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

Abstract

The invention belongs to the technical field of hydrogel preparations, and particularly relates to double-responsiveness hydrogel and a preparation method and application thereof.The cross-linked polycation hydrogel is synthesized and composed of phenylboronic acid modified sodium alginate, protocatechuic acid-polyethyleneimine graft polymer and bevacizumab slow-release nanoparticles, the invention relates to an OSPPB hydrogel, called a dual-responsive OSPPB hydrogel, which blocks neurovascularization at the osteochondral interface by locally removing extracellular RNA and releasing bevacizumab, thereby alleviating OA pain and disease progression.

Description

Technical Field

[0001] The 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, or OA, is the most common musculoskeletal disease with a high incidence rate. OA often leads to pain, disability, and functional impairment. To treat OA, medication, physical therapy, and exercise therapy are widely used. Medication is usually used as an initial therapeutic intervention to quickly relieve acute symptoms, while the latter two methods play an auxiliary and continuous role in the overall treatment strategy of OA. Medication, including nonsteroidal anti-inflammatory drugs, paracetamol, opioids, and corticosteroids, focuses on suppressing inflammation and relieving pain, but does not address pathological changes and cannot avoid final joint replacement surgery. Currently, the most important pathological change in joint pain is the neurovascularization at the osteochondral interface, however, treatment and analgesic strategies specifically targeting neurovascularization have not been fully utilized. A large number of nerves and blood vessels emerge from the subchondral bone, penetrate the osteochondral interface, and invade the avascular cartilage through vertical microcracks. This neurovascularization transmits inflammatory mediators to the cartilage, accelerates matrix degradation and mineralization, leads to the formation of condylar osteophytes and aggravates 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 intervention in osteoarthritis.

[0003] Currently, a variety of cytokines, such as tumor necrosis factor α and interleukin-1β, as well as growth factors, including nerve growth factor, vascular endothelial growth factor, abbreviated as VEGF, and platelet-derived growth factor BB, have been found to promote neurovascularization in OA. In addition, previous studies have shown that extracellular RNA, abbreviated as exRNA, as an organic polyanion recruitment factor, can bind and enhance the function of polycationic neurovascular factors in OA. Further studies have also confirmed that intraarticular injection of nucleic acids can induce arthritis through angiogenesis, and the controlled release of RNA can promote angiogenesis and osteogenesis. Similarly, the key role of exRNA in neurovascularization has also been found in cardiovascular diseases, where it can regulate the function of VEGF. Based on these facts, a strategy to clear exRNA may be superior to simply inhibiting neurovascular factors. To date, the exRNA clearance strategy has 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 been proven to be effective in various diseases, it is difficult to achieve responsive release of polycationic formulations to mitigate toxicity. 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. Substantial changes in the osteochondral microenvironment in OA are considered to be ideal conditions for achieving controlled drug delivery.

[0004] The temporomandibular joint, or TMJ, is particularly prone to osteoarthritis, with an incidence of 14.56% in people under 30 years old and increasing to 28-32% in people aged 30-80 years old. Previous studies have confirmed that temporomandibular arthritis, or TMJOA, is a common disease in the temporomandibular joint. ， Pathological neurovascularization at the condylar osteochondral interface of patients is closely related to maxillofacial pain, but existing hydrogels fail to effectively inhibit neurovascularization. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a dual-responsive hydrogel and application thereof.

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

[0007] The mass ratio of phenylboronic acid modified sodium alginate, protocatechuic acid-polyethyleneimine grafted 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] Polyethyleneimine and protocatechuic acid are subjected to an amidation reaction to synthesize the protocatechuic acid-polyethyleneimine graft polymer; 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 are dissolved in PBS to obtain solution A, phenylboronic acid-modified sodium alginate is dissolved in solution A to obtain solution B, protocatechuic acid-polyethyleneimine grafted polymer is dissolved in solution A to obtain solution C, and solution B and solution C are 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; 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; 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; 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; 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 h to 25 h.

[0018] Preferably, when polyethyleneimine and protocatechuic acid undergo amidation reaction, the activated protocatechuic acid solution is mixed with the polyethyleneimine solution, stirred for 23h to 25h in the dark, dialyzed with a molecular weight cutoff of 1800Da, 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 a. In order to construct OSPPB hydrogel, the present invention cross-links phenylboronic acid modified sodium alginate with the main chain of protocatechuic acid-polyethyleneimine grafted polymer molecules by forming dynamic boronate bonds and Schiff base bonds, and adds bevacizumab sustained-release nanoparticles through coordination. The boronate bond is established by cross-linking the boric acid group of phenylboronic acid modified sodium alginate with the catechol group of protocatechuic acid-polyethyleneimine grafted polymer. At the same time, the amino group of protocatechuic acid-polyethyleneimine grafted polymer is cross-linked with the aldehyde group of phenylboronic acid modified sodium alginate to form a Schiff base bond.

[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 hydrogel has the following advantages: (1) polycationic OSPPB hydrogel can remove polyanionic exRNA; (2) its pH / ROS dual responsiveness enables on-demand release of active ingredients; (3) sequential delivery of active ingredients promotes structural recovery. Subsequently, its inhibitory function on neurons and vascular endothelial cells was evaluated in vitro, and its effects on condylar nerve vascularization and joint pain were studied in a mouse unilateral anterior crossbite TMJOA model. The OSPPB hydrogel of the present invention removes exRNA, inhibits nerve vascularization, and treats TMJOA. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Preparation and characterization of OSPPB hydrogel, a is a 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 there is uniformly distributed BGN@Be on the surface of OSPPB hydrogel, and the red circles represent BGN, the scale bar in the first column = 200μm, and the scale bar in the second column = 5μm, e is the SEM-EDS of OSPPB hydrogel, showing the Ca, P and Si elements on 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 degraded for 21 days, 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 3 shows the pH / ROS dual responsiveness, degradation, antioxidant and positive charge of OSPPB hydrogel. a is an image of OSPPB hydrogel after incubation with PBS and / or H2O2 at pH = 5. b is a frequency scan analysis. c is a SEM image. Scale bar = 200 μm. d is a statistical analysis of the SEM image. e is the degradation of OSPP and OSPPB hydrogel in PBS. f is the variation characteristics of S concentration within 14 days. g is a photo and statistical results after incubation of OSPPB extracts with different concentrations with DPPH for 30 min. h is the Zeta potential of OSPP and OSPPB hydrogel. i is a plate incubation experiment of OSPPB hydrogel binding to RNA. j is the statistical results of the plate incubation experiment. k is a schematic diagram of the plate incubation experiment, showing the binding of OSPPB to RNA. Scale bar = 50 μm. l is the molecular dynamics simulation and quantification of RNA (light blue), VEGF (red) and PEI (purple). The data are expressed 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 OSPPB hydrogel inhibits angiogenesis and neurogenesis, a is a representative microscope image of EPCs after 24 hours of treatment, b is a representative image of TG cells after 24 hours of treatment, c is the statistical result of the number of EPCs migrating cells, d is the statistical result of the migration distance of EPCs, e is the statistical result of TG synaptic bifurcation, f is the statistical result of synapse length, scale bar = 100 μm, data are expressed as mean and standard deviation, n = 6.

[0036] Fig. 9Figure 4 shows that 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-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, 5 in c = 500 μm. d is OARSI semi-quantification. 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, and 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] Fig.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 the atomic force microscope, 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] Fig.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 the mice entered the open arms in the elevated cross test, d is the statistical analysis of the time spent in the open arms, e is the statistical analysis of the total number of times the mice entered the arms, f is the statistical analysis of the total distance traveled by the 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] Fig.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, and 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 exRNA statistical analysis, f is anterograde tracer EGFP statistical analysis, g is CGRP statistical analysis, h is CD31 statistical analysis, i is COX2 statistical analysis, j is DCC statistical analysis, k is SP statistical analysis, 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 protection scope 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 creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are 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 Microscope 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] 1 gram of sodium alginate was dissolved in 100 mL of deionized water. After complete dissolution, 0.985 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, abbreviated as EDC·HCl, was added. After 15 minutes, 0.49 g of 3-aminophenylboronic acid was added. The mixture was stirred for 24 hours and then dialyzed with water at room temperature for 7 days to remove unreacted substances. The molecular weight cutoff during dialysis was 3500 Da, and the dialysis water was replaced every 8 hours. After freeze-drying, the intermediate product SAP was obtained. Then, 1 g of SAP was dissolved in 100 mL of deionized water for 2 hours to 3 hours to obtain a SAP solution. 210 mg of NaIO4 was dispersed in 5 mL of deionized water, and then the SAP solution was dropped in the dark. After two and a half hours, 1 mL of ethylene glycol was added and reacted for 1 hour to neutralize the unreacted NaIO4 to obtain OSAP. SAP and OSAP were stored at 4°C before further use. SAP and OSAP were characterized by ATR-FTIR.

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

[0050] Dissolve 4.5g of polyethyleneimine in 20mL of MES buffer solution to obtain polyethyleneimine solution. Dissolve 308.24mg of protocatechuic acid and 1.54g of EDC·HCl in 80mL of MES buffer for 0.5h. Then, add 920.72mg of N-hydroxysuccinimide and stir for 12h to activate the carboxyl group in the dark. Then add the solution to the polyethyleneimine solution, stir and react in the dark at room temperature for 24h, dialyze for 7 days, with a molecular weight cutoff of 1800Da, and replace the dialysis water every 8h. The dialysate was freeze-dried to obtain PPCA and stored at 4℃ for further analysis. PPCA was characterized by FTIR technology.

[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, and then washed three times with 0.01 M PBS, pH 7.4. BGN@Be powder was obtained by freeze-drying and 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 techniques, respectively. 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. Then, solution B and solution C were thoroughly mixed to obtain OSPPB hydrogel.

[0057] Example 2

[0058] Physicochemical characterization of OSPPB hydrogels

[0059] Morphological characteristics

[0060] To characterize the morphology of the hydrogels, 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. In addition, energy dispersive X-ray spectroscopy was used to characterize the mineral element composition and the distribution of BGN@Be in the OSPPB hydrogels.

[0061] ATR-FTIR

[0062] The samples were analyzed by ATR-FTIR at 4000-400 cm -1 The scan 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, with 3 mL of hydrogel used for each test at 25 °C. Time sweep sequences for OSPP and OSPPB hydrogels were performed at 1 Hz and 1% fixed strain. In addition, frequency sweep sequences were performed between 0.1 Hz and 10 Hz at 2% fixed strain. In the shear thinning tests, 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. The hydrogel samples were soaked in PBS after being weighed and recorded (M0, mg). Then, the samples were taken out at 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] 20 μL of hydrochloric acid was used to adjust the pH of PBS to 5 to apply pH stimulation. In addition, 20 μL of 1 mM H2O2 was used to increase ROS in PBS to apply ROS stimulation. Before taking pictures, 1 mL of OSPPB hydrogel was subjected to the corresponding stimulation and placed at 37°C for 4 h.

[0069] Anti-inflammatory evaluation

[0070] The DPPH free radical scavenging activity test was conducted. Hydrogel extracts of different concentrations were obtained by completely dissolving OSPPB hydrogel in PBS. The hydrogel extracts of different concentrations were dispersed in 3 mL of DPPH methanol solution with a concentration of 0.1 mg / mL. DPPH methanol solution without hydrogel extract and vitamin C were selected as antioxidant negative and positive controls, respectively. Before taking pictures, the mixture was shaken vigorously and allowed to stand at 37°C for 30 minutes. The absorbance of the mixture was then measured at 528 nm.

[0071] Zeta Potential

[0072] The transmittance of OSPP and OSPPB extracts was first detected using a LiteSizer 500 particle analyzer, and 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 Cy3-RNA at a concentration of 150 μg / mL and 50 μL of diethyl pyrocarbonate water were incubated at 22°C for 2 h. In group 2, 50 μL of OSPPB hydrogel replaced the diethyl pyrocarbonate water. In group 3, the wells obtained in group 1 were then incubated with 50 μL of OSPPB hydrogel for another 2 h at 22°C. Confocal laser scanning microscopy was then used for observation, where the residual Cy3 fluorescence refers to the bound RNA.

[0075] Hemolysis evaluation

[0076] 1 mL of heparinized mouse blood was suspended in 9 mL of PBS and centrifuged at 257 × g for 5 min to separate red blood cells. Then, this process was repeated three times until the supernatant became clear. Red blood cells were incubated with different concentrations of extracts at 37°C for 1 h, and then centrifuged at 714 × g for 5 min at 4°C. PBS and Triton X-100 were used as negative and positive controls, respectively. Photos were taken and the absorbance of the supernatant 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. 4The cells were cultured on hydrogels at a density of 10 cells / mL at 37°C and 5% CO2, and the medium was 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 detected using CCK-8. For the scratch assay, the monolayer of EPCs was cultured in different conditioned media for 24 h after scratching. For the Transwell cell migration assay, the bottom surface of the Transwell insert was stained with crystal violet after 24 h of incubation. For the tubule formation assay, Matrigel was used according to the manufacturer's instructions. Primary trigeminal ganglion cells were isolated from 1-5 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 on poly-lysine salt-coated cell culture plates and cultured using Neurobasal medium containing 0.5 mM L-glutamine, 1% penicillin / streptomycin, and 2% B-27 supplement. Cells were cultured at 37 °C and 5% carbon dioxide, and the medium was changed every two days. Four hours later, TG neurons were divided into seven groups as described above. Immunofluorescence staining of β3-tubulin and crystal violet was used to reveal its synaptic morphology.

[0079] result

[0080] 3-Aminophenylboronic acid was grafted onto sodium alginate to obtain SAP polymer, and then 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, 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 with 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 BGNs have 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 show 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, and X-ray diffraction confirmed that bevacizumab loading did not affect the structure of BGN, such as 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 Figure d and e, the Si content and bevacizumab content of BGN@Be increased with the 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 vortex oscillator. The hydrogel was formed within 3 s due to the formation of dynamic phenylboronic acid-diol ester bonds and Schiff base bonds. The chemical structure of the hydrogel was determined by ATR-FTIR spectroscopy. Figure 1 As shown in c, the new -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, the microstructure and distribution law in the hydrogel were evaluated using scanning electron microscopy and energy chromatography techniques, such as Figure 1 As shown 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 about 200 μm. However, their surface morphologies are different, where 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 favorable properties, such as plasticity, injectability, self-healing, and pH / ROS dual responsiveness. OSPPB hydrogels have good plasticity and injectability, such as Figure 1 As shown in 1-2 of b, to demonstrate this, the hydrogel was injected with a 26G needle to write the letter "J". The self-healing properties of the OSPPB hydrogel were tested. The OSPPB hydrogel formed a circle, then was cut into two equal parts and the broken ends were reconnected at 37°C for 10 minutes. The two parts of the hydrogel gradually merged together to form a whole hydrogel with sufficient mechanical rigidity to be picked up with tweezers without any fractures along the contact interface, as shown in Fig. Figure 1 In addition, the multifunctional rheological properties of the hydrogel were tested using a rheometer, such as Figure 1 fh, OSPP and OSPPB hydrogels showed stable storage modulus and loss modulus in the time scanning sequence, the storage modulus was denoted as G', and the loss modulus was denoted 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, confirming its enhanced gel formation properties, indicating that it has a stronger gel network. Adding 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, since the viscosity decreases with increasing shear rate, thus showing a 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 hydrogel pH and ROS responsiveness. 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 cd in the 200 μg / mL 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 dual pH / ROS responsiveness, which enabled it to completely decompose within 6 days. Figure 5 e in the supernatant, moreover, the gradual increase in sulfur content in the supernatant was entirely attributed to the bevacizumab in the hydrogel, implying that bevacizumab was sustainedly released from the hydrogel matrix, e.g. Figure 5 In f, the dissociation of the Schiff base bond and the boronate ester bond 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 0 μg / mL to 300 μg / mL were similar, indicating that when the concentration was lower than 300 μg / 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 scavenging experiments with 2,2-diphenyl-1-pyridine hydrazine, abbreviated as DPPH. Vitamin C was used as a positive control, and the DPPH scavenging rate of 25 μg / mL extract was similar to that of vitamin C. Figure 5 g in the experiment. A low concentration was chosen for this experiment because both the hydrogel and the extract itself are dark in color, so the low concentration has the least effect on the results. However, in the 50 μg / mL group, a significant darkening phenomenon occurred, which cannot be clearly attributed to the increase in OSPPB concentration or the decrease in antioxidant capacity. Therefore, this experiment only qualitatively confirmed the antioxidant properties of 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. The positive charge determines its nucleic acid removal properties because their binding depends on electrostatic interactions, and the zeta potential of OSPPB confirms that it is positively charged, such as 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 simulations were performed to verify the results. Figure 5 As shown in Figure 1, in a system with a pH of approximately 6.5, PEI exhibits a stronger RNA binding ability for VEGF. The significant nucleic acid clearance properties will enable OSPPB hydrogel to effectively capture exRNA in the osteoarthritic 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 scratch and Transwell assays. RNA-VEGF treatment shortened the scratch and increased the number of migrating cells. However, PPCA and BGN@Be treatments showed a slight inhibitory effect on the process, while OSPPB hydrogels displayed stronger inhibitory function. In addition, hydrogel treatment also prevented the tubule formation of EPCs in Matrigel under RNA-VEGF stimulation. The morphology and synapses of TG cells, which are trigeminal ganglion cells, were measured by crystal violet staining and immunofluorescence staining of β3-tubulin. Compared with TG cells treated with RNA-VEGF, cells treated with PPCA, BGN@Be, and OSPPB hydrogels showed shorter synapse length, fewer synaptic bifurcations, and simpler morphology. Experiments with different components of the hydrogels 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 hydrogels. These results indicate that OSPPB hydrogels are able to inhibit angiogenesis and neurogenesis in vitro.

[0089] The effects of TMJOA on condylar histological changes, maxillofacial pain, and nerve vascularization were studied in a mouse unilateral anterior crossbite model. Figure 9-12 The results showed that there were randomly scattered neovascularization in the UAC+Veh and UAC+Celecoxib groups, but not in the UAC+OSPPB group, such as Fig. 9 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 Fig. 9 As shown in c1, c2, dg, the abbreviation of Osteoarthritis Research Society International is OARSI. The glycine silver staining results showed that the number of nerve fibers decreased after OSPPB treatment, as shown in Fig. 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. Fig. 9 As shown in c4 and h. Through these microcracks, nerves, blood vessels, inflammatory factors and enzymes invade the cartilage, establish "osteoclast-chondrocyte interaction", and ultimately accelerate the development of OA. Micro-CT three-dimensional reconstruction showed that the joint surface was smoother in the OSPPB group, without erosion or irregularity, such as Fig. 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. Fig. 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. Fig. 9 As shown in Figure 1.

[0090] The osteochondral interface, which is about 50 μm thick, 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 by atomic force microscopy and scanning electron microscopy-energy dispersive spectrometer, where atomic force microscopy is abbreviated as AFM and scanning electron microscopy-energy dispersive spectrometer is abbreviated as SEM-EDS. Fig.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±0.22MPa to 9.06±0.53MPa. However, in the UAC group, the gradient of tissue modulus was disordered, forming a stiffer matrix with a hardness of up to 17.04MPa, which was restored after OSPPB treatment, but not after Celecoxib treatment, as shown in Figure 2 . Fig.10 As shown in a. Figure 5 As shown in Fig. 2a, 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 distribution of Ca and P elements close to that of the control group. The present invention distinguishes condylar cartilage from 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 demonstrates 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 include von Frey and electroencephalogram. Traditionally, EPM and OFT have been used to assess anxiety in rodents. Given that persistent chronic pain will eventually lead to anxiety, the present invention indirectly characterizes 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 the EPM confirmed that Celecoxib and OSPPB hydrogel alleviated anxiety-like behaviors, such as Fig.11 The results of OFT are consistent with this observation, as shown in a, ce. Fig.11 As shown in b, fh. Based on these observations, it was speculated that the administration of Celecoxib and OSPPB hydrogel may have relieved the anxiety caused by the ongoing 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 Fig.11 In addition, the EEG of the primary sensory cortex S1BF was captured immediately after the brush stimulation, as shown in Fig.11 The lower timestamp accuracy resulted in some interference around the expected time points. However, the present invention observed a decrease in spectral energy in the 0-20 Hz low frequency band after Celecoxib and OSPPB treatment, similar to the control group. These preliminary results indicate that mice showed significant relief of maxillofacial pain after treatment with Celecoxib and OSPPB hydrogel.

[0092] To verify the significant anatomical changes associated with pain relief, immunofluorescence experiments were performed. The exRNAs at the osteochondral interface were identified using the RNA-specific stain SYTO RNASelect Green Fluorescent Cell Dye and the cell membrane marker E-cadherin. Extracellular flocculently arranged RNAs away from the E-cadherin-positive area were defined as exRNAs in immunofluorescence staining. Fig.12 As shown in a and d, the amount of exRNA, which is 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 Fig.12In addition, the confocal laser scanning microscopy results of PGP 9.5 and CGRP are consistent with the above findings, as shown in Fig.12 In addition, compared with the UAC+Celecoxib group, the levels of vascular and neurovascular factor VEGF positive for platelet and endothelial cell adhesion molecule 1, also known as PECAM1, also known as CD31, decreased after OSPPB hydrogel treatment. Fig.12 Several factors closely related to bone intraocular perception and pain were further examined, including COX2, DCC, and SP, and the results showed that pain mediators were reduced after OSPPB hydrogel treatment, such as Fig.12 c, ik. Taken together, these data show that Celecoxib exhibits efficacy primarily in relieving pain, but its effect on the regenerative microenvironment was found to be rather limited. However, OSPPB hydrogel effectively impeded neurovascularization at the osteochondral interface and relieved pain in osteoarthritis.

[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 a preferred embodiment.

[0094] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other 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 can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

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 grafted polymer and bevacizumab sustained-release nanoparticles is 80-120:80-120:0.8-1.

2.

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; Polyethyleneimine and protocatechuic acid are subjected to an amidation reaction to synthesize the protocatechuic acid-polyethyleneimine graft polymer; 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 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; Bevacizumab sustained-release nanoparticles are dissolved in PBS to obtain solution A, phenylboronic acid-modified sodium alginate is dissolved in solution A to obtain solution B, protocatechuic acid-polyethyleneimine grafted polymer is dissolved in solution A to obtain solution C, and solution B and solution C are 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 to 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 to 25h in the dark, dialyzed with a molecular weight cutoff of 1800Da, and freeze-dried to obtain a 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 bioactive glass nanoparticles include SiO2, CaO and P2O5, and 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 arthritis.

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

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