Hydrogel based on ROS (reactive oxygen species) response type hyperbranched polymer and preparation method thereof

Abstract

The invention provides a preparation method of hydrogel based on ROS (reactive oxygen species) response type hyperbranched polymer and hydrogel, and belongs to the technical field of hydrogel, the preparation method comprises the following steps: firstly, carrying out Michael addition reaction on double bonds on PEGDA (polyethylene glycol diacrylate) and amido of chain-like aliphatic diamine with ROS scavenging groups in the structure to obtain a hyperbranched polymer PBAE as a cross-linking agent; then, methacrylic anhydride and HA are subjected to an esterification reaction to obtain HAMA, finally, HAMA, AM and a PBAE solution are mixed, and the AHP hydrogel is formed through a free radical polymerization reaction one-step method. The AHP hydrogel is simple in preparation method, wide in material source, high in production efficiency, good in mechanical property, oxidation resistance and biocompatibility and easy to produce and prepare on a large scale.

Description

Technical Field

[0001] The invention belongs to the technical field of hydrogels, and in particular relates to a preparation method of a ROS-responsive hyperbranched polymer hydrogel and the hydrogel. Background Art

[0002] Hydrogel is a type of extremely hydrophilic three-dimensional network structure gel that swells rapidly in water and can retain a large volume of water in this swollen state without dissolving.

[0003] Hyaluronic acid (HA) is a polysaccharide naturally present in human tissues. The hydroxyl groups in its side chains can undergo esterification reaction with methacrylic anhydride to form methacryloyl hyaluronic acid (HAMA) with good biosafety. The carbon-carbon double bonds in the HAMA structure can be used as monomers to form hydrogels with various properties through chemical cross-linking with other components, which can then be applied in a variety of fields (Advanced Functional Materials 2020, 30, 2004709; Bioactive Materials 2021, 6, 1689-1698).

[0004] However, the mechanical properties and antioxidant properties of the hydrogel formed by HAMA cross-linking alone through free radical polymerization are weak. It is not only easy to break, but also the hydrogel will collapse after drying and aging due to oxidation if exposed to the air for a long time. Summary of the invention

[0005] The problem to be solved by the present invention is to provide a preparation method and hydrogel based on ROS responsive hyperbranched polymer hydrogel.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is: a preparation method based on ROS-responsive hyperbranched polymer hydrogel, comprising the following steps: S1, the double bond on polyethylene glycol diacrylate (PEGDA) and the amine group of the chain fatty diamine with ROS scavenging groups (such as TK bond, disulfide bond, diselenide bond) in the structure undergo Michael addition reaction, and the obtained hyperbranched polymer (PBAE) is used as a crosslinker; 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.

[0007] Furthermore, in step S1, the molecular weight of the PEGDA is in the range of 575-700.

[0008] Further, in step S1, the chain fatty diamine having a ROS scavenging group in the structure is cystamine dihydrochloride, which can be replaced by other chain fatty diamines having a disulfide bond, selenocystamine dihydrochloride, other chain fatty diamines having a diselenide bond, and chain fatty diamines having a thioketal structure.

[0009] Furthermore, in step S1, the molar ratio of the carbon-carbon double bond in the PEGDA to the active hydrogen of the amino group in cystamine dihydrochloride is (1.2-1.25):1.

[0010] Further, in step S1, cystamine dihydrochloride and PEGDA are added to a round-bottom flask respectively, a magnetic stirrer is added, and triethylamine is added as a catalyst, and the mixture is reacted at 60-100° C. for 4-10 hours, and then precipitated with glacial ether to obtain a yellow oily substance, namely PBAE.

[0011] Further, in step S2, methacrylic anhydride is added to HA with a solution concentration of 10-20 mg / mL, and the molar ratio of HA to methacrylic anhydride is (0.16-0.2):1. The reaction is maintained for 14-18 hours, and the pH value is 8-9. The entire reaction process is carried out on ice and protected from light. After the reaction is completed, the reaction solution is precipitated with acetone, washed with methanol, and then dialyzed and freeze-dried to obtain 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 through physical blending, carbon-carbon double bonds generate free radicals under the action of ultraviolet light initiation, and the free radicals polymerize to form AHP hydrogel.

[0013] Furthermore, in step S3, the solid content of HAMA in the gel precursor solution is 1-2%; the solid content of PBAE is 0.1-1%; the solid content of AM is 10-25%; the photoinitiator is 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone, and the content of the photoinitiator accounts for 0.1-0.2% of the gel precursor components.

[0014] Furthermore, in step S3, a thermal initiator may be used to generate free radicals from carbon-carbon double bonds, and the thermal initiator is ammonium persulfate.

[0015] Another object of the present invention is to provide the AHP hydrogel obtained by the above preparation method.

[0016] Due to the adoption of the above technical solution, the present invention has the following beneficial effects: The invention solves the problem of preparing AHP hydrogel. HAMA and AM are used as monomers, PBAE is used as a crosslinking agent, and ultraviolet light and a photoinitiator are used as initiating conditions for preparation. The carbon-carbon double bonds in the structures of HAMA, AM and PBAE undergo free radical polymerization to form AHP hydrogel.

[0017] The present invention focuses on the mechanical properties (tensile and compressive properties), rheological behavior, ROS scavenging ability and biosafety of the prepared AHP hydrogel. The mechanical properties of the AHP hydrogel are successfully enhanced by introducing AM monomers and hyperbranched polymers PBAE. AM can enhance the tensile and compressive mechanical properties of the hydrogel, making the hydrogel structure more stable and easy to apply to various fields; PBAE gives the hydrogel ROS scavenging ability, so that the hydrogel exhibits excellent antioxidant effects; the prepared AHP hydrogel has good biocompatibility, and the hydrogel does not show cytotoxicity under L929 cell culture. When the solid content of the hyperbranched polymer PBAE is 1%, the hydrogel has a good antioxidant effect.

[0018] It can be seen that the preparation method of the AHP hydrogel of the present invention is simple, the material source is wide, the production efficiency is high, it has good mechanical properties, antioxidant properties and biocompatibility, and it is easy to prepare on a large scale. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 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 It is the hydrogen nuclear magnetic resonance spectrum of the PBAE of the present invention.

[0020] Figure 2 It is the hydrogen nuclear magnetic resonance spectrum of HAMA and HA of the present invention.

[0021] Figure 3 3 is a tensile stress-strain curve diagram of the hydrogels with different PBAE contents of the present invention.

[0022] Figure 4 3 is a compressive stress-strain curve diagram of the hydrogels with different PBAE contents of the present invention.

[0023] Figure 5 This is a frequency scanning rheological characterization diagram of the hydrogels with different PBAE contents of the present invention.

[0024] Figure 6 This is a time-scan rheological characterization diagram of the hydrogels with different PBAE contents of the present invention.

[0025] Figure 7It is a scanning electron microscope image of the AHP1 hydrogel of the present invention.

[0026] Figure 8 It is a diagram of the cytotoxicity of hydrogels with different PBAE contents of the present invention.

[0027] Fig. 9 This is a characterization diagram of the in vitro ABTS scavenging ability of the hydrogels with different PBAE contents of the present invention.

[0028] Fig.10 This is a characterization diagram of the intracellular ROS scavenging effect of hydrogels with different PBAE contents of the present invention. DETAILED DESCRIPTION

[0029] like Figures 1 to 10 As shown, the present invention provides a method for preparing a ROS-responsive hyperbranched polymer hydrogel, comprising the following steps: S1, the double bond on polyethylene glycol diacrylate (PEGDA) and the amine group of the chain fatty diamine with ROS scavenging groups (such as TK bond, disulfide bond, diselenide bond) in the structure undergo Michael addition reaction, and the obtained hyperbranched polymer (PBAE) is used as a crosslinker; S2, using methacrylic anhydride to react with hyaluronic acid (HA) to obtain methacryloyl hyaluronic acid (HAMA); S3. HAMA was used as the main monomer and acrylamide (AM) was used as the monomer to form the main skeleton of the gel to enhance the mechanical properties of the gel. HAMA, AM and PBAE solution were mixed to form an antioxidant hydrogel (AHP) in one step through a free radical polymerization reaction.

[0030] In step S1, the molecular weight of PEGDA is 700, and PEGDA 700 is used in this embodiment. Cystamine dihydrochloride provides a disulfide bond for scavenging ROS, which can be replaced by other chain fatty diamines with disulfide bonds, selenocystamine dihydrochloride and other chain fatty diamines with diselenide bonds, and chain fatty diamines with thioketal structures. Cystamine dihydrochloride is used in this embodiment. The molar ratio of carbon-carbon double bonds in polyethylene glycol diacrylate to active hydrogen of amino groups in cystamine dihydrochloride is 1.25:1.

[0031] In this embodiment, PEGDA700 and cystamine dihydrochloride were used to synthesize a 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) were added to a round-bottom flask, and a magnetic stirrer was added at a stirring speed of 200 rpm; 1 mL of triethylamine was added as a catalyst, and the mixture was reacted at 80°C for 4 hours, and then precipitated with glacial ether to obtain a yellow oily substance, which was PBAE.

[0032] like Figure 1 As shown, the obtained PBAE was subjected to nuclear magnetic resonance hydrogen spectrum detection ( 1 H NMR), in which a, b, c, d, e, f, i, j, k, and m represent peaks at different positions of PBAE. 1 In the H NMR results, an obvious peak appeared at 5.9-6.4 ppm, which was derived from the double bond of acrylate.

[0033] In step S2, the hyaluronic acid in this embodiment uses hyaluronic acid with a molecular weight of 10w.

[0034] In this embodiment, 1 g of hyaluronic acid (HA) was dissolved in 100 mL of ultrapure water, and then 3.7 mL of methacrylic anhydride was added. The reaction was maintained for 16 hours, and the pH value was 8.5. The entire reaction process was carried out on ice and protected from light. After the reaction, the solution was precipitated with acetone, washed with methanol, and then dialyzed for 3 days (molecular weight cutoff was 7000 Da) and freeze-dried to obtain the product HAMA.

[0035] like Figure 2 As shown, HA and the obtained HAMA were subjected to nuclear magnetic resonance hydrogen spectrum detection. Compared with HA, HAMA 1 H NMR characterization showed that two new peaks appeared at 5.7 ppm and 6.1 ppm. These two new peaks were caused by the C=C bonds of methacrylate, indicating the successful synthesis of HAMA.

[0036] In step S3, HAMA, PBAE and AM are dissolved in deionized water to form a gel precursor solution, a photoinitiator is added, and AHP hydrogel is formed by physical blending under the action of ultraviolet light.

[0037] In this embodiment, 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 components.

[0038] Another object of the present invention is to provide the AHP hydrogel obtained by the above preparation method.

[0039] like Figure 3 As shown, the hydrogels with different PBAE contents (AH, AHP 0.1 , AHP 0.5The tensile stress-strain curves show that the tensile strain of AHP1 hydrogel increased significantly from 169.2% to 410.2%, and the tensile stress increased from 23.55 kPa to 267.3 kPa. The microscopic morphology of AHP1 hydrogel is shown in Figure 1. Figure 7 As shown (scale bar: 20 μm).

[0040] like Figure 4 As shown, the hydrogels with different PBAE contents (AH, AHP 0.1 , AHP 0.5 , AHP1) was subjected to compressive stress-strain test. The test sample was cylindrical with a height of 5 mm and a diameter of 1 cm. The compressive stress-strain curve showed that when the compressive strain was 80%, AHP with a PBAE content of 0.5% 0.5 The compressive stress of the hydrogels was similar to that of the AHP1 hydrogels with 1% PBAE content. 0.5 The compressive stress of the hydrogel is 1.22MPa, and the compressive stress of the AHP1 hydrogel is 1.58MPa. The compressive stress of both is about twice that of the hydrogel without PBAE.

[0041] like Figure 5 As shown, the hydrogels with different PBAE contents (AH, AHP 0.1 , AHP 0.5 , AHP1) for frequency sweep rheological tests, the test sample is a disc with a diameter of 3cm and a thickness of 1mm, the test frequency sweep test temperature is 25℃, the fixed shear strain is 1%, and the storage modulus G' and loss modulus G'' values ​​under the conditions of frequency variation range of 0.01~10Hz are used to examine the structural stability of the hydrogel. The results show that within the frequency range of 0.01-10Hz, the G' of all hydrogels is greater than G'', which indicates that the above hydrogels can maintain a stable hydrogel state within this frequency range.

[0042] like Figure 6 As shown, the hydrogels with different PBAE contents (AH, AHP 0.1 , AHP 0.5 , AHP1) for time scanning rheology test, the test sample is a disc, its diameter is 3cm, thickness is 1mm, the test temperature is 25℃, the shear strain is fixed at 1%, the scanning time is 300s, the storage modulus G' and loss modulus G'' values ​​are measured, the purpose is to investigate the structural stability of the hydrogel. The results show that all hydrogels remain stable within 5 minutes, and G' is greater than the loss modulus G''.

[0043] like Figure 8As shown, the CCK-8 method was used to test the hydrogels with different PBAE contents (AH, AHP 0.5 , AHP1) were used for cytotoxicity test. L929 mouse fibroblasts (5×10 3 cells / well, 100 μL) were inoculated into a 96-well plate and cultured for 24 h before being washed with PBS, AH, and AHP. 0.5 , AHP1 hydrogel extract (100 μL) was cultured for another 24 h; then, the original culture medium was aspirated, 10 μL of CCK-8 solution and 90 μL of 1640 culture medium were counted in each well, and after culturing in the dark for 2 h, the absorbance of the final solution in the 96-well plate was measured at a wavelength of 450 nm using a full-wavelength plate reader (Thermo Scientific Varioskan Flash, USA); finally, the cell viability was calculated according to the following formula: Cell viability (%) = [(OD sample -OD blank ) / (OD PBS -OD blank )]×100%.

[0044] In the formula, OD sample OD is the optical density of the hydrogel; blank is the optical density value of the blank group; OD PBS is the optical density of PBS.

[0045] The results showed that AHP1 hydrogel had the highest cell survival rate.

[0046] like Fig. 9 As shown in the figure, the ABTS free radical (2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) scavenging experiment was performed on the hydrogels with different PBAE contents (AH, AHP 0.1 , AHP 0.5 , AHP1) for antioxidant capacity test. First, 2.84g / L ABTS ethanol solution and 0.664g / L potassium persulfate solution were mixed in a 1:1 ratio and then placed in the dark for 15h. For the hydrogel sample group, 100μL of hydrogel and 100μL of ABTS working solution were added to each well. For the control group, 100μL of ultrapure water and 100μL of ABTS working solution were added to each well. After incubation in the dark for 30min at room temperature, the absorbance of the hydrogel sample (As) and ABTS solution (Ac) at a wavelength of 734nm was measured using a microplate reader. The clearance rate was calculated as follows: Clearance rate (%) = (Ac-As) / Ac×100%.

[0047] The results showed that the ABTS free radical scavenging rate of AHP1 hydrogel could reach 80%, which was the best effect.

[0048] like Fig.10 As shown in the figure, the DCFH-DA probe was used to test the intracellular ROS clearance effect, and the intracellular ROS clearance was studied by laser confocal microscopy. At the same time, the intracellular ROS clearance was compared with PBS (negative control) and PBS + IR (positive control). First, L929 mouse fibroblasts (1.5 × 10 5 pcs / dish) were seeded in a confocal culture dish together with 1.5 mL of 1640 complete medium. 0.5 L929 mouse fibroblasts were cultured in 1640 complete medium with AHP1 hydrogel solution for 12 h. The cells were then rinsed 3 times with PBS. 1 mL of 1640 medium containing DCFH-DA probe (5 μL) was added to each dish, and the cells were cultured in a 37°C incubator for 20 min. The cells were then exposed to 6 Gy X-rays at a dose of 1 Gy / min for 6 min. Finally, the irradiated cells were rinsed 3 times with PBS and photographed using a laser confocal microscope.

[0049] The staining results of intracellular reactive oxygen species in L929 mouse fibroblasts treated with different methods after irradiation showed that AHP1 hydrogel had the best intracellular ROS scavenging effect.

[0050] 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. A method for preparing a ROS-responsive hyperbranched polymer hydrogel, characterized in that: The following steps are involved: S1, the double bond on PEGDA and the amine group of the chain fatty diamine with ROS scavenging group in the structure undergo Michael addition reaction, and the obtained hyperbranched polymer PBAE is used as a cross-linking agent; S2, using methacrylic anhydride and HA to undergo an esterification reaction to obtain HAMA; S3. Mix HAMA, AM and PBAE solution to form AHP hydrogel in one step through free radical polymerization.

2. The method for preparing a ROS-responsive hyperbranched polymer hydrogel according to claim 1, characterized in that: In step S1, the molecular weight of the PEGDA is in the range of 575-700.

3. The method for preparing a ROS-responsive hyperbranched polymer hydrogel according to claim 1, characterized in that: In step S1, the chain fatty diamine having a ROS scavenging group in its structure is cystamine dihydrochloride.

4. The method for preparing a ROS-responsive hyperbranched polymer hydrogel according to claim 3, characterized in that: In step S1, the molar ratio of the carbon-carbon double bond in the PEGDA to the active hydrogen of the amino group in cystamine dihydrochloride is (1.2-1.25):

1.

5. The method for preparing a ROS-responsive hyperbranched polymer hydrogel according to claim 4, characterized in that: In step S1, cystamine dihydrochloride and PEGDA are added to a round-bottom flask respectively, a magnetic stirrer is added, and triethylamine is added as a catalyst, and the mixture is reacted at 60-100° C. for 4-10 h, and then precipitated with glacial ether to obtain a yellow oily substance PBAE.

6. The method for preparing a ROS-responsive hyperbranched polymer hydrogel according to claim 1, characterized in that: In step S2, methacrylic anhydride is added to HA with a solution concentration of 10-20 mg / mL, and the molar ratio of HA to methacrylic anhydride is (0.16-0.2):

1. The reaction is maintained for 14-18 hours, and the pH value is 8-9. The entire reaction process is carried out on ice and protected from light. After the reaction is completed, the reaction solution is precipitated with acetone, washed with methanol, and then dialyzed and freeze-dried to obtain HAMA.

7. The method for preparing a ROS-responsive hyperbranched polymer hydrogel according to claim 1, characterized in that: In step S3, HAMA, PBAE, and AM are dissolved in deionized water to form a gel precursor solution, and a photoinitiator is added. Through physical blending, carbon-carbon double bonds generate free radicals under the action of ultraviolet light initiation, and the free radicals polymerize to form AHP hydrogel.

8. The method for preparing a ROS-responsive hyperbranched polymer hydrogel according to claim 7, characterized in that: In step S3, the solid content of HAMA in the gel precursor solution is 1-2%; the solid content of PBAE is 0.1-1%; the solid content of AM is 10-25%; the photoinitiator is 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone, and the content of the photoinitiator accounts for 0.1-0.2% of the gel precursor components.

9. A hydrogel, characterized in that: The hydrogel is manufactured by the preparation method of ROS-responsive hyperbranched polymer hydrogel according to any one of claims 1 to 8.

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