A dual-responsive hydrogel and its preparation method and application

Abstract

The present invention belongs to the technical field of hydrogel preparations, and specifically relates to a dual-responsive hydrogel, a preparation method, and an application thereof. The present invention synthesizes a cross-linked polycationic hydrogel composed of phenylboronic acid-modified sodium alginate, a protocatechuic acid-polyethyleneimine graft polymer, and bevacizumab sustained-release nanoparticles, referred to as a dual-responsive OSPPB hydrogel. This hydrogel blocks neurovascularization at the osteochondral interface by locally clearing extracellular RNA and releasing bevacizumab, thereby alleviating OA pain and disease progression.

Description

Technical Field

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

[0002] Osteoarthritis (OA) is the most common musculoskeletal disease with a high morbidity and often leads to pain, disability, and functional impairment. Pharmacological therapy, physical therapy, and exercise therapy are widely used to treat OA. Pharmacological therapy is typically used as an initial therapeutic intervention, aiming to rapidly relieve acute symptoms. The latter two modalities play an adjunctive and ongoing role in the overall treatment strategy for OA. Pharmacological treatments, including nonsteroidal anti-inflammatory drugs (NSAIDs), paracetamol, opioids, and corticosteroids, focus on suppressing inflammation and alleviating pain, but do not address the pathological changes that ultimately necessitate joint replacement surgery. Currently, the most prominent pathological change associated with joint pain is neurovascularization at the osteochondral interface. However, treatments and analgesic strategies specifically targeting this neurovascularization have been underutilized. Numerous nerves and blood vessels emerge from the subchondral bone, penetrate the osteochondral interface, and invade the avascular cartilage through vertical microcracks. This neurovascularization delivers inflammatory mediators to the cartilage, accelerating matrix degradation and mineralization, leading to the formation of condylar osteophytes and exacerbating OA-related pain. It also promotes mechanical and chemical osteoclast-chondrocyte interactions, leading to abnormal subchondral bone remodeling and pain. Therefore, neurovascularization of the osteochondral interface is a potential target for osteoarthritis intervention.

[0003] Currently, multiple cytokines, such as tumor necrosis factor α and interleukin-1β, as well as growth factors, including nerve growth factor, vascular endothelial growth factor (VEGF), and platelet-derived growth factor BB, have been found to promote neurovascularization in OA. Furthermore, previous studies have demonstrated that extracellular RNA (exRNA), as an organic polyanion recruitment factor, can bind to and enhance the function of polycationic neurovascular factors in OA. Further studies have confirmed that intraarticular injection of nucleic acids can induce arthritis through angiogenesis, and that controlled release of RNA can promote angiogenesis and osteogenesis. Similarly, a key role for exRNA in neurovascularization has been identified in cardiovascular disease, where it can regulate VEGF function. Based on these findings, a strategy to deplete exRNA may be superior to simply inhibiting neurovascular factors. To date, exRNA depletion strategies have been widely used to treat diseases such as periodontitis, inflammatory bowel disease, obesity, severe sepsis, psoriasis, acute kidney injury, and rheumatoid arthritis. Although this strategy has demonstrated efficacy in various diseases, achieving responsive release of polycationic agents to mitigate toxicity has been difficult. Responsive hydrogels can achieve environmentally triggered drug release and have been widely studied in the treatment of OA. They are expected to achieve responsive release of polycationic drugs and reduce toxicity. The substantial changes in the osteochondral microenvironment in OA are considered to be ideal conditions for achieving controlled drug delivery.

[0004] The temporomandibular joint, abbreviated as TMJ, is particularly prone to osteoarthritis. The incidence rate in people under 30 years old is 14.56%, and it increases to 28-32% in people aged 30-80 years old. Previous studies have confirmed that temporomandibular joint arthritis, abbreviated as TMJOA ， Pathological neurovascularization at the condylar bone-cartilage interface in patients is closely related to maxillofacial pain, but existing hydrogels have failed to effectively inhibit neurovascularization. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a dual-responsive hydrogel and applications thereof.

[0006] A dual-responsive hydrogel, wherein the dual-responsive hydrogel is made of phenylboronic acid-modified sodium alginate, protocatechuic acid-polyethyleneimine graft polymer, and bevacizumab sustained-release nanoparticles;

[0007] The mass ratio of phenylboronic acid modified sodium alginate, protocatechuic acid-polyethyleneimine graft polymer and bevacizumab sustained-release nanoparticles is 80-120:80-120:0.8-1.2.

[0008] Preferably, the method comprises the following steps:

[0009] Grafting 3-aminophenylboronic acid onto sodium alginate to obtain an intermediate product, and oxidizing the intermediate product with sodium periodate to obtain the phenylboronic acid-modified sodium alginate; the mass ratio of the 3-aminophenylboronic acid to the sodium alginate is 0.3-0.8:0.8-1.2;

[0010] The protocatechuic acid-polyethyleneimine graft polymer is synthesized by amidation reaction of polyethyleneimine and protocatechuic acid; the mass ratio of the polyethyleneimine to the protocatechuic acid is 10-15:0.8-1.2;

[0011] The bioactive glass nanoparticles and bevacizumab are reacted in an ice bath for 22 hours to 26 hours to obtain the bevacizumab sustained-release nanoparticles; the mass ratio of the bioactive glass nanoparticles to the bevacizumab is 15 mg to 25 mg: 45 μg to 55 μg;

[0012] Bevacizumab sustained-release nanoparticles were dissolved in PBS to obtain solution A, phenylboronic acid-modified sodium alginate was dissolved in solution A to obtain solution B, protocatechuic acid-polyethyleneimine grafted polymer was dissolved in solution A to obtain solution C, and solution B and solution C were mixed to obtain the dual-responsive hydrogel.

[0013] Preferably, the mass ratio of the 3-aminophenylboronic acid to the sodium alginate is 0.5:1; the mass ratio of the polyethyleneimine to the protocatechuic acid is 14.6:1; and the mass ratio of the bioactive glass nanoparticles to the bevacizumab is 20 mg:50 μg.

[0014] Preferably, the mass ratio of the 3-aminophenylboronic acid to the sodium alginate is 0.8:0.8; the mass ratio of the polyethyleneimine to the protocatechuic acid is 15:0.8; and the mass ratio of the bioactive glass nanoparticles to the bevacizumab is 15 mg:55 μg.

[0015] Preferably, the mass ratio of the 3-aminophenylboronic acid to the sodium alginate is 0.3:1.2; the mass ratio of the polyethyleneimine to the protocatechuic acid is 10:1.2; and the mass ratio of the bioactive glass nanoparticles to the bevacizumab is 15 mg:45 μg.

[0016] Preferably, the mass ratio of the 3-aminophenylboronic acid to the sodium alginate is 0.3:0.8; the mass ratio of the polyethyleneimine to the protocatechuic acid is 15:.2; and the mass ratio of the bioactive glass nanoparticles to the bevacizumab is 25 mg:55 μg.

[0017] Preferably, when 3-aminophenylboronic acid is grafted onto sodium alginate, the reaction time of 3-aminophenylboronic acid and sodium alginate is 23 hours to 25 hours.

[0018] Preferably, when polyethyleneimine and protocatechuic acid undergo amidation reaction, the activated protocatechuic acid solution is mixed with the polyethyleneimine solution, stirred for 23 to 25 hours in the dark, dialyzed with a molecular weight cut-off of 1800 Da, and freeze-dried to obtain a protocatechuic acid-polyethyleneimine graft polymer.

[0019] Preferably, when preparing the bevacizumab sustained-release nanoparticles, the specific reaction is: stirring for 23 hours to 25 hours, centrifuging to obtain a precipitate, then washing with PBS, and freeze-drying to obtain BGN@Be powder.

[0020] Preferably, the bioactive glass nanoparticles include SiO2, CaO and P2O5, and the molar percentage of SiO2, CaO and P2O5 is 80:16:4.

[0021] Application of dual-responsive hydrogels in the preparation of drugs for the treatment of osteoarthritis,

[0022] Preferably, the drug inhibits TMJOA.

[0023] Preferably, the medicament alleviates pathological neurovascularization.

[0024] The preparation process of OSPPB hydrogel is as follows Figure 1 As shown in Figure a. To construct an OSPPB hydrogel, the present invention crosslinks phenylboronic acid-modified sodium alginate with the main chain of a protocatechuic acid-polyethyleneimine graft polymer through the formation of dynamic boronate and Schiff base bonds. Bevacizumab sustained-release nanoparticles are then added through coordination. Boronic acid groups of the phenylboronic acid-modified sodium alginate are crosslinked with catechol groups of the protocatechuic acid-polyethyleneimine graft polymer to establish boronate bonds. Simultaneously, amino groups of the protocatechuic acid-polyethyleneimine graft polymer are crosslinked with aldehyde groups of the phenylboronic acid-modified sodium alginate to form Schiff base bonds.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] In order to inhibit TMJOA and alleviate pathological neurovascularization, the present invention constructed OSPPB hydrogel.

[0027] OSPPB hydrogels have the following advantages: (1) polycationic OSPPB hydrogels can scavenge polyanionic exRNA; (2) their pH / ROS dual responsiveness enables on-demand release of active ingredients; and (3) sequential delivery of active ingredients promotes structural recovery. Subsequently, their inhibitory function on neurons and vascular endothelial cells was evaluated in vitro, and their effects on condylar neurovascularization and joint pain were studied in a mouse model of unilateral anterior crossbite TMJOA. The OSPPB hydrogels of the present invention scavenge exRNA, inhibit neurovascularization, and treat TMJOA. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Preparation and characterization of OSPPB hydrogel, a is the simple preparation route of OSPPB hydrogel, b shows the gelation, injectability and self-healing properties of OSPPB hydrogel, where 1 shows gelation, 2 shows injectability, and 3-6 show self-healing properties, c is the ATR-FTIR of PPCA, OSAP, OSPP and OSPPB, showing that Schiff base bonds and boron ester bonds lead to gelation, d is the scanning electron microscopy of OSPP and OSPPB, showing that BGN@Be is uniformly distributed on the surface of OSPPB hydrogel, and the red circles represent BGN, the scale bar in the first column = 200 μm, and the scale bar in the second column = 5 μm, e is the SEM-EDS of OSPPB hydrogel, showing the Ca, P and Si elements on the OSPPB hydrogel, f is the frequency scanning sequence of OSPP and OSPPB hydrogels, g is the time scanning sequence of OSPP and OSPPB hydrogels, and h is the shear thinning test of OSPP and OSPPB hydrogels.

[0029] Figure 2 FTIR results of SA, SAP and OSAP, showing the synthesis of OSAP polymer.

[0030] Figure 3 FTIR results of PEI, PCA and PPCA, showing the synthesis of PPCA polymer.

[0031] Figure 4 Characterization diagrams of BGN and BGN@Be, where a is the TEM image of BGN, BGN@Be and BGN@Be after 21 days of degradation, scale bar = 100 nm, b is the size distribution of BGN and BGN@Be, c is the X-ray diffraction pattern of BGN and BGN@Be, d is the release of Si after PBS immersion, and e is the release of bevacizumab after PBS immersion.

[0032] Figure 5Figure 5. OSPPB hydrogels with dual pH / ROS responsiveness, degradation, antioxidant properties, and positive charge. (a) Image of OSPPB hydrogels after incubation with PBS and / or H2O2 at pH 5. (b) Frequency sweep analysis. (c) SEM image. Scale bar = 200 μm. (d) Statistical analysis of SEM images. (e) Degradation of OSPP and OSPPB hydrogels in PBS. (f) Changes in S concentration within 14 days. (g) Photos and statistical results after incubation of OSPPB extracts with DPPH at different concentrations for 30 min. (h) Zeta potential of OSPP and OSPPB hydrogels. (i) Plate incubation experiment of OSPPB hydrogels binding to RNA. (j) Statistical results of plate incubation experiment. (k) Schematic diagram of plate incubation experiment showing the binding of OSPPB to RNA. Scale bar = 50 μm. (l) Molecular dynamics simulation and quantification of RNA (light blue), VEGF (red), and PEI (purple). Data are presented as mean and standard deviation. (n = 6)

[0033] Figure 6 These are pictures and statistical results of the hemolysis experiment after OSPPB treatment.

[0034] Figure 7 We show that an injectable, self-healing, pH / ROS dual-responsive, polycationic hydrogel reverses TMJOA and alleviates chronic pain by inhibiting neurovascularization at the osteochondral interface.

[0035] Figure 8 Figure 3: OSPPB hydrogel inhibits angiogenesis and neuritogenesis. a is a representative microscopic image of EPCs after 24 hours of treatment, b is a representative microscopic image of TG cells after 24 hours of treatment, c is the statistical results of the number of EPCs migrating cells, d is the statistical results of the migration distance of EPCs, e is the statistical results of TG synaptic bifurcation, and f is the statistical results of synapse length. Scale bar = 100 μm. Data are expressed as mean and standard deviation, n = 6.

[0036] Figure 9Figure 3: OSPPB hydrogel alleviates TMJOA in mice. a is a schematic diagram of the in vivo experimental design. b is a top view of the condyle three weeks after intra-articular injection. Scale bar = 200 μm. c is a representative image of hematoxylin-eosin staining, safranin O-fast green staining, glycine silver staining, SEM, and micro-CT. Arrows in the SEM image indicate microcracks. Scale bars 1-3 in c = 100 μm, 4 in c = 10 μm, and 5 in c = 500 μm. d is semi-quantitative OARSI. e is the statistical result of cartilage thickness. f is the statistical result of proteoglycan. g is the statistical result of blood vessel number. h is the statistical result of nerve number. i is the statistical result of microcracks. j is the statistical result of BV / TV. k is the statistical result of Tb.Th. l is the qRT-PCR analysis of neurovascular factor gene expression in the condyle. Data are expressed as mean and standard deviation. n = 6.

[0037] Figure 10 Figure 3 shows that OSPPB hydrogel improves the changes in the osteochondral interface caused by TMJOA. a is the result of atomic force microscopy analysis of the TMJ osteochondral interface, where (1) is a schematic diagram of atomic force microscopy, and (2) is the atomic force microscopy results of CON, Veh, Celecoxib, and OSPPB. b is the result of scanning electron microscopy-energy dispersive spectroscopy analysis of the TMJ osteochondral interface, where (1) is the elemental analysis diagram of CON, Veh, Celecoxib, and OSPPB, and (2) is the elemental statistical diagram of CON, Veh, Celecoxib, and OSPPB.

[0038] Figure 11 It shows that celecoxib and OSPPB hydrogel exhibit significant analgesic function. a is the representative trajectory of the elevated cross test of mice three weeks after intra-articular injection, b is the representative trajectory of the open field test of mice three weeks after intra-articular injection, c is the statistical analysis of the number of times mice entered the open arm in the elevated cross test, d is the statistical analysis of the time spent in the open arm, e is the statistical analysis of the total number of times entered the arm, f is the statistical analysis of the total distance traveled by mice in the open field test, g is the statistical analysis of the activity time, h is the statistical analysis of the central time, i is the result of the von Frey test of mice, showing the pain relief, j is the EEG spectrum of mice before and after brush stimulation, and the data are expressed as mean and standard deviation, n=6.

[0039] Figure 12 OSPPB hydrogel effectively reduces neurovascularization at the osteochondral interface. a is a representative image of exRNA distribution at the osteochondral interface of the mouse condyle three weeks after different treatments. Scale bar in the first column = 50 μm, scale bar in the second, third, and fourth columns = 10 μm. b is anterograde tracing of the trigeminal ganglion, PGP9.5 + cells, CGRP + cells, CD31+ Cells and VEGF + Representative images of cell immunofluorescence staining, scale bar = 30 μm, c is COX2 + cells, DCC + cells, SP + Representative images of cell distribution, scale bar = 30 μm, d is the statistical analysis of exRNA, f is the statistical analysis of anterograde tracer EGFP, g is the statistical analysis of CGRP, h is the statistical analysis of CD31, i is the statistical analysis of COX2, j is the statistical analysis of DCC, k is the statistical analysis of SP, data are expressed as mean and standard deviation, n = 6. DETAILED DESCRIPTION

[0040] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0041] Table 1 Reagent information used in the present invention

[0042]

[0043] Table 2 Instrument information used in the present invention

[0044] instrument Model, manufacturer Field emission scanning electron microscopy FE-SEM, S-4800, Hitachi, Tokyo, Japan Energy dispersive X-ray spectroscopy Element EDS Systems, Ametek, PA, USA ATR-FTIR FTIR-8400S, Shimadzu, Tokyo, Japan Infrared Solution Software Shimadzu, Kyoto, Japan Rheometer Kinexus Lab+, Bayern, Germany Litesize 500 Particle Analyzer Anton Paar, Graz, Austria CLSM Nikon A1R, Nikon Corporation, Minato-ku, Tokyo, Japan

[0045] Example 1

[0046] Preparation of OSAP polymer, PPCA polymer and BGN@Be

[0047] Phenylboronic acid modified sodium alginate is abbreviated as OSAP. Preparation of OSAP

[0048] Dissolve 1 gram of sodium alginate in 100 mL of deionized water. Once completely dissolved, add 0.985 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl). After 15 minutes, add 0.49 g of 3-aminophenylboronic acid. Stir the mixture for 24 hours and then dialyze against water at room temperature for 7 days to remove unreacted substances. The dialysis water is replaced every 8 hours at a molecular weight cutoff of 3500 Da. After lyophilization, the intermediate product, SAP, is obtained. Next, dissolve 1 g of SAP in 100 mL of deionized water for 2–3 hours to obtain a SAP solution. Disperse 210 mg of NaIO4 in 5 mL of deionized water, and then add the SAP solution dropwise in the dark. After two and a half hours, add 1 mL of ethylene glycol and react for 1 hour to neutralize any unreacted NaIO4, yielding OSAP. Both SAP and OSAP are stored at 4°C until further use. SAP and OSAP were characterized by ATR-FTIR.

[0049] Protocatechuic acid-polyethyleneimine graft polymer is abbreviated as PPCA. Preparation of PPCA

[0050] 4.5 g of polyethyleneimine was dissolved in 20 mL of MES buffer to obtain a polyethyleneimine solution. 308.24 mg of protocatechuic acid and 1.54 g of EDC·HCl were dissolved in 80 mL of MES buffer for 0.5 h. Then, 920.72 mg of N-hydroxysuccinimide was added and stirred for 12 h. The carboxyl groups were activated in the dark. This solution was then added to the polyethyleneimine solution and stirred in the dark at room temperature for 24 h. The mixture was then dialyzed for 7 days with a molecular weight cutoff of 1800 Da, with the dialysis water replaced every 8 h. The dialyzate was lyophilized to obtain poly(propylene glycol) acetate (PPCA) and stored at 4°C for further analysis. PPCA was characterized using FTIR.

[0051] Bevacizumab sustained-release nanoparticles are abbreviated as BGN@Be. Preparation of BGN@Be

[0052] BGN nanoparticles, called bioactive glass nanoparticles, are synthesized by the sol-gel method and obtained by freeze-drying and high-temperature calcination. They include SiO2, CaO and P2O5. The specific preparation method refers to Niu, W. et al. A multifunctional bioactive glass-ceramic nanodrug for post-surgical infection / cancer therapy-tissue regeneration. ACS. Nano. 15, 14323-14337 (2021). The difference from the prior art is that the molar percentages of SiO2, CaO and P2O5 in the preparation process of the present invention are 80:16:4.

[0053] 20 mg of BGN nanoparticles were dispersed in 20 mL of PBS by ultrasonic treatment, 50 μg of bevacizumab was added, and the mixture was stirred in an ice bath for 24 h. The precipitate was obtained by centrifugation at 11433 × g for 15 min at 4°C. The precipitate was then washed three times with 0.01 M PBS, pH 7.4, and freeze-dried to obtain BGN@Be powder, which was stored at 4°C until further use.

[0054] The morphology, size distribution and phase structure of the nanoparticles were investigated by transmission electron microscopy, nanoparticle tracking analysis and XRD. The degradation was detected by inductively coupled plasma mass spectrometry and UV-visible spectrophotometry.

[0055] Synthesis of OSPPB hydrogel

[0056] 1 mg of BGN@Be powder was dispersed in 1 mL of PBS to obtain solution A, 100 mg of OSAP was dissolved in 2 mL of solution A to obtain solution B, and 100 mg of PPCA was dissolved in 2 mL of solution A to obtain solution C. Solution B and solution C were then thoroughly mixed to obtain OSPPB hydrogel.

[0057] Example 2

[0058] Physicochemical characterization of OSPPB hydrogels

[0059] Morphological characteristics

[0060] To characterize the hydrogel morphology, the samples were freeze-dried, sputter-coated with gold, and examined using a field-emission scanning electron microscope at an accelerating voltage of 5 kV. Furthermore, energy-dispersive X-ray spectroscopy was used to characterize the mineral element composition and the distribution of BGN@Be in the OSPPB hydrogel.

[0061] ATR-FTIR

[0062] The samples were analyzed by ATR-FTIR at 4000-400 cm -1 Scanning was performed within the range, with a total of 32 scans and an average resolution of 4 cm -1 Spectral analysis was performed using infrared spectrum analysis software.

[0063] Rheological measurements

[0064] The rheological properties of OSPP and OSPPB hydrogels were measured using a rheometer. 3 mL of hydrogel was used for each test at 25°C. Time sweep sequences were performed at 1 Hz and 1% fixed strain for OSPP and OSPPB hydrogels. In addition, frequency sweep sequences were performed between 0.1 Hz and 10 Hz at 2% fixed strain. In the shear thinning experiments, the shear rate range was 0 s -1~100s -1 , and the frequency and strain are kept at 10 rad·s -1 and 1%.

[0065] In vitro degradation of hydrogels

[0066] The in vitro degradation behavior of OSPP and OSPPB hydrogels was studied in PBS at 37°C. After weighing and recording the mass (M0, mg), the hydrogel samples were soaked in PBS. Samples were then removed after 1, 2, 3, 4, 5, and 6 days, dried with filter paper, weighed, and recorded (Mt, mg). The degradation rate was calculated as: Remaining hydrogel (%) = (Mt / M0) × 100%.

[0067] Response of OSPPB hydrogel to changes in pH and ROS

[0068] PBS was adjusted to pH 5 with 20 μL of hydrochloric acid to apply pH stimulation. Additionally, 20 μL of 1 mM H₂O₂ was used to increase ROS in the PBS to apply ROS stimulation. Before imaging, 1 mL of OSPPB hydrogel was exposed to the corresponding stimulation and incubated at 37°C for 4 hours.

[0069] Anti-inflammatory evaluation

[0070] A DPPH free radical scavenging activity assay was conducted. Hydrogel extracts of varying concentrations were obtained by completely dissolving OSPPB hydrogel in PBS. The hydrogel extracts of varying concentrations were dispersed in 3 mL of a 0.1 mg / mL DPPH methanol solution. A DPPH methanol solution without the hydrogel extract and vitamin C were used as negative and positive antioxidant controls, respectively. The mixtures were shaken vigorously and allowed to stand at 37°C for 30 minutes before imaging. The absorbance of the mixtures was then measured at 528 nm.

[0071] Zeta potential

[0072] The transmittance of OSPP and OSPPB extracts was first detected using a Litesize 500 particle analyzer. When the transmittance reached 80%, the zeta potential was tested.

[0073] Plate incubation experiment

[0074] Microtiter plate wells pre-coated with VEGF were obtained and blocked with Tris-buffered saline containing 3% bovine serum albumin. In group 1, 50 μL of 150 μg / mL Cy3-RNA was incubated with 50 μL of diethyl pyrocarbonate water at 22°C for 2 hours. In group 2, the diethyl pyrocarbonate water was replaced with 50 μL of OSPPB hydrogel. In group 3, the wells obtained in group 1 were then incubated with 50 μL of OSPPB hydrogel at 22°C for an additional 2 hours. Confocal laser scanning microscopy was then used to visualize the residual Cy3 fluorescent finger bound to the RNA.

[0075] Hemolysis evaluation

[0076] One mL of heparinized mouse blood was suspended in 9 mL of PBS and centrifuged at 257 × g for 5 minutes to isolate red blood cells. This process was repeated three times until the supernatant became clear. Red blood cells were incubated with various concentrations of the extract at 37°C for 1 hour and then centrifuged at 714 × g for 5 minutes at 4°C. PBS and Triton X-100 served as negative and positive controls, respectively. The supernatant was photographed and its absorbance was measured at 540 nm.

[0077] In vitro experiments

[0078] After disinfecting the hydrogel with 75% ethanol for 24 hours, it was thoroughly washed with PBS to remove the residual ethanol. EPCs were cultured in DMEM containing 1% penicillin / streptomycin and 10% fetal bovine serum. EPCs are endothelial progenitor cells. EPCs were cultured at 5×10 per ml. 4EPCs were cultured on hydrogels at a density of 10 cells / mL at 37°C in 5% CO2, with medium changed every two days. EPCs were divided into different groups: untreated, VEGF, RNA-VEGF complex, and RNA-VEGF-OSAP, RNA-VEGF-PPCA, RNA-VEGF-BGN@Be, and RNA-VEGF-OSPPB. EPC activity was assessed using CCK-8. For the wound wound assay, EPC monolayers were cultured in the various conditioned media for 24 hours after wound wounding. For the Transwell cell migration assay, the underside of the Transwell inserts was stained with crystal violet after 24 hours of incubation. For the tubule formation assay, Matrigel was used according to the manufacturer's instructions. Primary trigeminal ganglion cells were isolated from 1- to 5-day-old Sprague-Dawley rats. Trigeminal ganglia were dissected under sterile conditions and digested with an enzyme cocktail. After centrifugation, the pellet was resuspended and plated onto polylysine salt-coated cell culture plates. Cells were cultured in Neurobasal medium containing 0.5 mM L-glutamine, 1% penicillin / streptomycin, and 2% B-27 supplement. Cells were incubated at 37°C in 5% carbon dioxide, with medium replacement every two days. Four hours later, TG neurons were sorted into seven groups as described above. Immunofluorescence staining for β3-tubulin and crystal violet was used to reveal synaptic morphology.

[0079] result

[0080] 3-Aminophenylboronic acid was grafted onto sodium alginate to obtain SAP polymer. Subsequently, the hydroxyl groups of SAP were oxidized with sodium periodate to obtain OSAP polymer containing boronic acid groups and aldehyde groups, such as Figure 1 A. Attenuated total reflection Fourier transform infrared spectroscopy Figure 2 As shown, SA represents sodium alginate, and the peak of the OSAP polymer is at 708 cm -1 1485cm -1 , 1339cm -1 and 1735cm -1 represents m-substituted benzene, phenyl group, BO vibration and CH bending vibration of aldehyde group, respectively. PEI represents polyethyleneimine, PCA represents protocatechuic acid, and PPCA polymer is synthesized by amidation reaction, which involves the combination of carboxyl group of protocatechuic acid and amino group present in polyethyleneimine. The chemical structure of PPCA polymer was tested by ATR-FTIR spectroscopy, such as Figure 3 As shown, 1556cm -1 is due to the bending of the NH of the amine, at 1643 cm -1 There is a new absorption peak at , indicating the carboxyamide I band.

[0081] The prepared BGN has a uniform spherical morphology and a relatively uniform particle size of 240 nm. Figure 4 Figures a and b in Figure 2. Subsequently, based on the Ca relationship between BGN and bevacizumab 2+ BGN@Be was prepared by coordination interaction, and the transmission electron microscopy images clearly showed the beaded structure of BGN@Be, such as Figure 4 Figure a shows that bevacizumab was successfully loaded on BGN. Nanoparticle tracking analysis showed that BGN@Be had a larger size of about 424 nm. X-ray diffraction confirmed that bevacizumab loading did not affect the structure of BGN, as shown in Figure 3. Figure 4 Figures b and c in Figure 2. After immersion in PBS for 21 days, BGN@Be became very loose, indicating its degradability. The Si content of BGN@Be was 89% within 10 days, followed by a plateau, and the bevacizumab content was 90% within 7 days, followed by a plateau. Figure 4 As shown in Figures d and e, the Si content and bevacizumab content of BGN@Be increased with increasing immersion time, confirming its slow release performance.

[0082] To obtain OSPPB hydrogel, BGN@Be was dispersed in OSAP solution and PPCA solution, respectively, and the two were mixed by vortexing. Due to the formation of dynamic phenylboronic acid-diol ester bonds and Schiff base bonds, the hydrogel was formed within 3 seconds. The chemical structure of the hydrogel was determined by ATR-FTIR spectroscopy. Figure 1 As shown in c, the new one appears at 1431cm -1 The peak at 1735 cm -1 The disappearance of the characteristic peak at indicates that the aldehyde group participates in the Schiff base reaction. Then, scanning electron microscopy and energy chromatography techniques were used to evaluate the microstructure and distribution of the hydrogel. Figure 1 In Figure d, both OSPP and OSPPB hydrogels exhibit uniform porous three-dimensional structures, where OSPP is composed of OSAP polymer and PPCA polymer. OSPP is obtained by the following method: 100 mg OSAP is dissolved in 2 mL PBS to obtain solution A, and 100 mg PPCA is dissolved in 2 mL PBS to obtain solution B. Solution A is mixed with solution B to obtain OSPP hydrogel, and the micropores are approximately 200 μm. However, their surface morphologies are different, and the unique feature of the surface of OSPPB hydrogel is the dispersed distribution of BGN. In other words, the incorporation of BGN@Be does not affect the porous structure of the hydrogel. In addition, Figure 1 (e) is the SEM-EDS of OSPPB hydrogel, showing the Ca, P and Si elements on the OSPPB hydrogel. The presence of elemental Si, Ca and P in the gel matrix further confirms the incorporation of BGN@Be into OSPPB hydrogel.

[0083] The dynamic Schiff base bonds and boronate ester bonds endow OSPPB hydrogels with a variety of advantageous properties, such as plasticity, injectability, self-healing, and pH / ROS dual responsiveness. OSPPB hydrogels have good plasticity and injectability, such as Figure 1 To demonstrate this, a 26G needle was used to inject the hydrogel to create the letter "J," as shown in Figure 1-2 of Figure 2. The self-healing properties of the OSPPB hydrogel were tested by forming a circular shape, then cutting it into two equal parts and reconnecting the broken ends at 37°C for 10 minutes. The two halves of the hydrogel gradually merged together, forming a single, integrated hydrogel with sufficient mechanical rigidity to be picked up with tweezers, without any fractures along the contact interface. Figure 1 In addition, the multifunctional rheological properties of the hydrogel were tested using a rheometer, as shown in Figure 3-6 of Figure b. Figure 1 fh, OSPP and OSPPB hydrogels showed stable storage modulus and loss modulus in the time scanning sequence, the storage modulus was recorded as G', and the loss modulus was recorded as G", G'>G", which confirmed the successful formation of the hydrogel. Figure 1 f, the G' of OSPP hydrogel is 129.46 ± 3.09 Pa, and the G' of OSPPB is 354.42 ± 3.97 Pa. The G' of OSPPB is higher than that of OSPP hydrogel, which confirms its enhanced gel formation property and indicates that it has a stronger gel network. The addition of BGN@Be to the hydrogel increases its linear viscoelastic region, such as Figure 1 In addition, the viscosity curves of the two hydrogels are comparable, and since the viscosity decreases with increasing shear rate, they exhibit non-Newtonian shear thinning behavior, as shown in Figure 1 These properties facilitate intra-articular injection without affecting the structure of the hydrogel.

[0084] Considering its potential pH / ROS dual responsiveness, the present invention detected the changes in the pH and ROS responsiveness of the hydrogel. Figure 5 In a, hydrochloric acid and H2O2 were used to simulate a low pH and high ROS environment. Low pH or high ROS stimulation can cause the hydrogel to quickly disintegrate and transform into a liquid-like state, such as Figure 5 b, Rheological results show that the storage modulus G' of OSPPB hydrogel decreased significantly after low pH or ROS stimulation, and this decrease was more obvious under the combined action of the two stimuli. Figure 5The SEM results of the CDs in the samples showed that although pores were still visible in the groups stimulated by ROS or pH alone, the pores had collapsed in the group stimulated by ROS+pH together, indicating that the network structure had decomposed. These results strongly demonstrated that the OSPPB hydrogel had a pH / ROS dual responsiveness, which enabled it to completely decompose within 6 days. Figure 5 e in, In addition, the gradual increase of sulfur content in the supernatant was entirely attributed to bevacizumab in the hydrogel, implying that bevacizumab was sustainedly released from the hydrogel matrix, e.g. Figure 5 The dissociation of the Schiff base bond and the boronate ester bond in f endows it with pH / ROS dual responsiveness.

[0085] To test its blood compatibility, different concentrations of the extract were incubated with red blood cells. The optical results of the different concentration groups from 0μg / mL to 300μg / mL were similar, indicating that when the concentration was lower than 300μg / mL, a safe hemolysis rate of less than 5% was obtained, such as Figure 6 .

[0086] The antioxidant effect of the hydrogel was verified by 2,2-diphenyl-1-pyridinium hydrazine scavenging experiment. 2,2-diphenyl-1-pyridinium hydrazine is abbreviated as DPPH. Vitamin C was used as a positive control. The DPPH scavenging rate of 25 μg / mL extract was similar to that of vitamin C. Figure 5 A low concentration was chosen for this experiment because both the hydrogel and the extract are dark in color, minimizing the effect on the results. However, a significant darkening was observed in the 50 μg / mL group, which cannot be definitively attributed to the increased OSPPB concentration or the decreased antioxidant capacity. Therefore, this experiment only qualitatively confirmed the antioxidant properties of the OSPPB hydrogel. This property may help reduce inflammatory responses in the body and alleviate oxidative stress, thereby treating OA.

[0087] Positive charge and nucleic acid removal ability are important parameters of this hydrogel. Positive charge determines its nucleic acid removal properties because their binding depends on electrostatic interactions. The zeta potential of OSPPB confirms that it is positively charged, as shown in Figure 5 As shown in Figure 3, the plate incubation experiment showed that after OSPPB treatment, very little Cy3 remained in the culture dish, which confirmed that OSPPB hydrogel interfered with the binding between VEGF and RNA. Figure 5 In addition, molecular dynamics simulation verification shows that the results are as follows Figure 5 Figure 1 shows that in a system with a pH of approximately 6.5, PEI exhibits stronger RNA binding ability for VEGF. The significant nucleic acid clearance properties will enable OSPPB hydrogel to effectively capture exRNA in the osteoarthritis condyle, potentially inhibiting abnormal neurovascularization.

[0088] Its inhibitory function on neurons and vascular endothelial cells was evaluated in vitro. Figure 8 . The migration of EPCs was analyzed by wound healing assay and Transwell assay. RNA-VEGF treatment shortened the wound and increased the number of migrating cells. However, PPCA and BGN@Be treatment showed a slight inhibitory effect on this process, while OSPPB hydrogel displayed stronger inhibitory function. In addition, hydrogel treatment also prevented the tubule formation of EPCs in Matrigel under RNA-VEGF stimulation. The morphology and synaptic status of TG cells, which are trigeminal ganglion cells, were measured by crystal violet staining and immunofluorescence staining of β3-tubulin. Compared with RNA-VEGF-treated TG cells, cells treated with PPCA, BGN@Be, and OSPPB hydrogels showed shorter synaptic length, fewer synaptic branches, and simpler morphology. Experiments with different hydrogel components showed that OSAP lacked inhibitory activity, while PPCA and BGN@Be were considered to be the key bioactive components. Although PPCA and BGN@Be had inhibitory effects on neurovascularization, their effects were less than those of OSPPB hydrogel. These results indicate that OSPPB hydrogel can inhibit angiogenesis and neurogenesis in vitro.

[0089] The effects of TMJOA on condylar histological changes, maxillofacial pain, and neurovascularization were studied in a mouse unilateral anterior crossbite model. Figure 9-12 The results showed that there were randomly scattered new blood vessels in the UAC+Veh and UAC+Celecoxib groups, but not in the UAC+OSPPB group, as shown in Figure 2. Figure 9 As shown in middle b. The results of hematoxylin and eosin staining and safranin O-green staining further showed that the TMJ condyle treated with OSPPB was more normal, showing a lower International Osteoarthritis Research Society score, fewer capillaries, thicker cartilage layer, and more glycosaminoglycans, such as Figure 9 As shown in c1, c2, dg, the abbreviation of Osteoarthritis Research Society International is OARSI. Glycine silver staining results showed that the number of nerve fibers decreased after OSPPB treatment, as shown in Figure 9 In addition, the total length of microcracks increased in UAC mice, slightly decreased in the UAC+Celecoxib group, and significantly decreased after OSPPB hydrogel treatment, as shown in Figure 3 and Figure 4. Figure 9 As shown in Figures c4 and h. Through these microcracks, nerves, blood vessels, inflammatory factors, and enzymes invade the cartilage, establishing an "osteoclast-chondrocyte interaction" that ultimately accelerates the development of OA. Micro-CT three-dimensional reconstruction showed that the joint surface in the OSPPB group was smoother, without erosion or irregularities, as shown in Figure 4. Figure 9 The quantitative results further confirmed the increase in bone mineral density after OSPPB, while Celecoxib showed a more limited effect, as shown in Figure 5. Figure 9 As shown in j and k, qRT-PCR demonstrated that the injection of OSPPB hydrogel changed the pro-angiogenic and pro-neurogenic microenvironment in the osteoarthritic condyle. Figure 9 As shown in Figure 1.

[0090] The approximately 50 μm thick osteochondral interface physiologically ensures a smooth transition from condylar cartilage to subchondral bone. However, in OA, its microstructure, micromechanics, and biomolecular characteristics undergo significant changes. Therefore, the present invention examined the remodeling of the osteochondral interface using atomic force microscopy and scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS). Figure 10 As shown in Figure 2 . The results of atomic force microscopy showed that the Young's modulus of the osteochondral interface of the condyle in the control group gradually increased from 0.72 ± 0.22 MPa to 9.06 ± 0.53 MPa. However, in the UAC group, the gradient of tissue modulus was disordered, forming a matrix with a higher hardness of up to 17.04 MPa, which was restored after OSPPB treatment, but not after Celecoxib treatment, as shown in Figure 2 . Figure 10 As shown in a. Figure 5 As shown in Figure 2b, the distribution of Ca and P significantly extended to the condylar cartilage of the UAC group and the UAC+Celecoxib group. In contrast, the condyle treated with OSPPB hydrogel showed a Ca and P element distribution close to that of the control group. The present invention distinguishes between condylar cartilage and subchondral bone based on the differences in inorganic and organic components. Neurovascularization at the osteochondral interface ultimately leads to changes in its microstructure and micromechanical properties, which are closely related to the development of arthritis. The present invention confirms that OSPPB hydrogel can alleviate the microstructural and micromechanical changes caused by TMJOA.

[0091] The restorative effect of OSPPB on TMJOA was confirmed by histological and microstructural characterization; however, whether this treatment can alleviate osteoarthritis pain remains unknown. Since pain in rodents cannot be measured directly, "pain-like behavior" was tested. Spontaneous behavioral responses and evoked behavioral responses were evaluated. Spontaneous behavioral responses included elevated cross and open field tests. The elevated cross is abbreviated as EPM and the open field test is abbreviated as OFT. Evoked behavioral responses included von Frey and electroencephalogram. Traditionally, EPM and OFT are used to assess anxiety in rodents. Given that persistent chronic pain will eventually lead to anxiety, the present invention uses these two behavioral tests to indirectly characterize the analgesic effect of OSPPB hydrogel and Celecoxib on TMJOA-induced pain before sampling. The increase in the total number of arm entries, the number of open arm entries, and the open arm time in EPM confirmed that Celecoxib and OSPPB hydrogel alleviated anxiety-like behaviors, such as Figure 11 The results of OFT are consistent with this observation, as shown in a, ce. Figure 11 As shown in b, fh. Based on these observations, it is speculated that the administration of Celecoxib and OSPPB hydrogel may have alleviated the anxiety caused by persistent pain. To visually assess maxillofacial pain, mechanical hyperalgesia induced by von Frey fibers was tested. The withdrawal threshold of the UAC+Veh group decreased significantly, but returned to the control level after treatment with Celecoxib and OSPPB hydrogel, as shown in Figure 11 In addition, the EEG of the primary sensory cortex S1BF was captured immediately after the brush stimulation, as shown in Figure 11 The low timestamp accuracy resulted in some noise around the expected time points. However, the present invention observed a decrease in spectral energy in the low-frequency band of 0-20 Hz after Celecoxib and OSPPB treatment, similar to the control group. These preliminary results suggest that mice treated with Celecoxib and OSPPB hydrogels exhibited significant relief of maxillofacial pain.

[0092] To verify the significant anatomical changes associated with pain relief, immunofluorescence experiments were performed. ExoRNAs at the osteochondral interface were identified using co-staining with the RNA-specific stain SYTO RNASelect Green Fluorescent Cell Dye and the cell membrane marker E-cadherin. Extracellular flocculent RNAs away from E-cadherin-positive areas were defined as exoRNAs in immunofluorescence staining. Figure 12 As shown in (a) and (d), the amount of exRNA, a neurovascular recruitment factor, was reduced after treatment with OSPPB hydrogel but not Celecoxib. Anterograde nerve tracing showed that the distribution of axon terminals from the trigeminal ganglion was reduced in the UAC+OSPPB group, as shown in (a) and (d). Figure 12In addition, the confocal laser scanning microscopy results of PGP 9.5 and CGRP are consistent with the above findings, as shown in b and e. Figure 12 In addition, compared with the UAC+Celecoxib group, the levels of vascular and neurovascular factors VEGF, which are positive for platelet and endothelial cell adhesion molecule 1, were decreased after OSPPB hydrogel treatment. Platelet and endothelial cell adhesion molecule 1 is called PECAM1, also known as CD31, as shown in Figures b, f, and g. Figure 12 Further examination of several factors closely related to bone perception and pain, including COX2, DCC, and SP, showed that pain mediators decreased after OSPPB hydrogel treatment, as shown in Figure 3c and h. Figure 12 (c, ik) Taken together, these data suggest that Celecoxib primarily exhibits efficacy in alleviating pain, but its effects on the regenerative microenvironment were found to be quite limited. However, OSPPB hydrogel effectively impeded neurovascularization at the osteochondral interface and alleviated osteoarthritis pain.

[0093] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes preferred embodiments.

[0094] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0095] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A dual-responsive hydrogel, characterized in that: The dual-responsive hydrogel is made of phenylboronic acid-modified sodium alginate, protocatechuic acid-polyethyleneimine grafted polymer and bevacizumab sustained-release nanoparticles; The mass ratio of phenylboronic acid-modified sodium alginate, protocatechuic acid-polyethyleneimine graft polymer, and bevacizumab sustained-release nanoparticles was 80-120:80-120:0.8-1.2; Grafting 3-aminophenylboronic acid onto sodium alginate to obtain an intermediate product, and oxidizing the intermediate product with sodium periodate to obtain the phenylboronic acid-modified sodium alginate; reacting the bioactive glass nanoparticles with bevacizumab in an ice bath for 22 h to 26 h to obtain the bevacizumab sustained-release nanoparticles; The bioactive glass nanoparticles include SiO2, CaO and P2O5.

2. A method for preparing the dual-responsive hydrogel according to claim 1, characterized in that: The following steps are involved: Grafting 3-aminophenylboronic acid onto sodium alginate to obtain an intermediate product, and oxidizing the intermediate product with sodium periodate to obtain the phenylboronic acid-modified sodium alginate; the mass ratio of the 3-aminophenylboronic acid to the sodium alginate is 0.3-0.8:0.8-1.2; The protocatechuic acid-polyethyleneimine graft polymer is synthesized by amidation reaction of polyethyleneimine and protocatechuic acid; the mass ratio of the polyethyleneimine to the protocatechuic acid is 10-15:0.8-1.2; The bioactive glass nanoparticles and bevacizumab are reacted in an ice bath for 22 h to 26 h to obtain the bevacizumab sustained-release nanoparticles; the mass ratio of the bioactive glass nanoparticles to the bevacizumab is 15 mg to 25 mg: 45 μg to 55 μg; Bevacizumab sustained-release nanoparticles were dissolved in PBS to obtain solution A, phenylboronic acid-modified sodium alginate was dissolved in solution A to obtain solution B, protocatechuic acid-polyethyleneimine grafted polymer was dissolved in solution A to obtain solution C, and solution B and solution C were mixed to obtain the dual-responsive hydrogel.

3. The preparation method according to claim 2, characterized in that When 3-aminophenylboronic acid is grafted onto sodium alginate, the reaction time of 3-aminophenylboronic acid and sodium alginate is 23h~25h.

4. The preparation method according to claim 2, characterized in that When polyethyleneimine and protocatechuic acid undergo amidation reaction, the activated protocatechuic acid solution is mixed with the polyethyleneimine solution, stirred for 23h~25h under dark conditions, dialyzed with a molecular weight cutoff of 1800 Da, and freeze-dried to obtain the protocatechuic acid-polyethyleneimine graft polymer.

5. The preparation method according to claim 2, characterized in that When preparing the bevacizumab sustained-release nanoparticles, the specific reaction is: stirring for 23 hours to 25 hours, centrifuging to obtain a precipitate, washing with PBS, and freeze-drying to obtain BGN@Be powder.

6. The preparation method according to claim 2, characterized in that The molar percentage of SiO2, CaO and P2O5 is 80:16:

4.

7. Use of the dual-responsive hydrogel according to claim 1 in preparing a drug for treating osteoarthritis.

8. The use according to claim 7, characterized in that The drug inhibits temporomandibular joint arthritis.

9. The use according to claim 7, characterized in that The drug alleviates pathological neurovascularization.

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