Double-responsiveness hydrogel as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biomedical materials, in particular to double-responsiveness hydrogel as well as a preparation method and application thereof. The invention provides a dual-responsiveness injectable hydrogel. The dual-responsiveness injectable hydrogel comprises oxidized hyaluronic acid (OHA) and hyaluronic acid-3-aminophenylboronic acid (HA-PBA), the OHA and the HA-PBA are connected through a Schiff base bond; hA and PBA in the HA-PBA are connected through a boric acid ester bond. The hydrogel has ROS (reactive oxygen species) and pH (potential of hydrogen) double responsiveness due to existence of borate bonds and Schiff base bonds, and is suitable for being used as a carrier of a rheumatoid arthritis drug delivery system; hyaluronic acid is a main component of synovial fluid and is beneficial to relieving pain of a patient suffering from rheumatoid arthritis; the hydrogel is simple in used raw materials and simple and convenient in preparation process, and is expected to be applied to local drug delivery of rheumatoid arthritis and other pathological microenvironment diseases with high active oxygen or low pH.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a dual-responsive hydrogel and a preparation method and application thereof. Background Art

[0002] Rheumatoid arthritis (RA) is one of the most common and devastating autoimmune diseases, characterized by synovial inflammation and joint damage. Currently, the main routes of administration for RA treatment include oral administration (e.g., nonsteroidal anti-inflammatory drugs, methotrexate) and intravenous / subcutaneous injection (e.g., tocilizumab, methotrexate). These two routes of administration present numerous challenges. First, oral and intravenous administration result in systemic drug distribution, potentially causing systemic adverse reactions such as gastrointestinal discomfort and liver and kidney dysfunction. Second, oral or intravenous administration lacks targeting, resulting in insufficient drug accumulation in diseased joints, necessitating higher doses to achieve therapeutic efficacy, further exacerbating side effects. Third, oral administration presents challenges with first-pass effects and unstable absorption, while intravenous administration faces the challenges of rapid metabolism and clearance. Fourth, oral and intravenous administration require frequent re-dosing. Fifth, traditional drug delivery methods cannot regulate drug release rates according to the severity of joint inflammation. Studies have found that reactive oxygen species (ROS) levels in the RA microenvironment are higher than those in normal joints, and the pH of synovial tissue (approximately 6.0, sometimes even below 5.0) is lower than that in normal joints. Therefore, based on the particularity of the RA microenvironment, it is reasonable to develop an injectable hydrogel for intra-articular injection that can respond to ROS and pH stimulation to achieve on-demand drug release and provide lubrication to reduce joint wear to treat rheumatoid arthritis. This hydrogel can also be used for local drug delivery of diseases with high reactive oxygen species or low pH pathological microenvironment. Summary of the Invention

[0003] In view of this, the object of the present invention is to provide a dual-responsive injectable hydrogel and a preparation method and application thereof, wherein the dual-responsive injectable hydrogel has dual responsiveness to ROS and pH.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a dual-responsive injectable hydrogel comprising oxidized hyaluronic acid (OHA) and hyaluronic acid-3-aminophenylboronic acid (HA-PBA);

[0006] The oxidized hyaluronic acid and hyaluronic acid-3-aminophenylboronic acid interact with each other via a Schiff base bond;

[0007] In the hyaluronic acid-3-aminophenylboronic acid, hyaluronic acid and 3-aminophenylboronic acid are connected via a borate ester bond.

[0008] Preferably, the molar ratio of hyaluronic acid to 3-aminophenylboronic acid in the hyaluronic acid-3-aminophenylboronic acid is (1-1.5): (1-1.5);

[0009] The mass volume concentration of oxidized hyaluronic acid in the dual-responsive injectable hydrogel is 30% to 7.5%;

[0010] The mass volume concentration of hyaluronic acid-3-aminophenylboronic acid in the dual-responsive injectable hydrogel is 60% to 5%.

[0011] The present invention also provides a method for preparing the dual-responsive injectable hydrogel described in the above technical solution, comprising the following steps:

[0012] mixing the first hyaluronic acid solution and 3-aminophenylboronic acid to obtain HA-PBA;

[0013] Mixing the NaIO4 solution with the second hyaluronic acid solution and performing a light-protected reaction to obtain oxidized hyaluronic acid;

[0014] The oxidized hyaluronic acid, HA-PBA and a solvent are mixed to obtain the dual-responsive injectable hydrogel.

[0015] Preferably, the molar ratio of hyaluronic acid to 3-aminophenylboronic acid in the first hyaluronic acid solution is (1-1.5):(1-1.5).

[0016] Preferably, the mixing of the first hyaluronic acid solution and 3-aminophenylboronic acid is carried out under stirring;

[0017] The stirring time is 15 to 30 hours, and the stirring speed is 500 to 1000 rpm.

[0018] Preferably, the molar ratio of NaIO4 in the NaIO4 solution to hyaluronic acid in the second hyaluronic acid solution is (1-2): (1-2).

[0019] Preferably, the temperature of the light-proof reaction is 20° C. to 25° C., and the time is 12 to 36 hours.

[0020] Preferably, after the light-shielding reaction is completed, ethylene glycol is added to the product system obtained by the light-shielding reaction, followed by dialysis purification and freeze-drying;

[0021] The usage ratio of hyaluronic acid to ethylene glycol in the second hyaluronic acid solution is (4.0-12.0) g: (5-15) mL.

[0022] Preferably, the mass volume concentration of oxidized hyaluronic acid in the dual-responsive injectable hydrogel is 30% to 7.5%;

[0023] The mass volume concentration of HA-PBA in the dual-responsive injectable hydrogel is 60% to 5%.

[0024] The present invention also provides the use of the dual-responsive injectable hydrogel described in the above technical solution or the dual-responsive injectable hydrogel prepared by the preparation method described in the above technical solution in the preparation of drugs for rheumatoid arthritis and diseases with high reactive oxygen species or low pH pathological microenvironment.

[0025] The present invention provides a dual-responsive injectable hydrogel comprising oxidized hyaluronic acid (OHA) and hyaluronic acid-3-aminophenylboronic acid (HA-PBA); the OHA and HA-PBA are linked by a Schiff base bond; and the HA and PBA in the HA-PBA are linked by a boronate ester bond. Due to the presence of boronate and Schiff base bonds, the hydrogel exhibits dual responsiveness to ROS and pH, making it suitable as a carrier for a drug delivery system for rheumatoid arthritis. Hyaluronic acid, a major component of synovial fluid, helps alleviate pain in patients with rheumatoid arthritis. Finally, the mechanical properties of the dual-responsive injectable hydrogel can be adjusted based on the ratio of HA-PBA to oxidized hyaluronic acid.

[0026] The present invention also provides a method for preparing the dual-responsive injectable hydrogel described in the above technical solution, comprising the following steps: mixing a first hyaluronic acid solution and 3-aminophenylboronic acid to obtain HA-PBA; mixing a NaIO4 solution and a second hyaluronic acid solution, performing a light-protected reaction, to obtain oxidized hyaluronic acid; and mixing the oxidized hyaluronic acid, HA-PBA, and a solvent to obtain the dual-responsive injectable hydrogel. This preparation process is simple, using simple raw materials, and is expected to be applicable for localized drug delivery in rheumatoid arthritis and other diseases characterized by high reactive oxygen species or low pH microenvironments. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 FTIR spectra of HA, OHA, 3-APBA, HA-PBA and OHA-PBA-HA described in Example 1;

[0028] Figure 2 This is the acid responsiveness graph of OHA-PBA-HA described in Example 1;

[0029] Figure 3 This is the in vitro release profile of Rhodamine B from OHA-PBA-HA described in Example 1;

[0030] Figure 4 The diagrams show the effect of OHA-PBA-HA on FLS cell viability described in Example 1 (a diagram showing cytotoxicity test results, a diagram showing quantitative analysis of live and dead cell staining, and a diagram showing live and dead cell staining);

[0031] Figure 5 The graph and quantitative analysis of the effect of OHA-PBA-HA on ROS in FLS cells observed under an inverted fluorescence microscope as described in Example 1;

[0032] Figure 6 This is a graph showing the effect of OHA-PBA-HA on ROS in FLS cells detected by flow cytometry as described in Example 1 and a quantitative analysis graph;

[0033] Figure 7 is a curve showing the change of aldehyde content in the oxidized hyaluronic acid described in Examples 1 to 3;

[0034] Figure 8 Figure 2 is a diagram of the OHA-PBA-HA hydrogel described in Example 2 and Example 3;

[0035] Figure 9 This is the in vitro release graph of Rhodamine B from the OHA-PBA-HA hydrogel described in Example 2;

[0036] Figure 10 This is the in vitro release graph of Rhodamine B from the OHA-PBA-HA hydrogel described in Example 3. DETAILED DESCRIPTION

[0037] The present invention provides a dual-responsive injectable hydrogel comprising oxidized hyaluronic acid (OHA) and hyaluronic acid-3-aminophenylboronic acid (HA-PBA);

[0038] The OHA and HA-PBA interact with each other via Schiff base bonds;

[0039] The hyaluronic acid and 3-aminophenylboronic acid in the HA-PBA are connected via a boronate ester bond.

[0040] In the present invention, the molar ratio of hyaluronic acid to 3-aminophenylboronic acid in the HA-PBA is preferably (1-1.5):(1-1.5), more preferably (1.1-1.4):(1.1-1.4). In an embodiment of the present invention, the molar ratio of hyaluronic acid to 3-aminophenylboronic acid in the HA-PBA can be 1:1.5.

[0041] In the present invention, the mass volume concentration of oxidized hyaluronic acid in the dual-responsive injectable hydrogel is preferably 30% to 7.5%, more preferably 30%, 25%, 20%, 15%, 10%, or 7.5%. In an embodiment of the present invention, the mass volume concentration of oxidized hyaluronic acid in the dual-responsive injectable hydrogel can be 7.5%. In the present invention, the mass volume concentration is expressed in g, and the volume is expressed in mL.

[0042] In the present invention, the mass volume concentration of HA-PBA in the dual-responsive injectable hydrogel is preferably 60% to 5%, more preferably 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5%. In an embodiment of the present invention, the mass volume concentration of HA-PBA in the dual-responsive injectable hydrogel can be 60%. In the present invention, the unit of mass in the mass volume concentration is g, and the unit of volume is mL.

[0043] The present invention also provides a method for preparing the dual-responsive injectable hydrogel described in the above technical solution, comprising the following steps:

[0044] mixing the first hyaluronic acid solution and 3-aminophenylboronic acid to obtain HA-PBA;

[0045] Mixing the NaIO4 solution with the second hyaluronic acid solution and performing a light-protected reaction to obtain oxidized hyaluronic acid;

[0046] The oxidized hyaluronic acid, HA-PBA and a solvent are mixed to obtain the dual-responsive injectable hydrogel.

[0047] In the present invention, the preparation process of the dual-responsive injectable hydrogel is shown in Formula 1:

[0048]

[0049] In the present invention, unless otherwise specified, all preparation raw materials are commercially available products well known to those skilled in the art.

[0050] The present invention mixes the first hyaluronic acid solution and 3-aminophenylboronic acid to obtain HA-PBA.

[0051] In the present invention, the concentration of hyaluronic acid in the first hyaluronic acid solution is preferably 0.05 mol / L to 0.20 mol / L, more preferably 0.05 mol / L, 0.06 mol / L, 0.07 mol / L, 0.08 mol / L, 0.09 mol / L, 0.1 mol / L, 0.11 mol / L, 0.12 mol / L, 0.13 mol / L, 0.14 mol / L, 0.15 mol / L, 0.16 mol / L, 0.17 mol / L, 0.18 mol / L, 0.19 mol / L or 0.20 mol / L. In an embodiment of the present invention, the concentration of hyaluronic acid in the first hyaluronic acid solution can be 0.08 mol / L.

[0052] In the present invention, the solvent in the first hyaluronic acid solution is preferably a buffer solution with a pH of 8.5 to 9.6. In embodiments of the present invention, the solvent in the first hyaluronic acid solution can be a buffer solution with a pH of 8.5. The present invention does not have any particular limitations on the type of the buffer solution, and any type familiar to those skilled in the art can be used. The present invention does not have any particular limitations on the preparation of the first hyaluronic acid solution, and any process familiar to those skilled in the art can be used.

[0053] In the present invention, the molar ratio of hyaluronic acid to 3-aminophenylboronic acid in the first hyaluronic acid solution is preferably (1-1.5):(1-1.5), more preferably (1.1-1.4):(1.1-1.4). In an embodiment of the present invention, the molar ratio of hyaluronic acid to 3-aminophenylboronic acid in the first hyaluronic acid solution can be 1:1.5.

[0054] In the present invention, the mixing is preferably carried out under stirring conditions, the stirring speed is preferably 500-1000 rpm, more preferably 500 rpm, 600 rpm, 700 rpm, 800 rpm, 900 rpm or 1000 rpm; the stirring time is preferably 15-30 hours, more preferably 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours or 30 hours. In an embodiment of the present invention, the stirring speed can be 600 rpm, and the stirring time can be 30 hours, 15 hours or 24 hours.

[0055] After the mixing is completed, the present invention further preferably includes drying. In the present invention, the drying is preferably freeze drying. The present invention has no particular limitation on the freeze drying process, and the freeze drying process can be carried out using a process well known to those skilled in the art.

[0056] In the present invention, after the first hyaluronic acid solution and 3-aminophenylboronic acid are mixed, the vicinal diol in the hyaluronic acid forms a borate ester bond with the boronic acid group in the 3-aminophenylboronic acid to obtain 3-aminophenylboronic acid modified hyaluronic acid (ie, HA-PBA).

[0057] The preparation method of the present invention further comprises mixing the NaIO4 solution and the second hyaluronic acid solution, and performing a light-proof reaction to obtain oxidized hyaluronic acid.

[0058] In the present invention, the concentration of NaIO4 in the NaIO4 solution is preferably 0.1mol / L to 1.0mol / L, more preferably 0.1mol / L, 0.2mol / L, 0.3mol / L, 0.4mol / L, 0.5mol / L, 0.6mol / L, 0.7mol / L, 0.8mol / L, 0.9mol / L or 1.0mol / L. In an embodiment of the present invention, the concentration of NaIO4 in the NaIO4 solution may be 0.7mol / L. In the present invention, the solvent in the NaIO4 solution is preferably water.

[0059] In the present invention, the concentration of hyaluronic acid in the second hyaluronic acid solution is preferably 0.05mol / L to 0.15mol / L, more preferably 0.05mol / L, 0.06mol / L, 0.07mol / L, 0.08mol / L, 0.09mol / L, 0.10mol / L, 0.11mol / L, 0.12mol / L, 0.13mol / L, 0.14mol / L or 0.15mol / L. In an embodiment of the present invention, the concentration of hyaluronic acid in the second hyaluronic acid solution may be 0.05mol / L. In the present invention, the solvent in the second hyaluronic acid solution is preferably a buffer solution with a pH of 8.5 to 9.6; the present invention does not have any special restrictions on the type of the buffer solution, and any type familiar to those skilled in the art can be used. The present invention does not have any special restrictions on the preparation of the second hyaluronic acid solution, and any process familiar to those skilled in the art can be used. The present invention does not have any special limitation on the preparation process of the second hyaluronic acid solution. The hyaluronic acid can be fully dissolved in the buffer solution with a pH of 8.5 by using a mixing process well known to those skilled in the art.

[0060] In the present invention, the molar ratio of NaIO4 in the NaIO4 solution to the hyaluronic acid in the second hyaluronic acid solution is preferably (1-2):(1-2), more preferably (1-1.5):(1-1.5). In an embodiment of the present invention, the molar ratio of NaIO4 in the NaIO4 solution to the hyaluronic acid in the second hyaluronic acid solution can be 1:2.

[0061] The present invention does not have any special limitation on the mixing process of the NaIO4 solution and the second hyaluronic acid solution, and the mixing process can be carried out using a process well known to those skilled in the art.

[0062] In the present invention, the temperature of the light-shielding reaction is preferably 20°C to 25°C, more preferably 20°C, 21°C, 22°C, 23°C, 24°C or 25°C; the time is preferably 12 to 36 hours, more preferably 12 hours, 15 hours, 18 hours, 21 hours, 24 hours, 27 hours, 30 hours, 33 hours or 36 hours. In an embodiment of the present invention, the temperature of the light-shielding reaction can be 25°C, and the time can be 12 hours or 24 hours.

[0063] After the light-shielding reaction is completed, the present invention preferably further comprises adding ethylene glycol to the product system obtained by the light-shielding reaction, followed by dialysis purification and freeze-drying.

[0064] In the present invention, the ratio of hyaluronic acid to ethylene glycol in the second hyaluronic acid solution is preferably (4.0-12.0) g: (5-15) mL, more preferably (4.0-8.0) g: (5-10) mL. In an embodiment of the present invention, the ratio of hyaluronic acid to ethylene glycol in the second hyaluronic acid solution can be 4.0 g: 5 mL.

[0065] In the present invention, the role of adding the ethylene glycol is to remove excess NaIO4.

[0066] After adding the ethylene glycol, the present invention also preferably terminates the oxidation. The terminating oxidation is preferably carried out under stirring. The stirring speed is preferably 400-800 rpm, more preferably 400 rpm, 500 rpm, 600 rpm, 700 rpm, or 800 rpm. The stirring time is preferably 30-60 minutes, more preferably 30 minutes, 40 minutes, 50 minutes, or 60 minutes. In an embodiment of the present invention, the stirring speed can be 800 rpm and the stirring time can be 30 minutes.

[0067] In the present invention, the dialysis purification is preferably carried out in ultrapure water. The present invention does not have any special limitation on the process of the dialysis purification, and the process well known to those skilled in the art can be used.

[0068] The present invention does not have any special limitation on the freeze-drying process, and the freeze-drying process may be carried out using a process well known to those skilled in the art.

[0069] In the present invention, during the process of preparing oxidized hyaluronic acid, the NaIO4 oxidizes the hydroxyl groups of the hyaluronic acid to obtain aldehyde-modified hyaluronic acid; the NaIO4 is light-sensitive, so it needs to be used under light-proof conditions to ensure the accuracy and stability of the reaction; the hydroxyl groups in the ethylene glycol can react with the NaIO4 to terminate the hyaluronic acid oxidation reaction.

[0070] After obtaining the oxidized hyaluronic acid and HA-PBA, the present invention mixes the oxidized hyaluronic acid, HA-PBA and a solvent to obtain the dual-responsive injectable hydrogel.

[0071] In the present invention, the solvent is preferably a buffer solution with a pH of 8.5 to 9.6.

[0072] In the present invention, the mixing of the oxidized hyaluronic acid, HA-PBA, and solvent preferably includes mixing the oxidized hyaluronic acid with a portion of the solvent to obtain an oxidized hyaluronic acid solution; mixing the HA-PBA with the remaining solvent to obtain an HA-PBA solution; and mixing the oxidized hyaluronic acid solution with the HA-PBA solution. In the present invention, the mass volume concentration of the oxidized hyaluronic acid in the oxidized hyaluronic acid solution is 30% to 7.5%, more preferably 30%, 25%, 20%, 15%, 10%, or 7.5%. In embodiments of the present invention, the mass volume concentration of the oxidized hyaluronic acid in the oxidized hyaluronic acid solution may be 7.5%. In the present invention, the mass volume concentration of the HA-PBA in the HA-PBA solution is preferably 60% to 5%, more preferably 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%. In embodiments of the present invention, the mass volume concentration of the HA-PBA in the HA-PBA solution may be 60%. In the present invention, the unit of mass in the mass volume concentration is g, and the unit of volume is mL.

[0073] In the present invention, the mass volume concentration ratio of the oxidized hyaluronic acid and HA-PBA in the dual-responsive injectable hydrogel is preferably (30% to 7.5%): (60% to 5%), more preferably 30%:5%, 30%:10%, 30%:15%, 30%:20%, 30%:40%, 30%:60%, 15%:5%, 15%:10%, 15%:15%, 15%:20%, 15%:40%, 15%:60%, 7.5%:5%, 7.5%:10%, 7.5%:15%, 7.5%:20%, 7.5%:40% or 7.5%:60%. In an embodiment of the present invention, the mass volume concentration ratio of the oxidized hyaluronic acid and HA-PBA can be 7.5%:60%.

[0074] The present invention also provides the use of the dual-responsive injectable hydrogel described in the above technical solution or the dual-responsive injectable hydrogel prepared by the preparation method described in the above technical solution in the preparation of an anti-rheumatoid arthritis drug. The present invention does not impose any particular limitations on the method of application, and methods well known to those skilled in the art can be used.

[0075] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0076] Example 1

[0077] 1.5 g of hyaluronic acid (HA, molar weight 0.004 mol) was dissolved in 50 mL of a buffer solution having a pH value of 8.5 to obtain a hyaluronic acid solution; 0.8 g of 3-aminophenylboronic acid (3-APBA, 0.006 mol) was added to the hyaluronic acid solution, stirred for 30 h (at a speed of 600 rpm), and freeze-dried to obtain HA-PBA (yield 75%);

[0078] 4.0 g of hyaluronic acid was added to 200 mL of a pH 8.5 buffer solution and stirred until completely dissolved to obtain a hyaluronic acid solution; 4.5 g of NaIO4 was dissolved in 30 mL of water to obtain a NaIO4 solution; the NaIO4 solution was added to the hyaluronic acid solution and oxidized in the dark for 24 h (at a speed of 800 rpm); 5 mL of ethylene glycol was added and stirred for 30 min to terminate the oxidation; the resulting reaction mixture was dialyzed against ultrapure water and freeze-dried to obtain oxidized hyaluronic acid (OHA, with a yield of 94.28%);

[0079] 30 mg of the oxidized hyaluronic acid was dissolved in 400 μL of a buffer solution having a pH of 8.5 to obtain an oxidized hyaluronic acid solution; 30 mg of the HA-PBA was dissolved in 50 μL of a buffer solution having a pH of 8.5 to obtain an HA-PBA solution; the oxidized hyaluronic acid solution and the HA-PBA solution were mixed to obtain the dual-responsive injectable hydrogel (OHA-PBA-HA).

[0080] Fourier transform infrared spectroscopy: HA, OHA, 3-APBA, HA-PBA and OHA-PBA-HA described in Example 1 were mixed and ground evenly with a small amount of KBr, and pressed into thin slices. The samples were analyzed by Fourier transform infrared spectroscopy at a wavelength of 500-4000 cm -1 Scan within the range; Figure 1 The Fourier transform infrared spectra of HA, OHA, 3-APBA, HA-PBA and OHA-PBA-HA described in Example 1 are shown in FIG. Figure 1 It can be seen that the infrared spectrum of OHA is different from that of HA at 1730 cm -1 The stretching vibration absorption peak of aldehyde group appeared at 1380cm -1The stretching vibration absorption peak of BO appears at 706 cm -1 The out-of-plane deformation vibration absorption peak of the meta-phenyl ring appeared at 1730 cm, indicating that the vicinal diol of HA formed a borate ester bond with the boronic acid group of 3-APBA, and HA-PBA was successfully prepared. The infrared spectrum of OHA-PBA-HA was compared with that of OHA. -1 The absorption peak of the aldehyde group disappeared, and the absorption peak at 1645 cm -1 The stretching vibration absorption peak of the imine bond (C=N) appeared at 1380 cm -1 The stretching vibration absorption peak of BO appeared at , indicating that OHA-PBA-HA was successfully prepared.

[0081] Acid responsiveness of the dual-responsive injectable hydrogel described in Example 1: 1.5 mL of the dual-responsive injectable hydrogel (OHA-PBA-HA described in Example 1) was prepared in a beaker. 1 mol / L HCL and 1 mol / L NaOH were added, respectively. The changes in sol and gel were observed by the inverted bottle method as the pH value of the system was changed. Since the imine bond will hydrolyze and break under acidic conditions, the hydrogel may swell rapidly or even dissolve when the pH of the system is changed. The results are shown in Figure 2. Figure 2 As shown, after adding 1 mol / L hydrochloric acid, OHA-PBA-HA changes from a gel state to a sol state, and after adding 1 mol / L sodium hydroxide, OHA-PBA-HA returns to a gel state.

[0082] In vitro release of rhodamine B loaded by the dual-responsive injectable hydrogel (OHA-PBA-HA) described in Example 1: Rhodamine B was selected as a model drug. OHA-PBA-HA loaded with rhodamine B was prepared at the bottom of a glass bottle, and then equal amounts of pH = 6.0 phosphate buffer + 1mM H2O2, pH = 7.4 phosphate buffer + 1mM H2O2, pH = 6.0 phosphate buffer, and pH = 7.4 phosphate buffer were added to each bottle, and the dissolution of the hydrogel over time and the release of rhodamine B were observed and recorded. At regular intervals, the absorbance curve was recorded using a UV spectrophotometer. The Rhodamine B in PBS 7.4 was established. 2 = 0.99930(Abs(554nm) = 0.0387×[Rhodamine B](μg·mL -1 )-0.0905) and R in PBS 6.0 2 = 0.99901(Abs(554nm) = 0.0375×[Rhodamine B](μg·mL -1 )-0.0235) was used as a calibration curve to determine the release amount of rhodamine B in OHA-PBA-HA. Figure 3Figure A (A is the release test of Rhodamine B in different environments) is the release test diagram of OHA-PBA-HA loaded with Rhodamine B in (1) pH = 6.0 phosphate buffer + 1mM H2O2, (2) pH = 7.4 phosphate buffer + 1mM H2O2, (3) pH = 6.0 phosphate buffer, and (4) pH = 7.4 phosphate buffer. It can be seen that a large amount of Rhodamine B in (1) enters into PBS by diffusion, followed by (2) and (3), and a small part diffuses into PBS in (4). Figure 3 B (B represents the release rate of rhodamine B in different environments) represents the release rate of rhodamine B from the rhodamine B-loaded OHA-PBA-HA in the above experiment. It can be seen that before 10 hours, the release rate of rhodamine B was nearly identical under various environmental conditions. This is attributed to the fact that the amount of rhodamine B released under the influence of pH and H₂O₂ was not sufficient to significantly alter the release rate. After 72 hours, the release rate of rhodamine B in pH = 6.0 phosphate buffer + 1 mM H₂O₂ was 93.42 ± 5.61%, indicating that rhodamine B was essentially completely released. The release rates of rhodamine B in pH = 7.4 phosphate buffer + 1 mM H₂O₂ and pH = 6.0 phosphate buffer were 74.26 ± 4.84% and 67.53 ± 7.38%, respectively, both exceeding the release rate of 52.14 ± 2.22% in pH = 7.4 phosphate buffer. The above experimental results show that the drug release efficiency is higher in acidic and H2O2 environments, and the drug release rate of hydrogel-loaded rhodamine B decreases with increasing pH value and decreasing H2O2;

[0083] Biocompatibility study:

[0084] Cytotoxicity assay: FLS cells were cultured at a rate of 1.0 × 10 4 The cells were seeded at a density of 10 cells / well in a 96-well plate and incubated at 37°C, 5% CO2 for 24 hours. After the FLS cells were treated with hydrogel for 24 hours, 10 μL of CCK-8 reagent was added and the cells were incubated for another 30 minutes. The absorbance (A450nm) was detected using a microplate reader at a wavelength of 450 nm. Figure 4 A in the figure is the result of cytotoxicity experiment. Figure 4 As shown in Figure A, OHA-PBA-HA allowed more than 80% of FLS cells to survive after 24 h, showing negligible cytotoxicity.

[0085] Live and dead cell staining experiment: FLS cells were cultured in 24-well plates for 24 h (6 × 10 4 cells / well), and then incubated for 24 hours with complete medium and OHA-PBA-HA described in Example 1. They were stained with AM / PI and observed under a fluorescence microscope. Figure 4 B in the figure is the quantitative analysis of live and dead cell staining. Figure 4C in the figure is the live-dead cell staining of FLS by OHA-PBA-HA. Figure 4 As shown in Figures B to C, FLS grew well on the surface of OHA-PBA-HA, most living cells were stained green by Calcein-AM, and only a few dead cells were stained red by PI, indicating that the cytotoxicity of OHA-PBA-HA was low.

[0086] ROS scavenging experiment: An inverted fluorescence microscope was used to observe the effects of different drug treatments on the ROS levels in FLS cells. The specific operation was as follows: FLS cells were plated at 6×10 4 The cells were seeded at a density of 10 cells / well in a 24-well plate and cultured overnight. The original culture medium was removed, and complete culture medium was added to the blank group. The experimental group and the control group were added with an equal amount of Rosup (final concentration of 50 μg / mL) to stimulate the cells and incubated for 4 hours. The whole culture medium was aspirated, and the cells were gently rinsed twice with PBS. The experimental group was added with 0.5 mL of complete culture medium containing OHA-PBA-HA described in Example 1, and the culture was continued for 12 hours. The old culture medium was removed and rinsed with PBS. Serum-free culture medium containing DCFH-DA (2,7-dichlorodihydrofluorescein diacetate) was added and incubated in a cell culture incubator at 37°C in the dark for 30 minutes. After rinsing with PBS, an appropriate amount of PBS was added to each well, and the effects of different drug treatments on the ROS level in FLS cells were observed using an inverted fluorescence microscope. The fluorescence intensity was quantified using ImageJ software. The results are shown in the figure. Figure 5 As shown in Figure A (A is the result of the effect of OHA-PBA-HA on intracellular ROS under an inverted fluorescence microscope), the green fluorescence signal in the OHA-PBA-HA treatment group decreased significantly compared with the control group. This shows that OHA-PBA-HA reduces intracellular ROS and has certain antioxidant properties. Figure 5 The statistical results of fluorescence quantification in Figure B (B is the quantitative analysis diagram) also verified this phenomenon.

[0087] To further study the ROS scavenging effect in cells, flow cytometry was used for further determination. FLS cells were cultured at 1x10 6 Cells were seeded at a density of 100 cells / well in a 6-well plate. After the same treatment method as above, serum-free culture medium was added for washing and centrifugation. The cells were then resuspended in serum-free culture medium. Finally, the cell suspension was transferred to a flow cytometer and the FITC channel of the flow cytometer was selected to detect the intracellular DCF fluorescence signal. Figure 6 A in the figure (A is the result of flow cytometry detection of the effect of OHA-PBA-HA on intracellular ROS) shows that compared with the control group, OHA-PBA-HA has the ability to clear ROS; Figure 6As shown in Figure B (B is a quantitative analysis graph), OHA-PBA-HA can eliminate the abnormally elevated ROS in FLS cells. This result is consistent with the ROS results detected by inverted fluorescence microscopy.

[0088] Example 2

[0089] 1.5 g of hyaluronic acid (HA, molar weight 0.004 mol) was dissolved in 50 mL of a buffer solution having a pH value of 8.5 to obtain a hyaluronic acid solution; 0.8 g of 3-aminophenylboronic acid (3-APBA, 0.006 mol) was added to the hyaluronic acid solution, stirred for 15 h (at a speed of 600 rpm), and freeze-dried to obtain HA-PBA (yield 75%);

[0090] 4.0 g of hyaluronic acid was added to 200 mL of a pH 8.5 buffer solution and stirred until completely dissolved to obtain a hyaluronic acid solution; 2.3 g of NaIO4 was dissolved in 30 mL of water to obtain a NaIO4 solution; the NaIO4 solution was added to the hyaluronic acid solution and oxidized in the dark for 24 h (at a speed of 800 rpm); 5 mL of ethylene glycol was added and stirred for 30 min to terminate the oxidation; the resulting reaction mixture was dialyzed against ultrapure water and freeze-dried to obtain oxidized hyaluronic acid (OHA, with a yield of 47.44%);

[0091] 30 mg of the oxidized hyaluronic acid was dissolved in 400 μL of a buffer solution having a pH of 8.5 to obtain an oxidized hyaluronic acid solution; 30 mg of the HA-PBA was dissolved in 75 μL of a buffer solution having a pH of 8.5 to obtain an HA-PBA solution; the oxidized hyaluronic acid solution and the HA-PBA solution were mixed to obtain the dual-responsive injectable hydrogel (OHA-PBA-HA).

[0092] The hydrogel (OHA-PBA-HA) formed in Example 2 is shown in FIG. Figure 8 From A (A is the hydrogel formed as described in Example 2), it can be seen that a hydrogel can be formed when the mass volume concentration of OHA is 7.5% and the mass volume concentration of HA-PBA is 40%.

[0093] In vitro release of rhodamine B from the dual-responsive injectable hydrogel (OHA-PBA-HA) described in Example 2: Rhodamine B was selected as a model drug. Rhodamine B-loaded OHA-PBA-HA was prepared at the bottom of a glass vial. Equal amounts of pH 6.0 phosphate buffer + 1 mM H₂O₂, pH 7.4 phosphate buffer + 1 mM H₂O₂, pH 6.0 phosphate buffer, and pH 7.4 phosphate buffer were then added to each vial, and the release of rhodamine B was recorded. The absorbance curves were recorded at regular intervals using a UV spectrophotometer. Figure 9The release rate of rhodamine B from OHA-PBA-HA loaded with rhodamine B in the above experiment. It can be seen that the release rate of rhodamine B is the fastest under the conditions of pH 6.0+H2O2. After 72 hours, the release rate of rhodamine B in pH = 6.0 phosphate buffer + 1mM H2O2 is 95.35±0.89%, indicating that rhodamine B is basically completely released; the release rates of rhodamine B in pH = 7.4 phosphate buffer + 1mM H2O2 and pH = 6.0 phosphate buffer are 78.40±2.06% and 70.43±1.24%, respectively, which are both greater than the release rate of rhodamine B in pH = 7.4 phosphate buffer of 53.90±1.15%. The above experimental results show that the release efficiency of drugs is higher in acidic environments and H2O2 environments, and the drug release rate of rhodamine B loaded in hydrogels decreases with increasing pH value and decreasing H2O2.

[0094] Example 3

[0095] 1.5 g of hyaluronic acid (HA, molar weight 0.004 mol) was dissolved in 50 mL of a buffer solution having a pH value of 8.5 to obtain a hyaluronic acid solution; 0.8 g of 3-aminophenylboronic acid (3-APBA, 0.006 mol) was added to the hyaluronic acid solution, stirred for 24 h (at a speed of 600 rpm), and freeze-dried to obtain HA-PBA (yield 75%);

[0096] 4.0 g of hyaluronic acid was added to 200 mL of a pH 8.5 buffer solution and stirred until completely dissolved to obtain a hyaluronic acid solution; 1.1 g of NaIO4 was dissolved in 30 mL of water to obtain a NaIO4 solution; the NaIO4 solution was added to the hyaluronic acid solution and oxidized in the dark for 24 h (at a speed of 800 rpm); 5 mL of ethylene glycol was added and stirred for 30 min to terminate the oxidation; the resulting reaction mixture was dialyzed against ultrapure water for 2 days and freeze-dried to obtain oxidized hyaluronic acid (OHA, with a yield of 48.8%);

[0097] 30 mg of the oxidized hyaluronic acid was dissolved in 400 μL of a buffer solution having a pH of 8.5 to obtain an oxidized hyaluronic acid solution; 30 mg of the HA-PBA was dissolved in 150 μL of a buffer solution having a pH of 8.5 to obtain an HA-PBA solution; the oxidized hyaluronic acid solution and the HA-PBA solution were mixed to obtain the dual-responsive injectable hydrogel (OHA-PBA-HA).

[0098] The hydrogel (OHA-PBA-HA) formed in Example 3 is shown in FIG. Figure 8 From B (B is the hydrogel formed as described in Example 3), it can be seen that a hydrogel can be formed when the mass volume concentration of OHA is 7.5% and the mass volume concentration of HA-PBA is 20%.

[0099] In vitro release of rhodamine B from the dual-responsive injectable hydrogel (OHA-PBA-HA) described in Example 3: Rhodamine B was selected as a model drug. Rhodamine B-loaded OHA-PBA-HA was prepared at the bottom of a glass vial. Equal amounts of pH 6.0 phosphate buffer + 1 mM H₂O₂, pH 7.4 phosphate buffer + 1 mM H₂O₂, pH 6.0 phosphate buffer, and pH 7.4 phosphate buffer were then added to each vial, and the release of rhodamine B was recorded. The absorbance curves were recorded at regular intervals using a UV spectrophotometer. Figure 10 The figure shows the release rate of rhodamine B from OHA-PBA-HA loaded with rhodamine B in the aforementioned experiment. After 72 hours, the release rate of rhodamine B in pH 6.0 phosphate buffer + 1 mM H2O2 was 96.61 ± 0.74%, indicating nearly complete release of rhodamine B. The release rates in pH 7.4 phosphate buffer + 1 mM H2O2 and pH 6.0 phosphate buffer were 83.82 ± 1.98% and 74.30 ± 1.20%, respectively, both exceeding the 58.74 ± 3.14% release rate in pH 7.4 phosphate buffer. These experimental results demonstrate that drug release efficiency is higher in acidic environments and H2O2 environments, and that the release rate of rhodamine B from hydrogels decreases with increasing pH and decreasing H2O2.

[0100] The hydroxylamine hydrochloride method was used to detect the aldehyde content in the oxidized hyaluronic acid described in Examples 1 to 3, wherein Figure 7 is the variation curve of the aldehyde content in the oxidized hyaluronic acid described in Examples 1 to 3, wherein Figure 7 wherein nHA:NaIO4=1:2 corresponds to Example 1; nHA:NaIO4=1:1 corresponds to Example 2; nHA:NaIO4=2:1 corresponds to Example 3. Figure 7 It can be seen that with the increase of the amount of NaIO4, the aldehyde content shows a significant increasing trend, indicating that under unchanged environmental conditions, the oxidation degree of the oxidation product (oxidized hyaluronic acid) can be controlled by controlling the amount of NaIO4.

[0101] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation thereto. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications shall also be considered within the scope of protection of the present invention.

Claims

1. A dual-responsive injectable hydrogel, characterized in that: including oxidized hyaluronic acid and hyaluronic acid-3-aminophenylboronic acid; The oxidized hyaluronic acid and hyaluronic acid-3-aminophenylboronic acid interact with each other via a Schiff base bond; In the hyaluronic acid-3-aminophenylboronic acid, hyaluronic acid and 3-aminophenylboronic acid are connected via a borate ester bond.

2. The dual-responsive injectable hydrogel according to claim 1, wherein The molar ratio of hyaluronic acid to 3-aminophenylboronic acid in the hyaluronic acid-3-aminophenylboronic acid is (1-1.5): (1-1.5); The mass volume concentration of oxidized hyaluronic acid in the dual-responsive injectable hydrogel is 7.5-30%; The mass volume ratio of hyaluronic acid-3-aminophenylboronic acid in the dual-responsive injectable hydrogel is 60% to 5%.

3. The method for preparing the dual-responsive injectable hydrogel according to claim 1 or 2, characterized in that: The following steps are involved: mixing the first hyaluronic acid solution and 3-aminophenylboronic acid to obtain HA-PBA; Mixing the NaIO4 solution with the second hyaluronic acid solution and performing a light-protected reaction to obtain oxidized hyaluronic acid; The oxidized hyaluronic acid, HA-PBA and a solvent are mixed to obtain the dual-responsive injectable hydrogel.

4. The preparation method according to claim 3, wherein The molar ratio of hyaluronic acid to 3-aminophenylboronic acid in the first hyaluronic acid solution is (1-1.5):(1-1.5).

5. The preparation method according to claim 3, wherein The mixing of the first hyaluronic acid solution and 3-aminophenylboronic acid is carried out under stirring; The stirring time is 15 to 30 hours, and the stirring speed is 500 to 1000 rpm.

6. The preparation method according to claim 3, wherein The molar ratio of NaIO4 in the NaIO4 solution to hyaluronic acid in the second hyaluronic acid solution is (1-2): (1-2).

7. The preparation method according to claim 3, wherein The temperature of the light-proof reaction is 20° C. to 25° C., and the time is 12 to 36 hours.

8. The preparation method according to claim 3, 6 or 7, characterized in that: After the light-shielding reaction is completed, ethylene glycol is added to the product system obtained by the light-shielding reaction, followed by dialysis purification and freeze-drying; The usage ratio of hyaluronic acid to ethylene glycol in the second hyaluronic acid solution is (4.0-12.0) g: (5-15) mL.

9. The preparation method according to claim 3, wherein The mass volume concentration of oxidized hyaluronic acid in the dual-responsive injectable hydrogel is 30% to 7.5%; The mass volume concentration of HA-PBA in the dual-responsive injectable hydrogel is 60% to 5%.

10. Use of the dual-responsive injectable hydrogel according to claim 1 or 2 or the dual-responsive injectable hydrogel prepared by the preparation method according to any one of claims 3 to 9 in the preparation of drugs for rheumatoid arthritis and diseases with high reactive oxygen species or low pH pathological microenvironment.

Images