An anti-inflammatory hydrogel and methods of making and using the same

Abstract

The application discloses an anti-inflammatory hydrogel as well as a preparation method and application thereof; the anti-inflammatory hydrogel is obtained by cross-linking four-arm polyethylene glycol thiol, eight-arm polyethylene glycol maleimide and metal coordination nanoparticles in a PBS buffer solution; the metal coordination nanoparticles are modified with diphosphonate, minocycline hydrochloride and Mg 2+ The metal coordination is composed of magnesium ions, phenolic hydroxyl groups on the minocycline hydrochloride and diphosphonate groups on the diphosphonate. The anti-inflammatory hydrogel has the performances of responding to a spinal cord injury microenvironment, releasing the anti-inflammatory drug minocycline hydrochloride, inhibiting the polarization of macrophages / microglia to M1 type and the like, can better play the drug effect of anti-inflammation and promoting spinal cord injury repair, has the advantages of convenient operation, no harmful by-products and the like, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the technical field of biomedicine, specifically relating to an anti-inflammatory hydrogel, its preparation method, and its uses. Background Technology

[0002] The pathological mechanism of spinal cord injury involves both primary and secondary injury. Inflammation plays a crucial role in secondary injury, exacerbating the damage. Microglia and macrophages are important immune cells involved in the inflammatory response following spinal cord injury, and the various factors they secrete directly regulate the inflammatory microenvironment. Microglia are innate immune cells present in the nervous system, while macrophages originate from monocytes that enter the injury site from the vascular endothelium. Due to their similar morphological characteristics and surface protein markers, they are indistinguishable at the site of spinal cord injury, and this group of cells is usually referred to as the microglia / macrophage population. In the injury area, microglia / macrophages exist in two basic subtypes: the "classical activated" type, or M1, which secretes pro-inflammatory factors and exhibits significant neurotoxicity; and the "alternative activated" type, or M2, which secretes anti-inflammatory factors and neurotrophic factors, promoting spinal cord functional remodeling. Microglia and macrophages play crucial roles in inflammatory responses, and regulating their phenotypes to improve the inflammatory microenvironment and promote tissue regeneration has become a research hotspot in spinal cord injury repair. During secondary pathophysiological responses, intrinsic microglia and infiltrating macrophages in the spinal cord transition from a quiescent M0 state to a pro-inflammatory M1 phenotype. M1 microglia / macrophages induce neuroinflammation by continuously expressing and releasing inflammatory chemokines and pro-inflammatory cytokines (such as inducible nitric oxide synthase (iNOS), interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), thereby triggering an inflammatory cascade that leads to increased inflammation and prolonged pro-inflammatory responses, further exacerbating neuronal damage and hindering spinal cord repair. Therefore, inhibiting the polarization of microglia / macrophages towards M1 may be an effective strategy for treating spinal cord injury.

[0003] Minocycline hydrochloride (MH) can selectively inhibit microglial polarization towards M1 both in vitro and in vivo. Minocycline hydrochloride belongs to the phenolic class and is a second-generation semi-synthetic tetracycline derivative, possessing antioxidant, anti-inflammatory, and neuroprotective activities, and can cross the blood-brain barrier. Studies have shown that minocycline hydrochloride can inhibit lipopolysaccharide (LPS)-mediated macrophage production of inflammatory cytokines and suppress the expression of key inflammation-related proteins such as iNOS. However, in previous minocycline delivery systems, the release of minocycline lacks microenvironment responsiveness, which can lead to overdose or subtherapeutic doses of minocycline release. After spinal cord injury, the microenvironment at the injury site is slightly acidic due to persistent ischemia and hypoxia. CN103797160A discloses a minocycline hydrochloride nano-sustained-release gel for periodontitis, but this gel lacks microenvironment responsiveness, and its preparation requires the use of organic solvents and involves cumbersome steps.

[0004] The literature “Wang Z, Nong J, Shultz RB, et al. Local delivery of minocycline from metal ion-assisted self-assembled complexes promotes neuroprotection and functional recovery after spinal cord injury[J]. Biomaterials, 2017, 112:62-71” describes a hydrogel delivery system composed of magnesium chloride, 2X HBSS, agarose, and minocycline, which has shown good efficacy in spinal cord repair. However, this delivery system requires strict temperature control and is cumbersome to operate. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides an anti-inflammatory hydrogel, and further provides a preparation method and uses for the anti-inflammatory hydrogel. The anti-inflammatory hydrogel of this invention can produce a sustained-release system that releases minocycline in response to acidic conditions. This system can release the anti-inflammatory drug minocycline in response to the acidic microenvironment of spinal cord injury, improve the immune microenvironment at the site of spinal cord injury, reduce and increase scar tissue at the site of spinal cord injury, promote the regeneration and growth of nerve axons, and thus promote the recovery of motor function. Furthermore, the preparation of this anti-inflammatory hydrogel is convenient and produces no harmful byproducts.

[0006] In a first aspect, the present invention provides an anti-inflammatory hydrogel, which is obtained by dissolving or dispersing four-armed polyethylene glycol thiol, eight-armed polyethylene glycol maleimide, and metal coordination nanoparticles respectively in PBS buffer, and then mixing and crosslinking them; wherein the metal coordination nanoparticles are composed of bisphosphonates modified with thiol groups, minocycline hydrochloride, and Mg...2+ Metal coordination composition; the preparation method of the thiol-modified bisphosphonate includes the following steps:

[0007] S1. Preparation of bisphosphonates modified with double bonds:

[0008] First, aminobisphosphonate is dissolved in NaOH solution, then acryloyl chloride is added, and after ice bath reaction, extraction, concentration, precipitation, centrifugation collection, and drying are performed to obtain bisphosphonate modified with double bonds;

[0009] S2. Preparation of bisphosphonates modified with thiol groups:

[0010] The bisphosphonate modified with double bonds obtained in step S1 was dissolved in deionized water, DL-dithiothreitol was added and the pH was adjusted to alkaline. The solution was stirred overnight at room temperature. After concentration, precipitation, centrifugation and drying, the bisphosphonate modified with thiol groups was obtained.

[0011] During the anti-inflammatory hydrogel molding process, the thiol groups on the four-arm polyethylene glycol thiol group and the maleimide groups on the eight-arm polyethylene glycol maleimide group crosslink through a click chemistry reaction (Michael addition reaction) to form a hydrogel; the thiol groups on the surface of the metal coordination nanoparticles and the maleimide groups on the eight-arm polyethylene glycol maleimide group form covalent bonds through a click chemistry reaction (Michael addition reaction) to load the metal nanoparticles into the hydrogel.

[0012] Further, in step S1, the mass-to-volume ratio of aminobisphosphonate, NaOH solution, and acryloyl chloride is 400mg-600mg: 20mL-30mL: 460μL-650μL, the mass concentration of the NaOH solution is 1%-3%, the ice bath reaction time is 1h, the reaction after adding acryloyl chloride is carried out at room temperature for 60min-120min, the extraction is performed using ethyl acetate, and the precipitation is treated with methanol.

[0013] Further, in step S2, the mass-to-volume ratio of the modified bisphosphonate with double bonds, deionized water, and DL-dithiothreitol is 200mg-300mg: 20mL-30mL: 750mg-1500mg, the alkaline pH value is 7.5-9.0, and the reaction after adjusting the pH value is carried out at room temperature for 8-15 hours.

[0014] Secondly, the present invention also provides a method for preparing the above-mentioned anti-inflammatory hydrogel, comprising the following specific steps:

[0015] S3. Preparation of metal coordination nanoparticles:

[0016] The thiol-modified bisphosphonates, magnesium chloride, and minocycline hydrochloride obtained in step S2 were dissolved in PBS buffer and mixed. After stirring at room temperature for a period of time, the precipitate was collected by centrifugation. The synthesized product was then freeze-dried in a freeze dryer to obtain metal coordination nanoparticles.

[0017] In metal-coordinated nano-ions, magnesium ions will bind to the phenolic hydroxyl groups on minocycline hydrochloride through metal coordination, and the bisphosphonate groups on bisphosphonates will bind to magnesium ions through metal coordination.

[0018] S4. Preparation of hydrogel:

[0019] The four-armed polyethylene glycol thiol, the eight-armed polyethylene glycol maleimide, and the metal coordination nanoparticles prepared in step S3 are dissolved or dispersed in PBS buffer and mixed evenly to prepare an anti-inflammatory hydrogel.

[0020] Further, in step S3, the mass ratio of the thiol-modified bisphosphonate, magnesium chloride, and minocycline hydrochloride is 200 mg–400 mg: 200 mg–400 mg: 2 mg–4 mg; the concentration of the thiol-modified bisphosphonate in PBS buffer is 0.075 mol / L–0.125 mol / L; the concentration of the magnesium chloride in PBS buffer is 0.075 mol / L–0.125 mol / L; the stirring time is 30 min–60 min; the centrifugation speed is 3500 r / min–4000 r / min; and the centrifugation time is 5 min–15 min.

[0021] Further, in step S4, the mass-to-volume ratio of the four-armed polyethylene glycol thiol group to the PBS buffer is 3 mg–10 mg: 30 μL–100 μL, the mass-to-volume ratio of the eight-armed polyethylene glycol maleimide to the PBS buffer is 3 mg–10 mg: 30 μL–100 μL, and the mass-to-volume ratio of the metal coordination nanoparticles to the PBS buffer is 2 mg–10 mg: 10 μL–20 μL.

[0022] Furthermore, the volume ratio of the four-armed polyethylene glycol thiol-PBS solution, the eight-armed polyethylene glycol maleimide-PBS solution, and the metal coordination nanoparticle-PBS suspension is 1–1.5:1–1.5:0.25–0.75.

[0023] Thirdly, the present invention further provides the use of the above-mentioned anti-inflammatory hydrogel in the preparation of anti-inflammatory drugs for spinal cord injury.

[0024] Compared with the prior art, the advantages of the present invention are as follows:

[0025] In previous minocycline delivery systems, the release of minocycline lacked microenvironment responsiveness, leading to overdose or subtherapeutic doses of minocycline release. Following spinal cord injury, the persistent ischemia and hypoxia at the injury site result in a slightly acidic microenvironment. This application constructs a metal-coordinated nanoparticle formed with the anti-inflammatory drug minocycline, loading these nanoparticles into a neuroprotective polyethylene glycol hydrogel to obtain an acid-responsive sustained-release system for minocycline. This system can release minocycline in response to the acidic microenvironment of spinal cord injury, improving the immune microenvironment at the spinal cord injury site, reducing and increasing scar tissue, promoting the regeneration and growth of nerve axons, and thus promoting the recovery of motor function. Furthermore, the minocycline delivery system constructed in this invention not only reduces TNF-α gene expression but also reduces the gene expression of inflammatory factors such as IL-6 and IL-1β. Attached Figure Description

[0026] Figure 1 The particle size and morphology results of the metal coordination nanoparticles provided by this invention include: A) particle size measurement results of MH@BP NPs; B) potential measurement results of MH@BP NPs; and C) morphology results of MH@BP NPs observed by scanning electron microscopy.

[0027] Figure 2 Scanning electron microscopy results for the anti-inflammatory hydrogel provided by this invention.

[0028] Figure 3 The results of minocycline release from the anti-inflammatory hydrogel provided by the present invention include: A) in vitro release results of different hydrogels; and B) release results of MH@BP Gel under different pH conditions.

[0029] Figure 4 The in vitro anti-inflammatory results of the anti-inflammatory hydrogel provided by the present invention include: A) observing the expression of iNOS after BV2 was treated with different hydrogel extracts by immunofluorescence; B) evaluating the expression of M1-related genes after BV2 was treated with different hydrogel extracts by qRT-PCR; and C) evaluating the expression of iNOS after BV2 was treated with different hydrogel extracts by Western blotting.

[0030] Figure 5 The in vivo anti-inflammatory results of the anti-inflammatory hydrogel provided by the present invention include: A) observing the expression of iNOS by immunofluorescence; B) evaluating the expression of iNOS by Western blotting; and C) evaluating the expression of CD86 by Western blotting.

[0031] Figure 6The in vivo effect of the anti-inflammatory hydrogel provided by this invention on promoting spinal cord injury repair includes: A) postoperative motor function score of SD rats; B) postoperative electrophysiological test results of SD rats; C) expression of GFAP at the spinal cord injury site; and D) expression of Tuj-1 at the spinal cord injury site. Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and specific preferred embodiments, but this does not limit the scope of protection of the present invention. It should be noted that the following detailed description is illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0033] Example 1: This example provides an anti-inflammatory hydrogel, which is obtained by dissolving or dispersing four-armed polyethylene glycol thiol, eight-armed polyethylene glycol maleimide, and metal coordination nanoparticles in PBS buffer, and then mixing and crosslinking them; the metal coordination nanoparticles are composed of thiol-modified bisphosphonates, minocycline hydrochloride, and Mg... 2+ The metal-coordinated composition is as follows: Specifically, the metal-coordinated nanoparticles are composed of bisphosphonates modified with thiol groups, magnesium chloride, and minocycline hydrochloride. Magnesium ions bind to the phenolic hydroxyl groups on minocycline hydrochloride via metal coordination, and the bisphosphonate groups on the bisphosphonates further bind to magnesium ions via metal coordination. This anti-inflammatory hydrogel material is in a hydrogel state.

[0034] Example 2: Based on Example 1, this example further provides a preparation method based thereon, including the following steps:

[0035] S1. Preparation of bisphosphonates modified with double bonds:

[0036] 593 mg of aminobisphosphonate (aBP) was dissolved in 20 mL of 2 wt% NaOH solution and reacted in an ice bath at 0°C. Acrylyl chloride was then added in four 45-minute portions, 162 μL each time. After the addition was complete, the mixture was removed from the ice bath and reacted at room temperature for 90 min. Ethyl acetate was then mixed with the sample solution at a 1:1 volume ratio and magnetically stirred for 6 min. The aqueous phase was extracted, and this process was repeated four times. Finally, the aqueous phase was rotary evaporated to a volume less than 10 mL. Upon addition of an appropriate amount of methanol, a large amount of white precipitate formed. After centrifugation at 4000 rpm for 10 min, the supernatant was discarded, and the precipitate was dried to obtain the double-bonded bisphosphonate (BP-acrylamide).

[0037] S2. Preparation of bisphosphonates modified with thiol groups:

[0038] 250 mg (0.87 mmol) of double-bonded bisphosphonate was dissolved in 25 mL (8.7 mmol) of deionized water, and 1335 mg of DL-dithiothreitol was added. After complete dissolution, the pH of the solution was adjusted to 8. The solution was stirred overnight at room temperature and concentrated using a vacuum rotary evaporator. Then, it was precipitated with acetone overnight, centrifuged (4000 rpm, 10 min), the supernatant was discarded, and the thiol-modified bisphosphonate was obtained after drying.

[0039] S3. Preparation of metal coordination nanoparticles:

[0040] 300 mg of thiol-modified bisphosphonate, 300 mg of magnesium chloride, and 300 mg of minocycline hydrochloride were dissolved in PBS buffer to prepare 200 mM, 200 mM, and 12.15 mM solutions, respectively. The solutions were mixed and stirred at room temperature for 1 hour. The precipitates were collected by centrifugation (4000 rpm, 5 minutes). Finally, the synthesized products were lyophilized in a freeze dryer to obtain metal coordination nanoparticles, named MH@BP NPs. Figure 1 A schematic diagram of the preparation of metal coordination nanoparticles and their characterization results.

[0041] S4. Preparation of hydrogel:

[0042] First, 5 mg of four-arm polyethylene glycol thiol and 5 mg of eight-arm polyethylene glycol maleimide were dissolved in PBS buffer to prepare 12.5% ​​solutions. Then, MH@BP nanoparticles were uniformly suspended in PBS buffer to prepare a 20% suspension. Finally, the three solutions were mixed thoroughly to obtain the anti-inflammatory hydrogel.

[0043] First, the particle size and morphology of the metal coordination nanoparticle material described in this invention are examined. Specifically, the metal coordination nanoparticles prepared in embodiment S3 are used, including the following steps:

[0044] The prepared metal coordination nanoparticles were resuspended in PBS, and the particle size and zeta potential of the MH@BP nanoparticles were measured by a nanoparticle size analyzer. The MH@BP nanoparticles were then resuspended in water and dropped onto a silicon wafer. After drying, the microstructure of the MH@BP nanoparticles was observed using a scanning electron microscope.

[0045] like, Figure 1 The image shows the particle size and morphology of the metal coordination nanoparticles obtained in step S3 of this embodiment. According to the nanoparticle size analyzer test results, the hydrated particle size of MH@BP NPs is 307.60 ± 8.80 nm. Figure 1 A) The polydispersity index (PDI) is 0.19 ± 0.02. Figure 1A); The zeta potential of MH@BP NPs is -20.73±0.50 Mv ( Figure 1 B), The negative charge of MH@BPNPs is mainly due to the negatively charged phosphate groups in the BP that form the nanoparticles; scanning electron microscopy (SEM) images show that MH@BP NPs have a uniform spherical morphology with a diameter of approximately 205.6 ± 23.2 nm. Figure 1 C).

[0046] Second, examine the morphology of the anti-inflammatory hydrogel material described in this invention, specifically using the anti-inflammatory hydrogel material prepared in embodiment S4, including the following steps:

[0047] First, the prepared hydrogel was pre-frozen in a -80°C freezer and then freeze-dried in a freeze dryer. Then, the freeze-dried hydrogel was subjected to liquid nitrogen brittle fracture to expose the fracture surface. After sputtering with gold for 60 seconds, the morphology of the freeze-dried hydrogel was observed using a scanning electron microscope.

[0048] like, Figure 2 The image shows the scanning electron microscope (SEM) results of the anti-inflammatory hydrogel prepared in step S4 of the present invention. As can be seen from the image, the freeze-dried hydrogels all exhibit a continuous porous structure, but only the inner walls of the anti-inflammatory hydrogel contain a large number of particulate MH@BP nanoparticles.

[0049] Third, the minocycline release of the anti-inflammatory hydrogel material of the present invention was examined, specifically using the anti-inflammatory hydrogel material prepared in embodiment S4, including the following steps:

[0050] The hydrogel was immersed in phosphate buffer solution and placed in a constant temperature shaker at 37°C; samples were taken at predetermined time points and its ultraviolet absorption at 244 nm was detected.

[0051] like, Figure 3 This document presents the minocycline release results of the anti-inflammatory hydrogel prepared in step S4 of this embodiment. In vitro drug release results show that minocycline hydrochloride, directly loaded onto the hydrogel, is almost completely released within 24 hours; while MH@BP NPs prepared from minocycline hydrochloride, loaded onto the hydrogel (MH@BP Gel), can maintain release for 11 days. This release result matches the acute phase (<48 hours) and subacute phase (48 hours to 14 days) after spinal cord injury, effectively regulating the inflammatory response after spinal cord injury. Furthermore, the release rate of minocycline is significantly accelerated under acidic conditions, indicating that the hydrogel is acid-responsive and can release minocycline in response to the slightly acidic environment of spinal cord injury.

[0052] Fourth, examine the in vitro anti-inflammatory properties of the anti-inflammatory hydrogel material in this embodiment. Specifically, the anti-inflammatory hydrogel material prepared in implementation scheme S4 ​​is used, including the following steps:

[0053] The anti-inflammatory activity of the hydrogel material was evaluated using BV2 cells. BV2 cells were seeded in well plates; after overnight incubation, the cells were treated with hydrogel extract in the presence of LPS. The anti-inflammatory activity of the hydrogel was evaluated 24 hours later by immunofluorescence, Western blotting, and qRT-PCR.

[0054] like, Figure 4 This presents the in vitro anti-inflammatory results of the anti-inflammatory hydrogel prepared in step S4 of the present invention. Immunofluorescence, RT-qPCR, and Western Blot results show that MH@BP Gel can significantly reduce the expression of genes and proteins related to the M1 type in microglia, and inhibit microglia polarization towards the M1 type.

[0055] Fifth, examine the in vivo anti-inflammatory properties of the anti-inflammatory hydrogel material of the present invention, specifically using the anti-inflammatory hydrogel material prepared in embodiment S4, including the following steps:

[0056] SD rats were anesthetized, their back fur was shaved, and the area was disinfected with 75% ethanol. The skin and subcutaneous muscles along the midline of the rat's back were incised to expose the T8-T10 vertebrae. A laminectomy was then performed at T9 to expose the spinal cord. The T9 segment of the spinal cord was clamped with 3mm-wide forceps for 10 seconds, and hemostasis was achieved with gelatin sponge. 50μL of hydrogel was applied to the injury site in the hydrogel group, while no treatment was given to the injury site in the control group. Finally, the muscles and skin were sutured together. SD rats were randomly assigned to four groups: a control group, a pure hydrogel group, a hydrogel-loaded drug-free group, and a hydrogel-loaded nanoparticle group, with six rats in each group. The control group received no treatment after spinal cord clamping, while the other groups used different hydrogel materials according to their grouping characteristics. Anti-inflammatory levels were assessed 3 days later.

[0057] like, Figure 5 This presents the in vivo anti-inflammatory results of the anti-inflammatory hydrogel prepared in step S4 of the present invention. Simultaneously, the in vivo anti-inflammatory effect of the hydrogel was also studied using a rat spinal cord injury model. Immunofluorescence and Western blotting results showed that MH@BP Gel significantly reduced the expression of M1 type proteins iNOS and CD86, indicating that MH@BP Gel can inhibit the polarization of microglia / macrophages to the M1 type in vivo.

[0058] Example 3: Based on Example 1, this example further provides a preparation method based thereon, including the following steps:

[0059] S1. Preparation of bisphosphonates modified with double bonds:

[0060] 400 mg of aminobisphosphonate (aBP) was dissolved in 30 mL of 1 wt% NaOH solution and reacted in an ice bath at 0°C. Acrylyl chloride was then added in four portions over 45 min each, 132 μL each time. After the addition was complete, the mixture was removed from the ice bath and reacted at room temperature for 90 min. Ethyl acetate was then mixed with the sample solution at a 1:1 volume ratio and magnetically stirred for 6 min. The aqueous phase was extracted, and this process was repeated four times. Finally, the aqueous phase was rotary evaporated to a volume less than 10 mL. Upon addition of an appropriate amount of methanol, a large amount of white precipitate formed. After centrifugation at 4000 rpm for 10 min, the supernatant was discarded, and the precipitate was dried to obtain the double-bonded bisphosphonate (BP-acrylamide).

[0061] S2. Preparation of bisphosphonates modified with thiol groups:

[0062] 200 mg (0.704 mmol) of double-bonded bisphosphonate was dissolved in 20 mL (7.04 mmol) of deionized water, and 750 mg of DL-dithiothreitol was added. After complete dissolution, the pH of the solution was adjusted to 8. The solution was stirred overnight at room temperature and concentrated using a vacuum rotary evaporator. Then, it was precipitated with acetone overnight, centrifuged (4000 rpm, 10 min), the supernatant was discarded, and the thiol-modified bisphosphonate was obtained after drying.

[0063] S3. Preparation of metal coordination nanoparticles:

[0064] 200 mg of thiol-modified bisphosphonate, 200 mg of magnesium chloride, and 2 mg of minocycline hydrochloride were dissolved in PBS buffer to prepare 200 mM, 200 mM, and 12.15 mM solutions, respectively. The solutions were mixed and stirred at room temperature for 1 hour. The precipitate was collected by centrifugation (4000 rpm, 5 minutes). Finally, the synthesized product was lyophilized in a freeze dryer to obtain metal-coordinated MH@BP nanoparticles.

[0065] S4. Preparation of hydrogel:

[0066] First, 3 mg of four-arm polyethylene glycol thiol and 3 mg of eight-arm polyethylene glycol maleimide were dissolved in PBS buffer to prepare 12.5% ​​solutions. Then, MH@BP nanoparticles were uniformly suspended in PBS buffer to prepare a 20% suspension. Finally, the three solutions were mixed thoroughly to obtain the anti-inflammatory hydrogel.

[0067] Example 4: Based on Example 1, this example further provides a preparation method based thereon, including the following steps:

[0068] S1. Preparation of bisphosphonates modified with double bonds:

[0069] 593 mg of aminobisphosphonate (aBP) was dissolved in 20 mL of 2 wt% NaOH solution and reacted in an ice bath at 0°C. Acrylyl chloride was then added in four 45-minute portions, 162 μL each time. After the addition was complete, the mixture was removed from the ice bath and reacted at room temperature for 90 min. Ethyl acetate was then mixed with the sample solution at a 1:1 volume ratio and magnetically stirred for 6 min. The aqueous phase was extracted, and this process was repeated four times. Finally, the aqueous phase was rotary evaporated to a volume less than 10 mL. Upon addition of an appropriate amount of methanol, a large amount of white precipitate formed. After centrifugation at 4000 rpm for 10 min, the supernatant was discarded, and the precipitate was dried to obtain the double-bonded bisphosphonate (BP-acrylamide).

[0070] S2. Preparation of bisphosphonates modified with thiol groups:

[0071] The double-bonded bisphosphonate (300 mg, 1.044 mmol) was dissolved in deionized water (30 mL, 10.44 mmol), and DL-dithiothreitol (1500 mg) was added. After complete dissolution, the pH of the solution was adjusted to 8. The solution was stirred overnight at room temperature and concentrated using a vacuum rotary evaporator. Then, it was precipitated with acetone overnight, centrifuged (4000 rpm, 10 min), the supernatant was discarded, and the thiol-modified bisphosphonate was obtained after drying.

[0072] S3. Preparation of metal coordination nanoparticles:

[0073] 400 mg of thiol-modified bisphosphonate, 400 mg of magnesium chloride, and 400 mg of minocycline hydrochloride were dissolved in PBS buffer to prepare 200 mM, 200 mM, and 12.15 mM solutions, respectively. The solutions were mixed and stirred at room temperature for 1 hour. The precipitates were collected by centrifugation (4000 rpm, 5 minutes). Finally, the synthesized products were lyophilized in a freeze dryer to obtain metal coordination nanoparticles, named MH@BP NPs. Figure 1 A schematic diagram of the preparation of metal coordination nanoparticles and their characterization results.

[0074] S4. Preparation of hydrogel:

[0075] First, 10 mg of four-arm polyethylene glycol thiol and 10 mg of eight-arm polyethylene glycol maleimide were dissolved in PBS buffer to prepare 12.5% ​​solutions. Then, MH@BP nanoparticles were uniformly suspended in PBS buffer to prepare a 20% suspension. Finally, the three solutions were mixed thoroughly to obtain the anti-inflammatory hydrogel.

[0076] It should be noted that the hydrogels prepared in Examples 3 and 4 have similar or identical phases and properties to those in Example 2.

[0077] Example 5: This example further provides an application of the anti-inflammatory hydrogel described in Example 1: the application of the anti-inflammatory hydrogel in anti-inflammatory drugs for spinal cord injury.

[0078] To investigate the in vivo effect of the anti-inflammatory hydrogel material of the present invention on promoting spinal cord injury repair, the anti-inflammatory hydrogel material prepared in embodiment S4 was used, including the following steps:

[0079] After anesthetizing SD rats, the fur on their backs was shaved and the area disinfected with 75% ethanol. The skin and subcutaneous muscles along the midline of the rat's back were incised to expose the T8-T10 vertebrae. Then, a laminectomy was performed at T9 to expose the spinal cord. The T9 segment of the spinal cord was clamped with 3mm forceps for 10 seconds, and hemostasis was achieved with gelatin sponge. 50μL of hydrogel was applied to the injury site in the hydrogel group, while no treatment was given to the injury site in the control group. Finally, the muscles and skin were sutured together. SD rats were randomly divided into a control group, a pure hydrogel group, a hydrogel-loaded drug-free group, and a hydrogel-loaded nanoparticle group, with 6 rats in each group. The spinal cord in the control group received no treatment after clamping, while the other groups used different hydrogel materials according to their grouping characteristics. After 6 weeks, tissue samples were collected to assess spinal cord repair and recovery of motor function.

[0080] like, Figure 6 This diagram illustrates the application of the anti-inflammatory hydrogel prepared in step S4 of the present invention to promote spinal cord injury repair in vivo. After surgery, the recovery of hind limb motor function in rats was scored. The results showed that the MH@BP Gel group exhibited better motor function recovery at all time points, indicating that MH@BP Gel can significantly promote the recovery of motor function in rats with spinal cord injury. Six weeks after surgery, motor evoked potentials in SD rats were detected using an electrophysiological instrument, yielding results consistent with the motor function scores, demonstrating that MH@BP Gel can significantly promote spinal cord injury repair, leading to better signal transduction in the injured spinal cord.

[0081] This invention further investigated the distribution and formation of glial fibrillary acidic protein and chondroitin sulfate after hydrogel treatment. The results showed that MH@BP Gel significantly reduced the expression of glial fibrillary acidic protein and chondroitin sulfate at the site of spinal cord injury, indicating that MH@BP Gel can reduce the formation of scar tissue after spinal cord injury. Functional recovery after spinal cord injury is often caused by neuronal regeneration, and the above results demonstrate that MH@BP Gel can improve the motor function of rats with spinal cord injury. Therefore, this invention also investigated the in vivo neuronal cell regeneration after MH@BP Gel treatment. Immunofluorescence results showed that MH@BP Gel can significantly increase the retention of mature neurons and promote the generation of new neurons, indicating that MH@BP Gel can promote neuronal regeneration.

[0082] In summary, this invention provides an anti-inflammatory hydrogel, its preparation method, and its uses. The anti-inflammatory hydrogel of this invention can produce a sustained-release system that releases minocycline in response to acid. This system can release the anti-inflammatory drug minocycline in response to the acidic microenvironment of spinal cord injury, improving the immune microenvironment at the site of spinal cord injury, reducing and increasing scar tissue at the site of spinal cord injury, promoting the regeneration and growth of nerve axons, and thus promoting the recovery of motor function. Furthermore, the preparation of this anti-inflammatory hydrogel is convenient and produces no harmful byproducts.

[0083] In existing technologies, the release of minocycline in conventional minocycline delivery systems lacks microenvironment responsiveness, leading to overdose or subtherapeutic doses of minocycline release. Following spinal cord injury, the microenvironment at the injury site is slightly acidic due to persistent ischemia and hypoxia. This application addresses this by loading nanoparticles into a neuroprotective polyethylene glycol hydrogel, resulting in an acid-responsive sustained-release system for minocycline. This system responds to the acidic microenvironment of spinal cord injury by releasing the anti-inflammatory drug minocycline, improving the immune microenvironment at the spinal cord injury site, reducing and increasing scar tissue, promoting nerve axon regeneration and growth, and thus promoting motor function recovery. Furthermore, the minocycline delivery system constructed from the anti-inflammatory hydrogel provided by this invention not only reduces TNF-α gene expression but also reduces the gene expression of inflammatory factors such as IL-6 and IL-1β, making this anti-inflammatory hydrogel a promising candidate for anti-inflammatory drugs in spinal cord injury treatment.

[0084] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention shall still fall within the protection scope of the technical solutions of the present invention.

Claims

1. An anti-inflammatory hydrogel, characterized in that, This anti-inflammatory hydrogel was prepared by dissolving or dispersing four-armed polyethylene glycol thiol, eight-armed polyethylene glycol maleimide, and metal coordination nanoparticles separately in PBS buffer, followed by cross-linking. The metal coordination nanoparticles consist of thiol-modified bisphosphonates, minocycline hydrochloride, and Mg... 2+ Metal coordination composition; wherein, the preparation method of the thiol-modified bisphosphonate includes the following steps: S1. Preparation of bisphosphonates modified with double bonds: First, aminobisphosphonate is dissolved in NaOH solution, then acryloyl chloride is added, and after ice bath reaction, extraction, concentration, precipitation, centrifugation collection, and drying are performed to obtain bisphosphonate modified with double bonds; S2. Preparation of bisphosphonates modified with thiol groups: The bisphosphonate modified with double bonds obtained in step S1 was dissolved in deionized water, DL-dithiothreitol was added and the pH was adjusted to alkaline. The solution was stirred overnight at room temperature. After concentration, precipitation, centrifugation and drying, the bisphosphonate modified with thiol groups was obtained.

2. The anti-inflammatory hydrogel according to claim 1, characterized in that, In step S1, the mass-to-volume ratio of aminobisphosphonate, NaOH solution, and acryloyl chloride is 400mg-600mg: 20mL-30mL: 460μL-650μL, the mass concentration of the NaOH solution is 1%-3%, the ice bath reaction time is 1h, the reaction after adding acryloyl chloride is carried out at room temperature for 60min-120min, the extraction is performed with ethyl acetate, and the precipitation is treated with methanol.

3. The anti-inflammatory hydrogel according to claim 2, characterized in that, In step S2, the mass-to-volume ratio of the modified bisphosphonate with double bonds, deionized water, and DL-dithiothreitol is 200mg-300mg: 20mL-30mL: 750mg-1500mg, the alkaline pH value is 7.5-9.0, and the reaction after adjusting the pH value is carried out at room temperature for 8-15 hours.

4. A method for preparing an anti-inflammatory hydrogel as described in any one of claims 1-3, characterized in that, The specific steps include the following: S3. Preparation of metal coordination nanoparticles: The thiol-modified bisphosphonates, magnesium chloride, and minocycline hydrochloride obtained in step S2 were dissolved in PBS buffer and mixed. After stirring at room temperature for a period of time, the precipitate was collected by centrifugation. The synthesized product was then freeze-dried in a freeze dryer to obtain metal coordination nanoparticles. S4. Preparation of hydrogel: The four-armed polyethylene glycol thiol, the eight-armed polyethylene glycol maleimide, and the metal coordination nanoparticles prepared in step S3 are dissolved or dispersed in PBS buffer and mixed evenly to prepare an anti-inflammatory hydrogel.

5. The method for preparing the anti-inflammatory hydrogel according to claim 4, characterized in that, In step S3, the mass ratio of the thiol-modified bisphosphonate solution, magnesium chloride solution, and minocycline hydrochloride is 200mg-400mg:200mg-400mg:2mg-4mg. The concentration of the thiol-modified bisphosphonate solution is 0.075mol / L-0.125mol / L, the concentration of the magnesium chloride solution is 0.075mol / L-0.125mol / L, the stirring time is 30min-60min, the centrifugation speed is 3500r / min-4000r / min, and the centrifugation time is 5min-15min.

6. The method for preparing the anti-inflammatory hydrogel according to claim 5, characterized in that, In step S4, the mass-to-volume ratio of the four-armed polyethylene glycol thiol group to the PBS buffer is 3 mg–10 mg: 30 μL–100 μL; the mass-to-volume ratio of the eight-armed polyethylene glycol maleimide to the PBS buffer is 3 mg–10 mg: 30 μL–100 μL; and the mass-to-volume ratio of the metal coordination nanoparticles to the PBS buffer is 2 mg–10 mg: 10 μL–20 μL.

7. The method for preparing the anti-inflammatory hydrogel according to claim 6, characterized in that, In step S4, the volume ratio of the four-armed polyethylene glycol thiol-PBS solution, the eight-armed polyethylene glycol maleimide-PBS solution, and the metal coordination nanoparticle-PBS suspension is 1–1.5:1–1.5:0.25–0.

75.

8. Use of the anti-inflammatory hydrogel according to any one of claims 1-3 in the preparation of anti-inflammatory drugs for spinal cord injury.

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