Preparation method of ros-responsive hyperbranched polymer hydrogel and hydrogel

Abstract

The application provides a preparation method of ROS-responsive hyperbranched polymer hydrogel and the hydrogel, and belongs to the technical field of hydrogel. Firstly, Michael addition reaction occurs between double bonds on PEGDA and amine groups of chain-like aliphatic diamine with ROS scavenging groups in the structure, and a hyperbranched polymer PBAE is obtained as a crosslinking agent; then esterification reaction occurs between methyl methacrylate and HA to obtain HAMA; finally, HAMA, AM and PBAE solution are mixed, and AHP hydrogel is formed through a one-step free radical polymerization reaction. The preparation method of the AHP hydrogel is simple, the material source is wide, the production efficiency is high, the AHP hydrogel has good mechanical properties, oxidation resistance and biocompatibility, and is easy to produce and prepare on a large scale.

Description

Technical Field

[0001] This invention belongs to the field of hydrogel technology, and in particular relates to a method for preparing a ROS-responsive hyperbranched polymer hydrogel and the hydrogel itself. Background Technology

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

[0003] Hyaluronic acid (HA) is a naturally occurring polysaccharide found in human tissues. The hydroxyl groups in its structural side chains can undergo esterification with methacrylic anhydride to form methacryloyl hyaluronic acid (HAMA), which has good biocompatibility. The carbon-carbon double bonds in the HAMA structure can act as monomers to form hydrogels with various properties through chemical cross-linking with other components, thus enabling its application in multiple fields (Advanced Functional Materials 2020, 30, 2004709; BioactiveMaterials 2021, 6, 1689-1698).

[0004] However, the hydrogels formed by HAMA through free radical polymerization and cross-linking have weak mechanical and antioxidant properties. They are not only fragile, but also prone to collapse after drying and aging due to oxidation when exposed to air for a long time. Summary of the Invention

[0005] The problem to be solved by the present invention is to provide a method for preparing a ROS-responsive hyperbranched polymer hydrogel and the hydrogel itself.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for preparing ROS-responsive hyperbranched polymer hydrogels, comprising the following steps:

[0007] S1, the double bonds on polyethylene glycol diacrylate (PEGDA) and the amino groups of chain aliphatic diamines with ROS scavenging groups (such as TK bonds, disulfide bonds, and diselenide bonds) undergo Michael addition reaction to obtain hyperbranched polymers (PBAE), which are used as crosslinking agents.

[0008] S2. Methacrylamide hyaluronic acid (HAMA) is obtained by esterification reaction of methacrylic anhydride and hyaluronic acid (HA).

[0009] S3. Using HAMA as the main monomer and acrylamide (AM) as the monomer forming the main gel skeleton, HAMA, AM and PBAE solution are mixed and AHP hydrogel is formed in one step through free radical polymerization.

[0010] Further, in step S1, the molecular weight range of the PEGDA is 575-700.

[0011] Further, in step S1, the chain aliphatic diamine having a ROS scavenging group in its structure is cystamine dihydrochloride. It can be replaced by other chain aliphatic diamines with disulfide bonds, selenocystamine dihydrochloride, and other chain aliphatic diamines with diselenate bonds, or chain aliphatic diamines with a thioketal structure.

[0012] Further, in step S1, the molar ratio of the carbon-carbon double bond in the PEGDA to the amino active hydrogen in the cystamine dihydrochloride is (1.2-1.25):1.

[0013] Further, in step S1, cystamine dihydrochloride and PEGDA are added to a round-bottom flask, a magnetic stir bar is added, and triethylamine is added as a catalyst. The mixture is reacted at 60-100℃ for 4-10 hours. After that, the yellow oily substance, PBAE, is obtained by precipitation with ice-cold diethyl ether.

[0014] Further, in step S2, methacrylic anhydride is added to HA solution with a 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 h at a pH of 8-9. The entire reaction is carried out on ice in the dark. After the reaction is completed, the solution is precipitated with acetone, washed with methanol, dialyzed, and freeze-dried to obtain HAMA.

[0015] Further, in step S3, HAMA, PBAE, and AM are dissolved in deionized water to form a gel precursor solution. A photoinitiator is added, and carbon-carbon double bonds generate free radicals under the action of ultraviolet light through physical blending. The free radicals polymerize to form AHP hydrogel.

[0016] Further, 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%; and the photoinitiator is 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylphenylacetone, the content of which accounts for 0.1-0.2% of the gel precursor components.

[0017] Furthermore, in step S3, a thermal initiator can be used to generate free radicals from the carbon-carbon double bond, wherein the thermal initiator is ammonium persulfate.

[0018] Another object of the present invention is to provide an AHP hydrogel obtained by the above preparation method.

[0019] By adopting the above technical solution, the present invention has the following beneficial effects:

[0020] This invention solves the problem of AHP hydrogel preparation. It uses HAMA and AM as monomers, PBAE as crosslinking agent, and ultraviolet light and photoinitiator as initiation conditions to prepare AHP hydrogel. The carbon-carbon double bonds in the structures of HAMA, AM and PBAE undergo free radical polymerization to form AHP hydrogel.

[0021] This 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 were successfully enhanced by introducing AM monomer and hyperbranched polymer PBAE. AM enhances the tensile and compressive mechanical properties of the hydrogel, making the hydrogel structure more stable and facilitating its application in various fields; PBAE imparts ROS scavenging ability to the hydrogel, enabling it to exhibit excellent antioxidant effects. The prepared AHP hydrogel has good biocompatibility and does not exhibit cytotoxicity in L929 cell culture. The hydrogel demonstrates good antioxidant effects when the solid content of the hyperbranched polymer PBAE is 1%.

[0022] As can be seen, the preparation method of the AHP hydrogel of the present invention is simple, the materials are widely available, the production efficiency is high, and it has good mechanical properties, antioxidant properties and biocompatibility, and is easy to produce on a large scale. Attached Figure Description

[0023] The present invention will be described in detail below with reference to the accompanying drawings and examples. The advantages and implementation methods of the present invention will become more apparent from this description. The accompanying drawings are for illustrative purposes only and do not constitute any limitation on the present invention. In the accompanying drawings:

[0024] Figure 1 This is the proton NMR spectrum of the PBAE of this invention.

[0025] Figure 2 These are the proton NMR spectra of HAMA and HA of this invention.

[0026] Figure 3 This is a tensile stress-strain curve of hydrogels with different PBAE contents according to the present invention.

[0027] Figure 4 This is a compressive stress-strain curve of hydrogels with different PBAE contents according to the present invention.

[0028] Figure 5 This is a frequency-scan rheological characterization diagram of hydrogels with different PBAE contents according to the present invention.

[0029] Figure 6 This is a time-scan rheological characterization diagram of hydrogels with different PBAE contents according to the present invention.

[0030] Figure 7 This is a scanning electron microscope image of the AHP1 hydrogel of the present invention.

[0031] Figure 8 This is a cytotoxicity diagram of hydrogels with different PBAE contents according to the present invention.

[0032] Figure 9 This is a characterization diagram of the in vitro ABTS scavenging ability of hydrogels with different PBAE contents according to the present invention.

[0033] Figure 10 This is a characterization diagram of the intracellular ROS scavenging effect of hydrogels with different PBAE contents in this invention. Detailed Implementation

[0034] like Figures 1 to 10 As shown, the present invention discloses a method for preparing a ROS-responsive hyperbranched polymer hydrogel, comprising the following steps:

[0035] S1, the double bonds on polyethylene glycol diacrylate (PEGDA) and the amino groups of chain aliphatic diamines with ROS scavenging groups (such as TK bonds, disulfide bonds, and diselenide bonds) undergo Michael addition reaction to obtain hyperbranched polymers (PBAE), which are used as crosslinking agents.

[0036] S2. Methacrylamide hyaluronic acid (HAMA) is obtained by esterification reaction of methacrylic anhydride and hyaluronic acid (HA).

[0037] S3. Using HAMA as the main monomer and acrylamide (AM) as the monomer that forms the main skeleton of the gel to enhance the mechanical properties of the gel, HAMA, AM and PBAE solution are mixed and an antioxidant hydrogel (AHP) is formed in one step through free radical polymerization reaction.

[0038] In step S1, the molecular weight of PEGDA is 700; in this embodiment, PEGDA700 is used. Cystamine dihydrochloride provides disulfide bonds for ROS scavenging and can be replaced by other chain aliphatic diamines with disulfide bonds, selenocystamine dihydrochloride and other chain aliphatic diamines with diselenate bonds, or chain aliphatic diamines with a thioketal structure; in this embodiment, cystamine dihydrochloride is used. The molar ratio of the carbon-carbon double bond in polyethylene glycol diacrylate to the active amino hydrogen in cystamine dihydrochloride is 1.25:1.

[0039] In this embodiment, PEGDA700 and cystamine dihydrochloride were used to synthesize the hyperbranched polymer PBAE via 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 round-bottom flasks, a magnetic stir bar was added, and the stirring speed was 200 rpm; then 1 mL of triethylamine was added as a catalyst, and the reaction was carried out at 80 °C for 4 h. After that, the yellow oily substance was precipitated with ice-cold diethyl ether, which was PBAE.

[0040] like Figure 1 As shown, the obtained PBAE was detected by proton nuclear magnetic resonance spectroscopy. 1 (H NMR), in the figure, a, b, c, d, e, f, i, j, k, and m represent peaks at different positions in the PBAE, in the PBAE 1 In the 1H NMR results, a distinct peak appeared in the range of 5.9–6.4 ppm, which originated from the double bonds of the acrylate.

[0041] In step S2, the hyaluronic acid used in this embodiment has a molecular weight of 10w.

[0042] In this embodiment, 1g of hyaluronic acid (HA) was dissolved in 100mL of ultrapure water, and then 3.7mL of methacrylic anhydride was added. The reaction was maintained for 16h at a pH of 8.5. The entire reaction was carried out on ice in the dark. After the reaction was completed, the solution was precipitated with acetone, washed with methanol, dialyzed for 3 days (with a molecular weight cutoff of 7000Da), and then freeze-dried to obtain the product HAMA.

[0043] like Figure 2 As shown, HA and the resulting HAMA were detected by proton nuclear magnetic resonance spectroscopy. Compared with HA, HAMA showed improvement through... 1 ¹H NMR characterization revealed two new peaks at 5.7 ppm and 6.1 ppm, which were attributed to the C=C bonds of methacrylate, indicating the successful synthesis of HAMA.

[0044] 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 through physical blending under the action of ultraviolet light initiation.

[0045] 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-methylphenylacetone, which accounts for 0.1% of the gel precursor components.

[0046] Another object of the present invention is to provide an AHP hydrogel obtained by the above preparation method.

[0047] like Figure 3 As shown, hydrogels with different PBAE contents (AH, AHP) 0.1 AHP 0.5 Tensile stress-strain tests were conducted on AHP1 hydrogel. The test sample was a sheet with a thickness of 1 mm, a length of 4 cm, and a width of 1 cm. The tensile stress-strain curves showed that the tensile strain of the AHP1 hydrogel increased significantly from 169.2% to 410.2%, and the tensile stress increased from 23.55 kPa to 267.3 kPa. The microstructure of the AHP1 hydrogel is as follows: Figure 7 As shown (scale bar: 20μm).

[0048] like Figure 4 As shown, hydrogels with different PBAE contents (AH, AHP) 0.1 AHP 0.5 Compressive stress-strain tests were conducted on AHP1. The test sample was cylindrical, with a height of 5 mm and a diameter of 1 cm. The compressive stress-strain curves showed that at a compressive strain of 80%, AHP1 with a PBAE content of 0.5%... 0.5 The compressive stress of the hydrogel and the AHP1 hydrogel with 1% PBAE content are similar. 0.5 The compressive stress of the hydrogel is 1.22 MPa, and the compressive stress of the AHP1 hydrogel is 1.58 MPa. Both of them have a compressive stress that is about twice that of the hydrogel without PBAE.

[0049] like Figure 5 As shown, hydrogels with different PBAE contents (AH, AHP) 0.1 AHP 0.5 Frequency-scanning rheological tests were conducted on the hydrogels using AHP1. The test samples were discs with a diameter of 3 cm and a thickness of 1 mm. The frequency scan was performed at a temperature of 25 °C, with a fixed shear strain of 1%. The storage modulus G' and loss modulus G'' were measured within the frequency range of 0.01–10 Hz to investigate the structural stability of the hydrogels. The results showed that within the frequency range of 0.01–10 Hz, the G' of all hydrogels was greater than G'', indicating that the hydrogels could maintain a stable hydrogel state within this frequency range.

[0050] like Figure 6 As shown, hydrogels with different PBAE contents (AH, AHP) 0.1 AHP 0.5Time-scan rheological tests were performed on the hydrogels using AHP1. The test samples were discs with a diameter of 3 cm and a thickness of 1 mm. The test temperature was 25 °C, the shear strain was fixed at 1%, and the scanning time was 300 s. The storage modulus G' and loss modulus G'' were measured to investigate the structural stability of the hydrogels. The results showed that all hydrogels remained stable within 5 min, and G' was greater than the loss modulus G''.

[0051] like Figure 8 As shown, the CCK-8 method was used to test hydrogels with different PBAE contents (AH, AHP). 0.5 Cytotoxicity tests were performed on L929 mouse fibroblasts (5 × 10⁻⁶ cells) using AHP1. 3 Cells (100 μL / well) were seeded into 96-well plates and cultured for 24 h. The cells were then inoculated with PBS, AH, and AHP. 0.5 100 μL of AHP1 hydrogel extract was added and cultured for another 24 h. Then, the original culture medium was aspirated, and 10 μL of CCK-8 solution and 90 μL of 1640 medium were counted in each well. After culturing in the dark for 2 h, the absorbance of the final solution in the 96-well plate was measured at 450 nm using a full-wavelength plate reader (Thermo Scientific Varioskan Flash, USA). Finally, cell viability was calculated using the following formula:

[0052] Cell viability (%) = [(OD)] sample -OD blank ) / (OD PBS -OD blank )]×100%.

[0053] In the formula, OD sample The optical density value of the hydrogel; OD blank The optical density value of the blank group; OD PBS This represents the optical density value of PBS.

[0054] The results showed that the AHP1 hydrogel had the highest cell survival rate.

[0055] like Figure 9 As shown, the scavenging effect of ABTS radicals (2,2′-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) on hydrogels (AH, AHP) with different PBAE contents was investigated. 0.1 AHP 0.5Antioxidant capacity was tested using AHP1. First, a 2.84 g / L ABTS ethanol solution and a 0.664 g / L potassium persulfate solution were mixed in a 1:1 ratio and then incubated in the dark for 15 hours. For the hydrogel sample group, 100 μL of hydrogel and 100 μL of ABTS working solution were added to each well. For the control group, 100 μL of ultrapure water and 100 μL of ABTS working solution were added to each well. After incubation in the dark at room temperature for 30 minutes, the absorbance of the hydrogel sample (As) and ABTS solution (Ac) at 734 nm was measured using a microplate reader. The scavenging rate was calculated using the following formula:

[0056] Sweep rate (%) = (Ac-As) / Ac×100%.

[0057] The results showed that the AHP1 hydrogel had the best ABTS free radical scavenging rate, reaching 80%.

[0058] like Figure 10 As shown, the DCFH-DA probe was used to test the intracellular ROS clearance effect, and the intracellular ROS clearance was studied using laser confocal microscopy. The clearance effect was compared with PBS (negative control) and PBS+IR (positive control). First, L929 mouse fibroblasts (1.5 × 10⁻⁶ cells) were used... 5 Seeds (each seed per tray) were sown in confocal culture dishes along with 1.5 mL of 1640 complete medium. Then, the culture was prepared using culture media containing AH and AHP, respectively. 0.5 L929 mouse fibroblasts were cultured in 1640 complete medium with AHP1 hydrogel solution for 12 h. The cells were then washed three times with PBS. 1 mL of 1640 medium containing the DCFH-DA probe (5 μL) was added to each culture dish, and the cells were cultured at 37°C 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 washed three times with PBS and photographed using laser confocal microscopy.

[0059] Staining results of intracellular reactive oxygen species in L929 mouse fibroblasts after irradiation under different treatments showed that AHP1 hydrogel had the best intracellular ROS scavenging effect.

[0060] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope 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 ROS-responsive hyperbranched polymer hydrogels, characterized in that: Includes the following steps: The double bond on S1 and the amino group of the chain aliphatic diamine with ROS scavenging group in the structure undergo a Michael addition reaction to obtain the hyperbranched polymer PBAE, which is used as a crosslinking agent; wherein, the molecular weight of the PEGDA is in the range of 575-700; the chain aliphatic diamine with ROS scavenging group in the structure is cystamine dihydrochloride; the molar ratio of the carbon-carbon double bond in the PEGDA to the active hydrogen of the amino group in the cystamine dihydrochloride is (1.2-1.25):1; S2. HAMA is obtained by esterification reaction of methacrylic anhydride and HA. Methacrylic anhydride is added to HA solution with a 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 at a pH of 8-9. The entire reaction is carried out on ice in the dark. After the reaction, the solution is precipitated with acetone, washed with methanol, dialyzed, and freeze-dried to obtain HAMA. S3. HAMA, AM, and PBAE solutions are mixed and AHP hydrogel is formed in one step via free radical polymerization. Specifically, 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 UV light initiation, leading to free radical polymerization and the formation of AHP hydrogel. The solid content of HAMA in the gel precursor solution is 1-2%; the solid content of PBAE is 0.1-1%; and the solid content of AM is 10-25%. The photoinitiator is 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylphenylacetone, and its content accounts for 0.1-0.2% of the gel precursor components.

2. The method for preparing ROS-responsive hyperbranched polymer hydrogels according to claim 1, characterized in that: In step S1, cystamine dihydrochloride and PEGDA are added to a round-bottom flask, a magnetic stir bar is added, and triethylamine is added as a catalyst. The mixture is reacted at 60-100℃ for 4-10 hours. After that, the yellow oily substance PBAE is obtained by precipitation with ice-cold diethyl ether.

3. A hydrogel, characterized in that, The hydrogel is manufactured by the method for preparing ROS-responsive hyperbranched polymer hydrogels as described in claim 1 or 2.

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