Application of ROS-based response type hydrogel as biomedical material

Abstract

The invention provides application of ROS-based response type hydrogel as a biomedical material, and belongs to the technical field of hydrogel, AHP hydrogel is used as a main raw material to prepare a skin wound dressing product, and the AHP hydrogel is prepared by a one-step method through free radical polymerization reaction by taking HAMA and AM as skeletons and a hyperbranched polymer PBAE synthesized by PEGDA and cystamine dihydrochloride as a cross-linking agent. As a promising skin wound dressing, the AHP hydrogel and the AHPE hydrogel act on the surface of damaged skin to treat various skin injuries, especially acute or difficult-to-heal skin injuries.

Description

Technical Field

[0001] The invention belongs to the technical field of hydrogels, and in particular relates to an application of a ROS-responsive hydrogel as a biomedical material. Background Art

[0002] ROS plays a key role in life processes and is a signaling molecule that regulates cellular physiological activities. However, when ROS levels increase or the antioxidant capacity of cells decreases, resulting in the inability of antioxidant defense to neutralize excess ROS, oxidative stress occurs. Excessive ROS under oxidative stress is extremely destructive and may cause oxidative damage to cellular macromolecules, induce inflammation, and accelerate the progression of various diseases (Nature Reviews Molecular Cell Biology 2022, 23, 499-515).

[0003] Taking the treatment of skin injury wounds as an example, the microenvironment of difficult-to-heal wounds such as radiation-induced skin injury and diabetic foot accumulates a large amount of ROS. The large amount of ROS accumulated in these wounds not only induces a strong inflammatory response and a high oxidative stress microenvironment, making the wound fragile, but also inhibits macrophages and endogenous stem cells, hindering the regeneration process of wound tissue, thereby greatly delaying the healing of skin injuries (Nature Communications 2019, 10, 2538).

[0004] As a hydrophilic material with a three-dimensional network structure, hydrogel has the advantages of high water content and good biocompatibility. In recent years, it has been widely used in the research and development of skin wound dressings. Compared with traditional dressings, hydrogel dressings can absorb wound exudate in a timely manner while maintaining a moist environment on the wound surface, and also have obvious advantages in bioadhesion and water vapor permeability (ACS Nano 2021, 15, 12687-12722).

[0005] However, most of the commercial radiation protection agents or skin injury dressings in the prior art contain easily degraded small molecule antioxidant drugs, which make their antioxidant function unstable and affect wound healing. Summary of the invention

[0006] The problem to be solved by the present invention is to provide an application of ROS-responsive hydrogel as a biomedical material.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is: an application of ROS-responsive hydrogel as a biomedical material, AHP hydrogel is used as the main raw material to prepare a skin wound dressing product, wherein the AHP hydrogel uses methacryloyl hyaluronic acid (HAMA) and acrylamide (AM) as the skeleton, and a hyperbranched polymer (PBAE) synthesized from polyethylene glycol diacrylate (PEGDA) and cystamine dihydrochloride as the cross-linking agent, and is prepared by a one-step free radical polymerization reaction.

[0008] Furthermore, the AHP hydrogel is loaded with growth factors to form an AHPE hydrogel.

[0009] Furthermore, the preparation method of the AHP hydrogel comprises the following steps: S1, the double bond on polyethylene glycol diacrylate (PEGDA) and the amine group of cystamine dihydrochloride undergo Michael addition reaction, and the resulting hyperbranched polymer (PBAE) is used as a cross-linking agent; PBAE has the function of scavenging ROS; S2, using methacrylic anhydride to react with hyaluronic acid (HA) to obtain methacryloyl hyaluronic acid (HAMA); S3. Using HAMA as the main monomer and acrylamide (AM) as the monomer forming the main skeleton of the gel, HAMA, AM and PBAE solution were mixed to form AHP hydrogel in one step through free radical polymerization reaction.

[0010] Further, in step S1, the PEGDA and cystamine dihydrochloride are reacted by Michael addition to synthesize a hyperbranched polymer PBAE. Specifically, cystamine dihydrochloride and PEGDA are added to a round-bottom flask and stirred, and triethylamine is added as a catalyst, and reacted at 80° C. for 4 hours, and then precipitated with glacial ether to obtain a yellow oily substance, namely PBAE.

[0011] Further, in step S2, hyaluronic acid (HA) is dissolved in ultrapure water, and methacrylic anhydride is added. The reaction is maintained for 16 hours, and the pH value is 8.5. The entire reaction process is carried out on ice and protected from light. After the reaction, the solution is precipitated with acetone, washed with methanol, and then dialyzed for 3 days and freeze-dried to obtain the product HAMA.

[0012] Furthermore, in step S3, HAMA, PBAE and AM are dissolved in deionized water to form a gel precursor solution, a photoinitiator is added, and the AHP hydrogel is formed by physical blending under the action of ultraviolet light.

[0013] Due to the adoption of the above technical solution, the present invention has the following beneficial effects: The AHP hydrogel of the invention has the effect of reducing cell DNA damage caused by radiation. Growth factor EGF is added during the preparation of the AHP hydrogel, and the obtained AHPE hydrogel has better ability to promote cell proliferation and migration.

[0014] The AHP hydrogel and AHPE hydrogel of the present invention can be used as the main raw material of skin wound dressings for wound treatment in mice, and have a healing promoting effect on common wounds and radiation skin injury wounds in mice.

[0015] The AHP hydrogel and AHPE hydrogel of the present invention both have good biosafety, and in vitro experiments show that they have no hemolytic effect. After being used as the main raw material for skin wound dressings and applied to wound treatment of mice, they have no obvious toxic and side effects on the organs of the mice, and the hematological indicators of the mice are all normal.

[0016] The skin wound dressing prepared with AHP hydrogel and AHPE hydrogel as the main raw materials of the present invention is applied to the difficult-to-heal wound surface. Taking radioactive skin injury as an example, this hydrogel based on ROS-responsive hyperbranched polymer can reduce the oxidative damage caused by radiation, effectively promote the healing of radioactive skin injury wounds, and because it does not contain easily degradable small molecule antioxidant drugs but directly has antioxidant function through the change of gel structure, it is more stable and more conducive to wound healing. It can be seen that the AHP hydrogel and AHPE hydrogel of the present invention are used as a promising skin wound dressing, acting on the surface of damaged skin, and treating various skin injuries, especially acute or difficult-to-heal skin injuries, such as diabetic foot, radioactive skin injury, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The present invention will be described in detail below with reference to the accompanying drawings and in combination with examples, and the advantages and implementation methods of the present invention will become more obvious. The contents shown in the accompanying drawings are only used to explain the present invention and do not constitute any limitation to the present invention in any sense. In the accompanying drawings: Figure 1 This is an immunofluorescence staining diagram of DNA damage after radiation irradiation of hydrogels with different PBAE contents in the present invention.

[0018] Figure 2 It is a calcein staining diagram showing the promotion of cell proliferation by different hydrogels of the present invention.

[0019] Figure 3 It is a scratch test diagram of the ability of different hydrogels of the present invention to promote cell migration.

[0020] Figure 4 It is a hemolysis experiment diagram of the hydrogels with different PBAE contents of the present invention.

[0021] Figure 5It is a bar graph of the hemolysis rate of the hydrogels with different PBAE contents of the present invention.

[0022] Figure 6 This is an H&E staining image of the main organs of mice after different hydrogels of the present invention were applied to mice.

[0023] Figure 7 It is a bar graph of routine blood indexes of mice after different hydrogels of the present invention are applied to mice.

[0024] Figure 8 It is a schematic diagram of the mouse radiation skin injury animal model of the present invention.

[0025] Fig. 9 This is a diagram showing the progression of skin damage in mice after different hydrogels of the present invention were applied to mice irradiated by X-rays.

[0026] Fig.10 It is a schematic diagram of the rating of radiation skin damage of mice after different hydrogels of the present invention are applied to mice irradiated with X-rays. DETAILED DESCRIPTION

[0027] like Figures 1 to 10 As shown, the present invention is based on the application of ROS-responsive hydrogel as a biomedical material, and AHP hydrogel is used as the main raw material to prepare a skin wound dressing product (the preparation method of the dressing product is a prior art and is not a necessary technical feature for solving the technical problem of the present invention, and the present invention will not describe it again).

[0028] Among them, AHP hydrogel is prepared by a one-step free radical polymerization reaction with methacryloyl hyaluronic acid (HAMA) and acrylamide (AM) as the skeleton and a hyperbranched polymer (PBAE) synthesized from polyethylene glycol diacrylate (PEGDA) and cystamine dihydrochloride as the cross-linker.

[0029] The preparation method of AHP hydrogel comprises the following steps: S1, the double bond on polyethylene glycol diacrylate (PEGDA) and the amine group of cystamine dihydrochloride undergo Michael addition reaction, and the resulting hyperbranched polymer (PBAE) is used as a cross-linking agent; PBAE has the function of scavenging ROS; S2, using methacrylic anhydride to react with hyaluronic acid (HA) to obtain methacryloyl hyaluronic acid (HAMA); S3. Using HAMA as the main monomer and acrylamide (AM) as the monomer forming the main skeleton of the gel, HAMA, AM and PBAE solution were mixed to form AHP hydrogel in one step through free radical polymerization reaction.

[0030] In step S1, polyethylene glycol diacrylate uses PEGDA700. PEGDA700 and cystamine dihydrochloride are used to synthesize the hyperbranched polymer PBAE by the A2+B4 Michael addition method. Specifically, 453.6 mg of cystamine dihydrochloride (10%, DMSO) and 3.506 g of PEGDA700 (30%, DMSO) are added to a round-bottom flask, and a magnetic stirrer is added at a stirring speed of 200 rpm; 1 mL of triethylamine is added as a catalyst, and the mixture is reacted at 80°C for 4 hours, and then precipitated with ice ether to obtain a yellow oily substance, which is PBAE.

[0031] In step S2, hyaluronic acid with a molecular weight of 10w is used. Specifically, 1g of hyaluronic acid (HA) is dissolved in 100mL of ultrapure water, and 3.7mL of methacrylic anhydride is added. The reaction is maintained for 16 hours, and the pH value is 8.5. The entire reaction process is carried out on ice and protected from light. After the reaction is completed, the solution is precipitated with acetone, washed with methanol, and then dialyzed for 3 days (molecular weight cutoff is 7000Da) and freeze-dried to obtain the product HAMA.

[0032] In step S3, HAMA, PBAE, and AM are dissolved in deionized water to form a gel precursor solution, a photoinitiator is added, and an AHP hydrogel is formed under the action of ultraviolet light initiation through physical blending. The solid content of HAMA in the gel precursor solution system is 1%; the solid content of PBAE is 1%; and the solid content of AM is 20%. The photoinitiator used is 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone, and its content accounts for 0.1% of the gel precursor component.

[0033] The disulfide bonds in the AHP hydrogel system of the present invention are conducive to responding to ROS breakage and removing ROS at the same time. AHP hydrogel can be used as an antioxidant hydrogel dressing to treat various skin injuries, especially acute or difficult to heal skin injuries, such as diabetic foot, radiation skin injury, etc.

[0034] The AHP hydrogel of the present invention can be used to prepare a dressing with multiple functions by encapsulating other components. For example, after encapsulating the growth factor EGF in the AHP hydrogel to form an AHPE hydrogel, the healing of skin injuries can be promoted from the two aspects of anti-oxidation and promoting tissue regeneration. After the AHPE hydrogel is applied to mice as a wound dressing, it has no obvious toxic side effects on the mice and has good biocompatibility. The preparation method of the monomer and the hydrogel is simple and easy, and it can be used as a promising wound dressing to achieve the treatment of difficult-to-heal skin injury wounds represented by radiation skin injury.

[0035] Example: like Figure 1As shown, γ-H2AX immunofluorescence staining was used to investigate the hydrogels with different PBAE contents (AH, AHP 0.5 , AHP1) against radiation protection. At the same time, the radiation protection ability was compared with PBS (negative control) and PBS + IR (positive control). 6GyX-ray irradiation (1Gy / min, 6min) was applied to L929 cells incubated with 100μL hydrogel for 24h in advance. After 2h of continuous culture, γ-H2AX immunofluorescence staining was performed. Observation under laser confocal microscopy showed that AHP hydrogel can reduce the double-strand DNA damage caused by radiation. Compared with other irradiation groups, AHP1 hydrogel can effectively reduce the expression of γ-H2AX after treatment, indicating that it can effectively weaken the DNA damage caused by radiation.

[0036] like Figure 2 As shown in the figure, the ability of different hydrogels (AH, AHP1, AHPE) to promote cell proliferation was investigated by calcein staining experiment. At the same time, the ability to promote cell proliferation was compared with PBS (control group). Before 100μL hydrogel was co-incubated with L929 cells, calcein staining was performed after 12h and 24h of incubation. After staining, laser confocal microscopy was used to observe cell proliferation. The L929 cells in the field of view of AHPE hydrogel were significantly more than those in other groups. The results showed that AHPE hydrogel loaded with growth factor EGF can effectively promote the proliferation of L929 cells and has the ability to promote cell proliferation.

[0037] like Figure 3 As shown in the figure, the scratch test was used to investigate the ability of different hydrogels (AH, AHP1, AHPE) to promote cell migration. At the same time, the ability to promote cell migration was compared with PBS (control group). The scratches were photographed before, after 12h, and after 24h of incubation of 100μL hydrogel and L929 cells. The results showed that compared with other groups, more cells migrated to the center of the scratch after 24h of AHPE hydrogel treatment. It can be seen that AHPE hydrogel loaded with growth factor EGF can effectively promote the migration of L929 cells.

[0038] like Figure 4 and Figure 5 As shown in the figure, the hemolysis experiment was used to investigate the hydrogels with different PBAE contents (AH, AHP 0.5 , AHP1) in vitro biosafety. 1 mL of 2% red blood cell suspension was mixed with PBS (negative control), PBS solution containing 0.1% Triton X-100 (positive control), AH hydrogel, AHP 0.5The hydrogel and AHP1 hydrogel were incubated together at 37°C for 4 h, then centrifuged at 3000 rpm for 3 min, the supernatant was taken for photographing, and the absorbance of the supernatant at 570 nm was measured. The hemolysis rate of each hydrogel was calculated using the following formula: Hemolysis rate (%) = (absorbance of sample – absorbance of negative control) / (absorbance of positive control – absorbance of negative control) × 100.

[0039] The results showed that the hemolysis rates of all hydrogels were below 5%, which would not cause hemolysis and had good in vitro biosafety.

[0040] like Figure 6 As shown, H&E staining of the main organs in the body was used to investigate the in vivo biosafety of different hydrogels (AH, AHP, AHPE). At the same time, the in vivo biosafety was compared with PBS (blank control) and SOD (superoxide dismutase, positive control group). The main organs (heart, liver, spleen, lung, and kidney) of the mice were collected after the treatment. After formalin fixation for 48 hours, the tissues were dehydrated, paraffin-embedded, sectioned, and routinely stained with H&E. The results showed that the hydrogels did not damage the main organs and had good in vivo biosafety.

[0041] like Figure 7 As shown, the in vivo biosafety of different hydrogels (AH, AHP, AHPE) was further investigated by blood routine test, and the in vivo biosafety was compared with PBS (blank control) and SOD (superoxide dismutase, positive control group). After the hydrogel treatment, the orbital blood of the mice was taken for blood routine test. The test indicators included hemoglobin, hematocrit, white blood cells, red blood cells, mean hematocrit, mean hemoglobin content, mean hemoglobin concentration and monocyte percentage. The results showed that according to the official data of Charles River Laboratories as the control, the blood routine indicators of each group of mice were within the normal range, and each hydrogel had no hematological side effects on mice.

[0042] like Figures 8 to 10As shown in the figure, an animal model of radiation skin injury in mice was established, and different hydrogel (AH, AHP, AHPE) dressings were applied to the right leg of mice. The mice were irradiated with 40GyX-rays (2Gy / min, 20min) for 10 days, and the hydrogel dressings were changed every day. After that, the changes of the wounds of the mice were continuously observed and photographed, and the changes of the wounds of the mice treated with PBS group (blank control) and SOD group (superoxide dismutase, positive control) were compared, and the RTOG acute radiation injury grading evaluation was established. The results showed that hydrogels can effectively promote the healing of radiation skin injury wounds. And the wounds were radiation-damaged, and the mice in the AHPE hydrogel treatment group could basically return to normal on the 21st day, with the fastest recovery speed. The acute radiation injury grading of mice in each group during the treatment cycle showed that the mice in the AHPE hydrogel treatment group had the least damage, recovered faster, and had better treatment effect.

[0043] The raw materials used in the present invention are all commercially available.

[0044] The embodiments of the present invention are described in detail above, but the contents are only preferred embodiments of the present invention and cannot be considered to limit the scope of implementation of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. An application of ROS-responsive hydrogel as a biomedical material, characterized in that: AHP hydrogel is used as the main raw material to prepare a skin wound dressing product. The AHP hydrogel uses HAMA and AM as the skeleton, and PBAE synthesized from PEGDA and cystamine dihydrochloride as the crosslinking agent, and is prepared in a one-step method through a free radical polymerization reaction.

2. The use of the ROS-responsive hydrogel as a biomedical material according to claim 1, characterized in that: The AHP hydrogel is loaded with growth factor EGF to form AHPE hydrogel.

3. The use of the ROS-responsive hydrogel as a biomedical material according to claim 1, characterized in that: The preparation method of the AHP hydrogel comprises the following steps: S1, the double bond on the PEGDA and the amine group of cystamine dihydrochloride undergo Michael addition reaction, and the obtained PBAE is used as a cross-linking agent; S2, using methacrylic anhydride and HA to undergo an esterification reaction to obtain HAMA; S3, mixing the HAMA, AM and PBAE solution, and forming an AHP hydrogel by a one-step free radical polymerization reaction.

4. The use of the ROS-responsive hydrogel as a biomedical material according to claim 3, characterized in that: In step S1, cystamine dihydrochloride and PEGDA are added to a round-bottom flask respectively and stirred, and triethylamine is added as a catalyst, and the mixture is reacted at 80° C. for 4 h, and then precipitated with glacial ether to obtain PBAE.

5. The use of the ROS-responsive hydrogel as a biomedical material according to claim 3, characterized in that: In step S2, HA is dissolved in ultrapure water, and methacrylic anhydride is added. The reaction is maintained for 16 hours, and the pH value is 8.

5. The entire reaction process is carried out on ice and protected from light. After the reaction, the solution is precipitated with acetone, washed with methanol, dialyzed for 3 days and freeze-dried to obtain the product HAMA.

6. The use of the ROS-responsive hydrogel as a biomedical material according to claim 3, characterized in that: In step S3, HAMA, PBAE and AM are dissolved in deionized water to form a gel precursor solution, a photoinitiator is added, and the AHP hydrogel is formed by physical blending under the action of ultraviolet light initiation.

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