without specific conditions in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments without indicating the manufacturer can be obtained as conventional products through commercial purchase.

[0049] Therefore, the following detailed description of the provided embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0050] The features and performance of the present invention will be further described in detail below in conjunction with embodiments.

[0051] Example 1

[0052] A functionalized adhesive hydrogel, comprising the following raw materials in parts by weight: 10 parts of methacrylated 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 part of LAP photoinitiator and 100 parts of water;

[0053] Among them, the methacrylated recombinant humanized type I collagen is prepared through the following steps:

[0054] Dissolve 2 g of recombinant humanized type I collagen in 15 mL of carbonate buffer solution with a pH of 8.5 - 9.5, maintain the temperature at 40°C, and stir and dropwise add 0.2 mL of methacrylic anhydride at a rotation speed of 600 rpm. Carry out the acylation reaction at 40°C for 4 h. After the reaction is completed, dialyze it in ultrapure water for 3 d and then perform freeze-drying to obtain methacrylated recombinant humanized type I collagen;

[0055] Among them, the recombinant humanized type I collagen modified with N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy) butyramide is prepared through the following steps:

[0056] Dissolve 1 mmol of N-hydroxysuccinimide and 1 mmol of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride in 50 mL of a 1% recombinant humanized type I collagen solution, adjust the pH to 6.0, then add 327.33 mg of N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy) butyramide, and carry out magnetic stirring reaction at 37°C at a rotation speed of 600 rpm for 6 h. Then dialyze it in ultrapure water for 3 d, take it out and centrifuge at 4000 rpm for 5 min, and take the supernatant and perform freeze-drying to obtain the recombinant humanized type I collagen modified with N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy) butyramide.

[0057] This example also provides a preparation method of the above functionalized adhesive hydrogel, including the following steps:

[0058] Dissolve methacrylated recombinant humanized type I collagen and the recombinant humanized type I collagen modified with N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy) butyramide in water, stir evenly, add LAP photoinitiator, and then irradiate and crosslink under 365 nm ultraviolet light for 40 s to obtain the functionalized adhesive hydrogel.

[0059] Example 2

[0060] A functionalized adhesive hydrogel, comprising the following raw materials in parts by weight: 10 parts of methacrylated recombinant humanized type I collagen, 5 parts of the recombinant humanized type I collagen modified with N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy) butyramide, 0.05 part of LAP photoinitiator, and 100 parts of water;

[0061] Among them, the preparation methods of methacrylated recombinant humanized type I collagen and N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy) butyramide-modified recombinant humanized type I collagen are the same as those in Example 1.

[0062] This example also provides a preparation method of the above functionalized adhesive hydrogel, including the following steps:

[0063] Dissolve methacrylated recombinant humanized type I collagen and N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy) butyramide-modified recombinant humanized type I collagen in water, add LAP photoinitiator after stirring evenly, and then irradiate and crosslink under 365 nm ultraviolet light for 40 s to obtain the functionalized adhesive hydrogel.

[0064] Example 3

[0065] A functionalized adhesive hydrogel, comprising the following raw materials in parts by weight: 10 parts of methacrylated 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 part of LAP photoinitiator, and 100 parts of water; among them, each milliliter of the functionalized adhesive hydrogel also contains 100 U of deoxyribonuclease I, 500 ng of clemastine free drug, and 13 μg of clemastine liposome;

[0066] Among them, the particle size of the clemastine liposome is 100-200 nm; the mass concentration of clemastine in the clemastine liposome is 200 μg / mL;

[0067] Among them, the clemastine liposome is prepared through the following steps:

[0068] (1) Dissolve 3 mg of phospholipid, 1 mg of cholesterol, 600 μg of octadecylamine, and 200 μg of clemastine in 1 mL of absolute ethanol to obtain a stock solution;

[0069] (2) Add 4 mL of PBS buffer solution to a 25 mL glass beaker containing a magnetic stir bar, heat to 45°C and stir at a speed of 700 rpm to obtain a buffer solution;

[0070] (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;

[0071] (4) Inject the stock solution in step (3) into the buffer solution at a speed of 8 μL / s by the ethanol injection method to obtain liposome vesicles;

[0072] (5) Transfer the liposome vesicles to a 25 mL round-bottom flask and rotary evaporate the residual ethanol at 35 °C for 1.5 h to obtain a suspension;

[0073] (6) Transfer the suspension to a 10 mL centrifuge tube and sonicate it with a probe-type ultrasonic processor at a power of 180 W. During the sonication, sonicate for 3 s and then let it stand for 3 s. The total intermittent sonication time is 2 min. After sonication, transfer the suspension to a 10 kDa ultrafiltration tube and centrifuge at 3000 rpm for 40 min. Repeat 3 times and then perform lyophilization to obtain the product;

[0074] Among them, the methacrylated recombinant humanized type I collagen is prepared through the following steps:

[0075] Dissolve 2 g of recombinant humanized type I collagen in 15 mL of carbonate buffer solution with a pH of 8.5 - 9.5, keep the temperature at 35 °C, and stir and dropwise add 0.2 mL of methacrylic anhydride at 600 rpm. Carry out acylation reaction at 35 °C for 3 h. After the reaction is completed, dialyze it in ultrapure water for 3 d and then perform freeze-drying to obtain methacrylated recombinant humanized type I collagen;

[0076] Among them, the N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide-modified recombinant humanized type I collagen is prepared through the following steps:

[0077] Dissolve 1 mmol of N-hydroxysuccinimide and 1 mmol of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride in 40 mL of a 1% recombinant humanized type I collagen solution, adjust the pH to 5.5, then add 327.33 mg of N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide, and magnetically stir and react at 600 rpm at 35 °C for 5 h. Then dialyze it in ultrapure water for 3 d, take it out and centrifuge at 4000 rpm for 5 min. Take the supernatant and perform freeze-drying to obtain N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide-modified recombinant humanized type I collagen.

[0078] This example also provides a preparation method of the above functionalized adhesive hydrogel, including the following steps:

[0079] Dissolve methacrylated recombinant humanized type I collagen and N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide-modified recombinant humanized type I collagen in water, add deoxyribonuclease I, clemastine free drug and clemastine liposome, stir evenly, then add LAP photoinitiator, and irradiate and crosslink under 365 nm ultraviolet light for 40 s to obtain the functionalized adhesive hydrogel.

[0080] Example 4

[0081] A functionalized adhesive hydrogel, comprising the following raw materials in parts by weight: 10 parts of methacrylated 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 part of LAP photoinitiator, and 100 parts of water; wherein, each milliliter of the functionalized adhesive hydrogel also contains 100 U of deoxyribonuclease I, 500 ng of clemastine free drug and 7 μg of clemastine liposome;

[0082] Among them, the particle size of the clemastine liposome is 100-200 nm; the mass concentration of clemastine in the clemastine liposome is 600 μg / mL;

[0083] Among them, the clemastine liposome is prepared through the following steps:

[0084] (1) Dissolve 5 mg of phospholipid, 1 mg of cholesterol, 1 mg of octadecylamine and 600 μg of clemastine in 1 mL of absolute ethanol to obtain a stock solution;

[0085] (2) Add 4 mL of PBS buffer solution to a 25 mL glass beaker containing a magnetic stir bar, heat to 45 °C and stir at a speed of 700 rpm to obtain a buffer solution;

[0086] (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;

[0087] (4) Inject the stock solution in step (3) into the buffer solution at a speed of 12 μL / s by the ethanol injection method to obtain liposome vesicles;

[0088] (5) Transfer the liposome vesicles to a 25 mL round-bottom flask, and use a rotary evaporator to rotary evaporate the residual ethanol at 38 °C for 0.5 h to obtain a suspension;

[0089] (6) Transfer the suspension to a 10 mL centrifuge tube, and ultrasonically treat it with a probe-type ultrasonic processor at a power of 220 W. During the ultrasonic treatment, ultrasonicate for 5 s and then let it stand for 5 s. The total intermittent ultrasonic treatment duration is 4 min. After ultrasonic treatment, transfer the suspension to a 10 kDa ultrafiltration tube, centrifuge at 3000 rpm for 40 min, repeat 3 times, and then perform lyophilization to obtain it;

[0090] Among them, the methacrylated recombinant humanized type I collagen is prepared through the following steps:

[0091] Dissolve 2 g of recombinant humanized type I collagen in 15 mL of carbonate buffer solution with a pH of 8.5 - 9.5, keep the temperature at 45 °C, and stir and dropwise add 0.2 mL of methacrylic anhydride at 600 rpm and carry out acylation reaction at 45 °C for 5 h. After the reaction is completed, dialyze it in ultrapure water for 3 d and perform freeze-drying to obtain methacrylated recombinant humanized type I collagen;

[0092] Among them, the N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide-modified recombinant humanized type I collagen is prepared through the following steps:

[0093] Dissolve 1 mmol of N-hydroxysuccinimide and 1 mmol of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride in 60 mL of a 1% recombinant humanized type I collagen solution, adjust the pH to 6.5, then add 327.33 mg of N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide, and magnetically stir and react at 38 °C at 600 rpm for 7 h. Then dialyze it in ultrapure water for 3 d, take it out and centrifuge at 4000 rpm for 5 min, and take the supernatant and perform freeze-drying to obtain N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide-modified recombinant humanized type I collagen.

[0094] This example also provides a preparation method of the above functionalized adhesive hydrogel, including the following steps:

[0095] Dissolve methacrylated recombinant humanized type I collagen and N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide-modified recombinant humanized type I collagen in water, add deoxyribonuclease I, clemastine free drug and clemastine liposome, stir evenly, then add LAP photoinitiator, and irradiate and crosslink under 365 nm ultraviolet light for 40 s to obtain the functionalized adhesive hydrogel.

[0096] Example 5

[0097] A functionalized adhesive hydrogel, comprising raw materials in the following parts by weight: 10 parts of methacrylated 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 part of LAP photoinitiator, and 100 parts of water; wherein, each milliliter of the functionalized adhesive hydrogel further contains 100 U of deoxyribonuclease I, 500 ng of clemastine free drug, and 8 μg of clemastine liposome;

[0098] Wherein, the particle size of the clemastine liposome is 100 - 200 nm; the mass concentration of clemastine in the clemastine liposome is 400 μg / mL;

[0099] Wherein, the clemastine liposome is prepared through the following steps:

[0100] (1) Dissolve 4 mg of phospholipid, 1 mg of cholesterol, 800 μg of octadecylamine, and 400 μg of clemastine in 1 mL of absolute ethanol to obtain a stock solution;

[0101] (2) Add 4 mL of PBS buffer solution to a 25 mL glass beaker containing a magnetic stirrer, heat to 45 °C and stir at a speed of 700 rpm to obtain a buffer solution;

[0102] (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;

[0103] (4) Inject the stock solution in step (3) into the buffer solution at a speed of 10 μL / s by the ethanol injection method to obtain liposome vesicles;

[0104] (5) Transfer the liposome vesicles to a 25 mL round-bottom flask, and use a rotary evaporator to rotary evaporate the residual ethanol at 37 °C for 1 h to obtain a suspension;

[0105] (6) Transfer the suspension to a 10 mL centrifuge tube, use a probe-type ultrasonic processor to perform ultrasonic treatment at a power of 200 W. During the ultrasonic treatment, ultrasonic for 5 s and then stand still for 5 s. The total intermittent ultrasonic duration is 3 min. After ultrasonic treatment, transfer the suspension to a 10 kDa ultrafiltration tube, centrifuge at a speed of 3000 rpm for 40 min, repeat 3 times, and then perform freeze-drying to obtain the product;

[0106] Among them, the preparation methods of methacrylated recombinant humanized type I collagen and N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide-modified recombinant humanized type I collagen are the same as those in Example 1.

[0107] This example also provides a preparation method of the above functionalized adhesive hydrogel, including the following steps:

[0108] Dissolve methacrylated recombinant humanized type I collagen, N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide-modified recombinant humanized type I collagen in water, add deoxyribonuclease I, clemastine free drug and clemastine liposome, stir evenly, then add LAP photoinitiator, and irradiate and crosslink under 365 nm ultraviolet light for 40 s to obtain the functionalized adhesive hydrogel.

[0109] Example 6

[0110] A functionalized adhesive hydrogel, comprising the following raw materials in parts by weight: 10 parts of methacrylated 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 part of LAP photoinitiator, 100 parts of water; among them, each milliliter of the functionalized adhesive hydrogel also contains 100 U of deoxyribonuclease I, 500 ng of clemastine free drug and 8 μg of clemastine liposome;

[0111] Among them, the particle size of the clemastine liposome is 100-200 nm; the mass concentration of clemastine in the clemastine liposome is 400 μg / mL;

[0112] Among them, the preparation method of the clemastine liposome is the same as that in Example 5, and the preparation methods of methacrylated recombinant humanized type I collagen and N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide-modified recombinant humanized type I collagen are the same as those in Example 1.

[0113] This example also provides a preparation method of the above functionalized adhesive hydrogel, including the following steps:

[0114] Dissolve methacrylated recombinant humanized type I collagen and N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy) butyramide-modified recombinant humanized type I collagen in water, add deoxyribonuclease I, clemastine free drug and clemastine liposomes, stir evenly, then add LAP photoinitiator, and irradiate and crosslink under 365 nm ultraviolet light for 40 s to obtain the functionalized adhesive hydrogel.

[0115] Comparative Example 1

[0116] A functionalized adhesive hydrogel, comprising the following raw materials in parts by weight: 10 parts of methacrylated recombinant humanized type I collagen, 0.05 part of LAP photoinitiator, and 100 parts of water;

[0117] Among them, the preparation method of methacrylated recombinant humanized type I collagen is the same as that in Example 1.

[0118] This comparative example also provides a preparation method of the above functionalized adhesive hydrogel, comprising the following steps:

[0119] Dissolve methacrylated recombinant humanized type I collagen in water, stir evenly, then add LAP photoinitiator, and irradiate and crosslink under 365 nm ultraviolet light for 40 s to obtain the functionalized adhesive hydrogel.

[0120] Comparative Example 2

[0121] A functionalized adhesive hydrogel, comprising the following raw materials in parts by weight: 15 parts of methacrylated 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 part of LAP photoinitiator, and 100 parts of water;

[0122] Among them, the preparation methods of methacrylated recombinant humanized type I collagen and N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy) butyramide-modified recombinant humanized type I collagen are the same as those in Example 1.

[0123] This comparative example also provides a preparation method of the above functionalized adhesive hydrogel, comprising the following steps:

[0124] Dissolve methacrylated recombinant humanized type I collagen and N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy) butyramide-modified recombinant humanized type I collagen in water, stir evenly, then add LAP photoinitiator, and irradiate and crosslink under 365 nm ultraviolet light for 40 s to obtain the functionalized adhesive hydrogel.

[0125] Experimental Example

[0126] 1. The functionalized adhesive hydrogels prepared in Examples 1-2 and Comparative Examples 1-2 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-plate rheometer at a constant strain of 1% and a frequency of 10 rad / s; the results are as Figure 1 shown.

[0127] Hydrogels with a loss modulus less than 1000 Pa can match the modulus of brain tissue. From Figure 1 it can be seen that when the ratio of methacrylated recombinant humanized type I collagen to N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide-modified recombinant humanized type I collagen is 10:0 (Comparative Example 1), the G′ of the hydrogel is 552 Pa; when the ratio of methacrylated recombinant humanized type I collagen to N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide-modified recombinant humanized type I collagen is 10:1 (Example 1), the G′ of the hydrogel is 710 Pa; when the ratio of methacrylated recombinant humanized type I collagen to N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide-modified recombinant humanized type I collagen is 10:5 (Example 2), the G′ of the hydrogel is 996 Pa; when the ratio of methacrylated recombinant humanized type I collagen to N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide-modified recombinant humanized type I collagen is 15:1, the G′ of the hydrogel is 3585 Pa; it can be seen therefrom that when the ratio of methacrylated recombinant humanized type I collagen is relatively high (Comparative Example 2), the prepared functionalized adhesive hydrogel has a low matching degree with the modulus of brain tissue and is not suitable for preparing materials for the treatment of cerebral hemorrhage.

[0128] 2. The functionalized adhesive hydrogels prepared in Examples 1-2 and Comparative Examples 1-2 were subjected to adhesion performance tests. The specific test method was as follows: the adhesion force of the functionalized adhesive hydrogel was tested using a universal testing machine. The hydrogel was adhered to the surface of pig skin, and the adhesion area was 1 cm 2 , and then a tensile force was gradually applied through the universal testing machine to measure the adhesion strength between the hydrogel and the surface of pig skin; the results are as Figure 2 shown.

[0129] From Figure 2It can be seen that the functionalized adhesive hydrogel prepared in Comparative Example 1 has the lowest adhesiveness. The adhesive performance of the hydrogel can be significantly enhanced by increasing the parts by weight of recombinant humanized type I collagen modified with N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy) butyramide. There is no significant difference between the functionalized adhesive hydrogels prepared in Example 1 and Example 2, while the functionalized adhesive hydrogel prepared in Comparative Example 2 has the strongest adhesive performance.

[0130] 3. The functionalized adhesive hydrogels prepared in Examples 1 to 2 and Comparative Example 2 were subjected to a brain tissue compatibility test. The specific test method was as follows: The prepared functionalized adhesive hydrogels were respectively implanted into the brain tissues of mice, and tissues were collected 28 days after implantation for histological analysis; GFAP was used to label astrocytes, and IBA-1 was used to label microglia; The results are as Figure 3 shown.

[0131] From Figure 3 it can be seen that the excellent degree of the proportion of the GFAP-positive region of the functionalized adhesive hydrogel is PBS > Example 1 > Example 2 > Comparative Example 2; the excellent degree of the proportion of the IBA-1-positive region is PBS > Example 1 > Comparative Example 2 > Example 2. Among them, the functionalized adhesive hydrogel prepared in Example 1 has the best tissue compatibility and causes the smallest astrocyte reaction and microglia reaction.

[0132] 4. The chlorpheniramine liposomes and the functionalized adhesive hydrogel were characterized. First, the size of the chlorpheniramine liposomes was measured: The liposomes were diluted 10 times with PBS buffer, and then a Nano ZS® Zetasizer was used for dynamic light scattering to measure the size of the liposomes. Blank liposomes were prepared. The difference between the preparation method and the preparation method of the chlorpheniramine liposomes in Example 5 was that: chlorpheniramine was not added in step (1), and the remaining steps and parameters were the same as those in the preparation method of the chlorpheniramine liposomes in Example 5; SEM characterization and particle size statistics were performed on the blank liposomes and the chlorpheniramine liposomes prepared in Example 5, and SEM characterization was performed on the functionalized adhesive hydrogels prepared in Example 1 and Example 5. The results are as Figures 4 - 5 shown.

[0133] From Figures 4 - 5 it can be seen that the particle size of the chlorpheniramine 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 chlorpheniramine liposomes are successfully coated in the functionalized adhesive hydrogel.

[0134] 5. The functionalized adhesive hydrogel was subjected to a coagulation and blood compatibility test. The specific test method was as follows:

[0135] (1) A mouse liver bleeding model was used. Liver bleeding was induced by applying standardized trauma at the liver incision site. After bleeding occurred, the functionalized adhesive hydrogel was applied to the bleeding site, and filter paper was used to measure the blood loss. The blood loss was statistically analyzed by recording the change in the weight of the filter paper before and after the operation.

[0136] (2) 6 mL of peripheral blood was collected from 1 healthy volunteer, anticoagulated with 3.2% sodium citrate by mass concentration, 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 completely covered with 1 mL of PPP. The PPP without the functionalized adhesive hydrogel was used as a blank control, and the PPP containing a glass slide was used as a positive control. All samples were incubated with shaking at 37°C and a rotation speed of 100 rmp for 60 min. After incubation, 100 µL of PPP was taken and added to a 96-well plate, and 100 µL of 25 mM calcium chloride solution was added. Immediately, the absorbance at 405 nm was monitored using an enzyme-linked immunosorbent assay (ELISA) reader at 37°C, at intervals of 1 min, continuously for 1 h, and a plasma recalcification kinetic curve was plotted. The results are as Figure 6 shown.

[0137] It can be seen from Figure 6 that the functionalized adhesive hydrogel prepared in the present invention has good in vivo and in vitro hemostatic abilities. When it is used as a treatment material for cerebral hemorrhage, it can have a good prognostic effect on the inflammatory response of cerebral hemorrhage.

[0138] 6. The functionalized adhesive hydrogel was subjected to gelation and animal experiment brain implantation. The implantation and in vitro gelation schematic diagrams are as Figure 7 shown, and the effect of the functionalized adhesive hydrogel on neutrophil extracellular traps (NETs) in mice with cerebral hemorrhage was studied. The cells labeled with NETs were Ly6g and Cit-H3. The specific test method was as follows: First, a semi-liquid hydrogel was injected at the ICH site in the mouse brain, and ultraviolet light was guided to the area around the hematoma using an ultraviolet light optical fiber to induce gelation of the hydrogel at the target site. Subsequently, the mouse brain tissue was extracted and fixed, and immunofluorescence staining was used to label Ly6g and Cit-H3 to detect the formation of NETs. The labeled results were observed through a microscope, and the number of NETs-positive cells was counted. The results are as Figure 8 shown.

[0139] It can be seen from Figure 7 that the functionalized adhesive hydrogel in the present invention was implanted by injecting a semi-liquid hydrogel combined with in-situ ultraviolet light-induced gelation. Ultraviolet light was guided to the area around the hematoma through a thin ultraviolet light optical fiber to induce gelation of the hydrogel at the target site, ensuring the precise positioning of the hydrogel and avoiding damage to the surrounding normal tissues. It can be seen from Figure 8 that the functionalized adhesive hydrogel prepared in the present invention can significantly reduce the production of NETs in mice with cerebral hemorrhage.

[0140] 7. Investigate the effect of the functionalized adhesive hydrogel on the inflammatory response in mice with intracerebral hemorrhage. Microglia are the main inflammatory response cells in the brain tissue, and IBA-1 is used to label the total microglia. The results are as Figure 9 shown.

[0141] As can be seen from Figure 9 , the functionalized adhesive hydrogel prepared by the present invention can reduce the positive area of IBA-1 in mice with intracerebral hemorrhage and reduce the inflammatory activation of microglia.

[0142] 8. Investigate the effect of the functionalized adhesive hydrogel on promoting myelin repair in mice with intracerebral hemorrhage. MOG is used to label myelin, and SOX10 is used to label oligodendrocyte precursor cells. The results are as Figure 10 shown.

[0143] As can be seen from Figure 10 , the functionalized adhesive hydrogel prepared by the present invention can enhance the myelin repair in the hydrogel implantation area of mice with intracerebral hemorrhage and promote the proliferation of oligodendrocyte precursor cells.

[0144] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included within 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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