Drug-loaded temperature-sensitive hydrogel and preparation method and use thereof

Abstract

The application discloses a drug-loaded temperature-sensitive hydrogel and a preparation method and application thereof. The ROS-sensitive crosslinking agent TK and a drug BETA are modified to hyaluronic acid to synthesize a HA-TK-BETA polymer, the HA-TK-BETA polymer can self-assemble in physiological saline, a hydrophobic cavity formed in the interior can load ROPI in a solution to form a drug-loaded particle, and the particle solution is mixed with a PLGA-PEG-PLGA temperature-sensitive polymer to form a temperature-sensitive polymer particle solution with a body temperature gel forming capacity. After injecting the mixed solution of the microparticles and the temperature-sensitive polymer around a vertebra, a gel can be formed around the protruding IVD. The drug-loaded temperature-sensitive gel can respond to ROS in an inflammatory environment, break the TK bond, release the drug in the hydrogel, relieve inflammation around the protruding IVD, effectively relieve pain, relieve LDH from the perspective of inhibiting inflammation, improve the curative effect, and have better patient acceptance.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a drug-loaded thermosensitive hydrogel, its preparation method, and its uses. Background Technology

[0002] Low back pain (LBP) is a global disease affecting almost all age groups. Due to its high prevalence and disability rate, the annual treatment cost in the United States exceeds $100 billion, placing a significant economic burden on society and families. The most common cause of LBP is lumbago (LDH). Inflammation plays a crucial role in the progression of LDH, primarily including inflammation around the intervertebral disc (IVD), inflammation of the nucleus pulposus (NPP) within the IVD, and neurological inflammation.

[0003] Currently, the main clinical treatment methods include traditional drug therapy, surgical treatment, and stem cell therapy. Traditional drug therapy is the most widely used due to its minimal invasiveness and low cost. Betamethasone (BETA) and ropivacaine (ROPI) are first-line drugs in clinical practice. BETA is a commonly used glucocorticoid, but long-term use can still lead to many serious side effects such as persistently elevated blood sugar, osteoporosis, and avascular necrosis of the femoral head. ROPI is a new type of long-acting amide local anesthetic with strong anesthetic efficacy. Compared with other analgesics in the same class, ROPI has lower toxicity and better hemodynamic properties, but its neurotoxicity may cause nerve cell damage.

[0004] Biomaterial-based drug delivery systems are increasingly being used in the treatment of IVD degenerative diseases. Among these, hydrogels are widely used due to their excellent water retention and ease of modification. Hydrogels can be used to deliver exosomes, gene therapy drugs, antibody drugs, and protein drugs, treating the condition by inhibiting inflammation, maintaining microenvironment stability, remodeling biological properties, and promoting cell regeneration. For example, loading growth hormone analogs into thermosensitive hydrogels allows for drug release in response to ROS (reactive oxygen species), inhibiting inflammation and NPP autophagy. Tannic acid nanoparticles and miRNA inhibitors are also loaded into thermosensitive hydrogels, where anti-inflammatory and ECM regeneration effects synergistically enhance the treatment of intervertebral disc degeneration. This invention stems from this. Summary of the Invention

[0005] In order to solve at least one of the technical problems in the background art of this invention, this invention provides a drug-loaded thermosensitive hydrogel, its preparation method and uses.

[0006] The technical solution of the present invention is as follows: One objective of the present invention is to provide a method for preparing drug-loaded thermosensitive hydrogels. The drug BETA is modified onto HA using a ROS-sensitive crosslinking agent TK to synthesize an HA-TK-BETA polymer. The HA-TK-BETA polymer can self-assemble in physiological saline, and the hydrophobic cavities formed internally can encapsulate ROPI in the solution, forming drug-loaded particles. The particle solution is mixed with a PLGA-PEG-PLGA thermosensitive polymer to form a drug-loaded thermosensitive polymer particle solution with body temperature gelation capability.

[0007] Another object of the present invention is to provide a drug-loaded thermosensitive hydrogel MP@Gel prepared by the above preparation method.

[0008] Another object of the present invention is to provide the use of the above-mentioned drug-loaded thermosensitive hydrogel in the preparation of anti-inflammatory drugs, especially in the preparation of long-acting drugs for the treatment of intervertebral disc herniation.

[0009] The drug-loaded thermosensitive hydrogel of the present invention is a thermosensitive polymer particle solution with body temperature gelation ability formed by mixing a drug-loaded particle solution of HA-TK-BETA polymer loaded with ROPI with a PLGA-PEG-PLGA thermosensitive polymer. It can not only relieve LDH from the perspective of inhibiting inflammation, but also effectively relieve patients' pain symptoms and improve efficacy. It solves the defects of existing technologies using anti-inflammatory drugs such as betamethasone and analgesics such as ropivacaine, which have many administration times, insufficient efficacy, short duration of action and large toxic side effects, and can be better accepted by patients. Attached Figure Description

[0010] Figure 1 A schematic diagram illustrating the preparation process and treatment method of drug-loaded thermosensitive hydrogels. Figure 1 A represents the preparation method; Figure 1 B is a schematic diagram of LDH treatment;

[0011] Figure 2 For infrared spectrum comparison ( Figure 2 A represents the infrared standard of HA; Figure 2 B is the infrared spectrum of HA-TK;

[0012] Figure 3 A schematic diagram of the characterization of BETA-1 ( Figure 3 A represents BETA and BETA-1. 1 Comparison of H NMR spectra; Figure 3 B represents BETA and BETA-1. 13 Comparison of C NMR spectra; Figure 3 C represents the HPLC-MS / MS spectrum of BETA-1;

[0013] Figure 4 A schematic diagram of the characterization of polymer HA35-TK-BETA ( Figure 4 A shows a comparison of the HPLC chromatograms of Beta-1 and HA-TK-BETA; Figure 4 B represents BETA-1 and HA-TK-BETA. 19 Comparison of F NMR spectra; Figure 4 C represents BETA, BETA-1, and HA-TK-BETA. 1 Comparison of H NMR spectra; Figure 4 D is a comparison of the infrared spectra of HA-TK and HA-TK-BETA;

[0014] Figure 5 The middle image shows a schematic diagram of the characterization of particles obtained from the synthesis of HA with different molecular weights. Figure 5 A represents the particle size distribution of H5TB; Figure 5 B represents the particle size distribution of H5TB / R; Figure 5 C represents the ZETA potential diagram of H5TB and H5TB / R; Figure 5 D represents the particle size distribution of H10TB; Figure 5 E represents the particle size distribution of H10TB / R; Figure 5 F represents the ZETA potential diagram of H10TB and H10TB / R; Figure 5 G is a SEM image of H35TB particles; Figure 5 H is the SEM image of H35TB / R particles; Figure 5 I represents the ZETA potential diagrams for H35TB particles and H35TB / R particles; Figure 5 J is a SEM image of H100TB particles; Figure 5 K is the SEM image of H100TB / R particles; Figure 5 L represents the ZETA potential diagram of H100TB and H100TB / R particles;

[0015] Figure 6 A schematic diagram illustrating the method for determining the BETA and ROPI loading in prepared particles. Figure 6 A shows the UV spectra of HA, HA-TK, HA-TK-BETA, and BETA in formamide; Figure 6 B represents the standard curve of the absorbance of BETA at 280 nm in formamide; Figure 6 C represents the UV spectra of HA, HA-TK, HA-TK-BETA, and ROPI in methanol; Figure 6 D is the standard curve of ROPI absorbance at 220 nm in methanol; Figure 6 E represents the in vitro release kinetics of H10TB / R; Figure 6 F represents the in vitro release kinetics of H35TB / R;

[0016] Figure 7 This is a schematic diagram illustrating the characterization of the thermosensitive hydrogel of the present invention. Figure 7 A represents the form of the thermosensitive polymer solution at different temperatures; Figure 7 B represents the phase transition curves of temperature-sensitive polymers at different concentrations; Figure 7 C is the SEM image of the thermosensitive hydrogel; Figure 7 D is the UV absorption spectrum of HTB / R(MP) in PBS; Figure 7 E represents the standard curve of the absorbance of MP at 240 nm; Figure 7 F represents the loading efficiency of the hydrogel for different masses of MP.

[0017] Figure 8 This is a schematic diagram illustrating the in vitro degradation and drug release kinetics characterization of the thermosensitive hydrogel of the present invention. Figure 8 A represents the degradation of NP@Gel in the in vitro environment; Figure 8 B represents the degradation of MP@Gel in the in vitro environment; Figure 8 C represents the in vitro drug release kinetics of NP@Gel; Figure 8 D represents the in vitro drug release kinetics of MP@Gel;

[0018] Figure 9 The in vitro anti-inflammatory ability of drug-loaded thermosensitive hydrogels ( Figure 9 A represents the drug-loaded thermosensitive hydrogel of the present invention that inhibits LPS-induced inflammatory responses in BV2 cells; Figure 9 B represents the inflammatory response of NPC cells; Figure 9 C represents PM cell inflammatory response, and the groups are arranged sequentially from left to right on the horizontal axis;

[0019] Figure 10 The drug-loaded thermosensitive hydrogel of the present invention alleviates inflammatory damage in NPC (NPC). Figure 10 A shows the effect of different concentrations of TNF-α on NPC activity after co-incubation with NPC for 24 hours. Figure 10 B represents the effect of different concentrations of TNF-α on NPC activity after co-incubation with NPC for 96 hours. Figure 10 The alleviating effect of the drug on TNF-α-induced NPC apoptosis at C = 24h; Figure 10 (The effect of the drug on TNF-α-induced NPC apoptosis when D is 96h);

[0020] Figure 11 The drug-loaded thermosensitive hydrogel of the present invention alleviates inflammatory damage of PC12;

[0021] Figure 12 Cytotoxicity study of the drug-loaded thermosensitive hydrogel of the present invention;

[0022] Figure 13 Construction and validation of LDH model ( Figure 13 A is a schematic diagram of the rat LDH model construction; Figure 13 B represents the mechanical pain sensitivity of rats after LDH modeling; Figure 13 C represents the change in thermal pain sensitivity; Figure 13 D represents the expression level of IL-6 protein in serum, IVD and surrounding tissues, and dorsal root ganglion of the Sham group and LDH modeling group. Figure 13 E represents the IL-1β protein expression levels in serum, IVD and surrounding tissues, and DRG of the Sham group and LDH modeling group; Figure 13 F represents the mRNA expression levels of IFN-γ, PGE-2, and NGF in the DRG of the Sham group and the LDH modeling group.

[0023] Figure 14 For in vivo efficacy comparison and behavioral verification ( Figure 14 A is an X-ray image of the injection site; Figure 14 B represents the analgesic effect of NP@Gel and MP@Gel; Figure 14 C is the experimental flowchart; Figure 14 D represents the behavioral changes related to mechanical pain; Figure 14 E represents behavioral changes related to heat pain.

[0024] Figure 15 For the in vivo release of the drug-loaded thermosensitive hydrogel of the present invention ( Figure 15 A shows IVIS fluorescence imaging images of rats at different time points; Figure 15 B represents the quantitative analysis of local fluorescence intensity.

[0025] Figure 16 The expression levels of pro-inflammatory cytokines and pain-related molecules 7 days after treatment ( Figure 16 A is the experimental flowchart; Figure 16 B represents the expression level of IL-6 protein in serum, IVD, and DRG; Figure 16 C represents the expression level of IL-1β protein in serum, IVD, and DRG; Figure 16 D represents the mRNA expression levels of IFN-γ, PEG-2, and NGF in DRG.

[0026] Figure 17 The expression levels of pro-inflammatory cytokines and pain-related molecules after 11 days of treatment ( Figure 17 A is the experimental flowchart; Figure 17 B represents the expression level of IL-6 protein in serum, IVD, and DRG; Figure 17 C represents the expression level of IL-1β protein in serum, IVD, and DRG; Figure 17 D represents the mRNA expression levels of IFN-γ, PEG-2, and NGF in DRG. Detailed Implementation

[0027] The above solution will be further explained below with reference to the accompanying drawings and specific embodiments.

[0028] 1.1 Experimental Animals: Adult male SD rats, 6-8 weeks old, weighing 200-250g, were housed in the temporary storage room of the animal experimental center and purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. The animal experimental handling and operations involved in this application all comply with the animal experimental ethics guidelines and laboratory animal welfare requirements of Soochow University.

[0029] 1.2 Reagents: Hyaluronic acid (HA, 5, 10, 35, 100 kDa) was purchased from Chongqing Yusi Pharmaceutical Technology Co., Ltd. DMAP, NHS, and EDC were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Lipopolysaccharide (LPS) was purchased from Shanghai Beyotime Biotechnology Co., Ltd. IFN-γ was purchased from Peprotech, USA. Mouse-derived IL-6, IL-1β, and TNF-α ELISA kits were purchased from Thermo Fisher Scientific, USA. H2O2 was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. TK-NH2 was purchased from Chongqing Yusi Pharmaceutical Technology Co., Ltd.

[0030] 1.3 Experimental Methods:

[0031] 1.3.1 Synthesis of HA-TK

[0032] Weigh 200 mg of hyaluronic acid (HA, molecular weight 35k, i.e., 0.53 mmol) and dissolve it in 10 mL of deionized water. Add 305.28 mg (1.59 mmol) of EDC (dichloroethane) and 182.85 mg (1.59 mmol) of NHS (N-hydroxysuccinimide). Activate in an oil bath at 45°C for 1 h. Add 103.01 mg (0.53 mmol) of TK (ROS-sensitive crosslinking agent, ketethiol). Stir and react at 45°C for 24 h. After 24 h, remove the sample and add it to a 3500 Da dialysis bag for dialysis. The dialysis medium is deionized water. Change the water every 12 h. Dialyze for three days. Finally, freeze-dry the obtained dialysate sample using a vacuum freeze dryer.

[0033] 1.3.2 Synthesis of BETA-1

[0034] 500 mg (1.27 mmol) of BETA, 152.4 mg (1.524 mmol) of DDSA (dodecyl succinic anhydride), and 186.19 mg (1.524 mmol) of DMAP (4-dimethylaminopyridine) were weighed and dissolved in 10 mL of a 1:1 mixture of DCM (dichloromethane) and CAN (acetonitrile). 211.8 μL (1.524 mmol) of TEA (triethylamine) was added, and the mixture was stirred overnight. After the reaction was complete, thin-layer chromatography (developing solvent: EA (ethyl acetate)-PE (petroleum ether), 3:2, 1 drop of formic acid) under a darkroom UV detector revealed the formation of a new product. The reaction solution was purified by column chromatography (EA-PE = 30:20). The collected sample was evaporated under reduced pressure using a rotary evaporator to obtain 596.19 mg of a white solid powder, with a yield of 95.31%.

[0035] 1.3.3 Synthesis of HA-TK-BETA

[0036] Dissolve 150 mg (0.25 mmol) of HA-TK in 10 mL of formamide. Weigh 123.14 mg (0.25 mmol) of BETA-1 and 284.43 mg (0.75 mmol) of HBTU (O-benzotriazole-tetramethylurea hexafluorophosphate) and dissolve them in 3 mL of DMSO (dimethyl sulfoxide). Add the DMSO solution to the formamide solution, and then add 162.93 μL (0.75 mmol) of DIPEA (N,N-diisopropylethylamine) at room temperature. Stir the reaction mixture at 45 °C for 24 h. Add the reaction solution to a 3500 Da dialysis bag and dialyze it against DMSO, anhydrous ethanol, 50% ethanol, and deionized water for 24 h each. Change the dialysate approximately every 12 h. Finally, freeze-dry the sample in the dialysis bag.

[0037] 1.3.4 Synthesis of HA-TK-BETA / ROPI particles (HTB / R particles)

[0038] HA-TK-BETA / ROPI was prepared using an emulsion solvent evaporation method. 100 mg of HA-TK-BETA was dissolved in 5 mL of deionized water in a round-bottom flask. 100 mg of ROPI was weighed and dissolved in 3 mL of DCM. The HA-TK-BETA solution was placed on a magnetic stirrer with a rotor at 100 rpm / min and stirred at 25°C. The DCM solution was slowly added dropwise to the HA-TK-BETA solution, resulting in a layered solution. The upper layer was a deionized aqueous solution of HA-TK-BETA, and the lower layer was a DCM solution of ROPI. The layered solutions were ultrasonicated at 40 kHz for 30 min at 4°C, yielding a milky white solution. The ultrasonicated solution was then placed in a fume hood at 100 rpm and 25°C with the opening open and stirred overnight to allow the DCM to evaporate. The next day, white, insoluble ROPI was found at the bottom of the flask. Add 4 mL of DCM to the reaction solution and shake well to dissolve the unreacted ROPI. Use a separatory funnel to release the lower layer of DCM solution and collect the upper layer solution. Freeze dry the solution using a vacuum freeze dryer to obtain the dried product.

[0039] 1.3.5 Preparation of drug-loaded thermosensitive hydrogel

[0040] Weigh 30 mg of HA-TK-BETA / ROPI particles and add PBS (phosphate buffer) to prepare a 2 mg / mL solution. Take 20%, 22%, and 24% polymer solutions and vortex thoroughly. Add an appropriate amount of water to the metal bath heating module and set the temperature to 25°C. Place the vial containing the polymer solution below the liquid surface. After the temperature stabilizes for 5 minutes, increase the temperature by 1°C each time. Invert the vial and observe whether the colloid flows down the tube wall. If no liquid flows down, a gel has formed. If a white substance appears in the colloid, the polymer has precipitated. Record the temperature points of each gelation and precipitation. Freeze the gelled polymer in liquid nitrogen to solidify it. Remove the solid and freeze-dry it in a freeze dryer. Observe the morphology and structure of the final sample under a scanning electron microscope.

[0041] 1.3.6 Evaluation of the in vitro anti-inflammatory efficacy of drug-loaded thermosensitive hydrogels

[0042] Since intervertebral disc herniation (LDH) can lead to macrophage infiltration, activation of nucleus pulposus cells (NPCs), and neuronal inflammation, this invention uses peritoneal macrophages (PMs) and NPCs extracted from rats, as well as the BV2 cell line, which are activated with LPS (lipopolysaccharide) to simulate local inflammation of IVD, NPC inflammation, and neuronal inflammation, thus comprehensively simulating the complex inflammation following LDH.

[0043] The anti-inflammatory effect of the gel was evaluated by measuring cytokine levels using ELISA. BV2, NPC, and PM cells were evenly seeded in flat-bottomed 24-well plates, with 1.0 mL of cell suspension and 2 × 10⁶ cells per well. 4 Cells were collected in 24-well plates and incubated overnight at 37°C with 5% CO2. Experimental groups included PBS, LPS, LPS + free drug, LPS + MP, and LPS + MP@Gel (n=3). After incubation for 8 hours, LPS was used to induce inflammation in cells. BV2 cells were induced with 1 μg / mL LPS, while NPC and PM cells were induced with 10 ng / mL LPS. Incubation was continued for 4 days. On day 2, 200 μL of cell supernatant was collected, centrifuged (1000g, 4 min, 4°C), and then frozen at -80°C. On day 4, 200 μL of cell supernatant was collected again. MP@Gel was a hydrogel containing H35TB / R microparticles.

[0044] 1.3.7 Alleviation of Cellular Inflammatory Damage by Drug-Loaded Thermosensitive Hydrogels

[0045] NPC cells: Seed NPC cells evenly into 96-well plates with 100 μL of cell suspension, 1 × 10⁶ cells per well. 4 Cells were incubated overnight at 37°C with 5% CO2. Then, culture media containing 0, 10, 100, 200, 400, 600, and 1000 ng / mL TNF-α were added to simulate the effect of exogenous inflammatory factors secreted by other cells. After co-incubation for 24 h and 96 h, the supernatant was aspirated, and culture media containing 10% CCK8 solution was added for co-incubation for 4 h. The absorbance at 450 nm was measured using a microplate reader. The IC50 value at 24 h was calculated based on the absorbance. 50 The concentration was 446.1 ng / mL, and the IC50 value was 96 h. 50 The concentration was 63.37 ng / mL. NPC cells were seeded into 96-well plates under the same seeding conditions as above, with measurements taken at 24 h and 96 h. Experimental groups included PBS, TNF-α, TNF-α+ free drug, TNF-α+MP, and TNF-α+MP@Gel. After incubation for 24 h with the added drug, IC50 was added... 50 TNF-α at various concentrations were collected, and the supernatant was aspirated at 24 h and 96 h, respectively, and the absorbance was measured by adding culture medium containing CCK8 reagent. MP@Gel was a hydrogel encapsulating H35TB / R microparticles.

[0046] PC12 cells: The CCK8 assay was used to verify that the drug-loaded thermosensitive hydrogel had a certain alleviating effect on inflammation-induced PC12 cell necrosis. First, it was verified whether LPS-induced BV2 cell therapy could lead to PC12 cell damage. BV2 cells were evenly seeded into 12-well plates, with 2 mL of cell suspension per well and 1 × 10⁶ cells per well.5 PC12 cells were incubated overnight at 37°C with 5% CO2. The supernatant was aspirated, and medium containing 0, 1, 10, 100, 1000, and 10000 ng / mL LPS was added. After 24 h of incubation, the medium was centrifuged (12000g, 4°C, 5 min), and the supernatant was collected as conditioned medium (CM) for PC12 cell culture. PC12 cells were evenly seeded into 96-well plates, 100 μL of cell suspension per well, with 5000 cells per well. After overnight incubation, the medium was replaced with CM and cultured for 24 h. The PC12 cell supernatant was discarded, and medium containing 10% CCK8 reagent was added. After 1 h of incubation, the absorbance at 450 nm was measured using a multi-mode microplate reader. Analysis of PC12 cell activity revealed that the ability of LPS to induce neuronal damage was dose-dependent. BV2 cells were treated with different groups of drugs (PBS, LPS, free drug, MP, MP@Gel) for 12 h, followed by stimulation with 100 ng / mL and 3 ng / mL LPS for 24 h and 96 h, respectively. The supernatant was collected as conditioned medium. PC12 cells were seeded into 96-well plates and incubated overnight. Then, CM medium (divided into 24 h and 96 h groups) was added and incubated for 24 h. The cell supernatant was aspirated, washed with PBS, and CCK8 reagent was added. The absorbance at 450 nm was measured on a multi-mode microplate reader.

[0047] 1.3.8 Safety evaluation of gel drug delivery system

[0048] The biocompatibility of the drug-loaded gel was evaluated using the CCK8 assay. PM, NPC, BV2, and PC12 cells were uniformly seeded into flat-bottomed clear 96-well plates, with 100 μL of cell suspension and 1×10⁶ cells per well. 4 Cells were incubated in 96-well plates at 37°C with 5% CO2 overnight. Drugs prepared according to the cell culture medium were then added (based on preliminary in vivo experiments, MP (H35TB / R particles) was more effective than NP (H10TB / R particles), therefore MP was used in all experiments of this invention). Experimental groups were: Gel, free drug, MP, and MP@Gel (n=6). The Gel group consisted of a 20% (w / v) polymer solution; the free drug group consisted of BETA and ROPI culture media solutions at concentrations of 1 mg / mL; MP consisted of an equal amount of BETA and ROPI; the MP group consisted of a 1 mg / mL culture medium solution; and the MP@Gel group consisted of a polymer solution prepared using the micronized particle group's culture medium. After incubation for 24 hours, the supernatant was aspirated, and the 96-well plates were rinsed with 100 μL of PBS. Then, 10% CCK solution was added, and after incubation for 2 hours, the absorbance at 450 nm was measured using a microplate reader.

[0049] 1.3.9 Comparison of the therapeutic effects of NP@Gel and MP@Gel

[0050] Weigh 42 mg of H10TB / R(NP) and H35TB / R(MP) respectively, add 2.1 mL of PBS to prepare a 20 mg / mL particle solution, and dissolve 210 mg of PLGA-PEG-PLGA copolymer (poly(lactide-glycolic acid)) in 1.05 mL of the solution. 1640 -b-poly(ethylene glycol) 1500 -b-poly(lactide-glycolic acid) 1640 Based on the drug loading capacities of NP and MP, PBS solutions containing the same concentrations of free BETA and ROPI were prepared. Seven days after rat modeling, paravertebral administration was performed in three groups: PBS, NP@Gel, and MP@Gel (n=7). Behavioral assessments were conducted before administration, and behavioral changes were monitored starting 4 hours after administration. Thermal pain and mechanical pain were tested daily thereafter. The interval between thermal pain and mechanical pain tests was at least 2 hours to eliminate interference between the two behavioral experiments. NP@Gel was a gel encapsulating H10TB / R particles, and MP@Gel was a hydrogel encapsulating H35TB / R micron-sized particles.

[0051] 1.3.10 Behavioral testing of rats

[0052] This invention uses Von Frey fibers and a plantar thermal analgesia device to measure pain sensation in rats. The Von Frey fibers consist of a series of 20 nylon filaments of varying thicknesses, with 20 needle sizes ranging from small to large. During use, the tip of the fiber is pressed against the rat's foot surface at a perpendicular angle. The applied force gradually increases with each slow press until the fiber bends. Continued pressing causes further bending of the fiber, but without generating more force. Smaller needle sizes result in thinner fibers and less force applied. When using the Von Frey fibers, a small needle is used to vertically stimulate the exposed skin in the middle of the left hind foot of the rat. The fiber bends significantly and is held for 5 seconds. If the rat does not withdraw its foot, it is considered a negative reaction, and a larger needle is used. If the rat withdraws its foot, spreads its toes, or turns its head to lick the sole, it is considered a positive reaction. If a positive reaction is observed, the needle size is reduced by one. The above operation needs to be repeated 2 to 3 times. In order to reduce frequent stimulation, a 3-minute interval is required after each stimulation. Finally, the intensity of the needle number that produces 3 or more positive reactions is expressed as the 50% mechanical stimulation withdrawal threshold (PWT) of rats.

[0053] During the plantar heat pain test, the heat source parameters were adjusted to 80W power and 20s maximum duration. The testing instrument was placed under the rat, and the light source was focused on the exposed skin of the rat's foot. The heat source button was pressed, and the instrument began to generate a 4×6 mm photothermal stimulation point and started automatic timing. When the rat exhibited the behavior of withdrawing its foot and turning its head to lick the sole of its foot, the heat source button was pressed again, and the heat source was turned off. At the same time, the instrument displayed the stimulation time and recorded it as the rat's heat pain paw withdrawal latency (PWL). Five measurements were taken for each rat each time, and the maximum and minimum values ​​were removed before taking the average value for statistical analysis.

[0054] 1.3.11 In vivo release of drug-loaded thermosensitive hydrogel

[0055] In in vivo efficacy studies, MP was found to be significantly more effective than NP; therefore, MP was used in subsequent experiments. 36 mg of MP-Cy5 was prepared to a concentration of 20 mg / mL with PBS. 180 mg of PLGA-PEG-PLGA polymer was weighed and added to 900 μL of the above solution to dissolve the polymer. The solution was then vortexed and stirred until completely dissolved. Seven days after rat modeling, rats were divided into four groups, each receiving a paravertebral injection of 150 μL of the sample: ① PBS; ② Cy5; ③ MP-Cy5 (HA35-TK-BETA-Cy5 / ROPI particles); ④ MP-Cy5@Gel (a gel encapsulating Cy5-labeled MP). Rats were anesthetized with isoflurane after administration, and the fluorescence intensity at the lumbar spine of prone rats was examined at different time points (0h, 4h, 1d, 3d, 6d, 9d, 12d) using a small animal in vivo imaging system (instrument parameters: Ex = 620nm, Em = 670nm, imaging height = 5cm, exposure time = 0.2s), and the fluorescence intensity was quantitatively analyzed.

[0056] 1.3.12 In vivo efficacy validation of drug-loaded thermosensitive hydrogels

[0057] Weigh 42 mg of MP and add 2.1 mL of PBS to prepare a 20 mg / mL solution. Dissolve 210 mg of PLGA-PEG-PLGA copolymer in 1.05 mL of PBS. Prepare BETA / ROPI PBS solutions of the same concentration based on the drug loading of BETA and ROPI in MP. Seven days after rat modeling, administer the drug to rats in the following groups: Sham, PBS, free drug, MP, and MP@Gel (n=7). Behavioral assessments were performed on rats before administration. Behavioral changes were monitored starting 4 hours after administration, and thermal and mechanical pain were tested daily thereafter. A time interval of at least 2 hours was maintained between thermal and mechanical pain tests to eliminate interference between the two behavioral experiments. The experiment was repeated twice, with rats euthanized on days 7 and 11, respectively. Protein or mRNA expression levels of pro-inflammatory factors and pain-related genes were analyzed. Protein expression levels were measured using relevant ELISA kits, and mRNA expression levels were measured according to the following steps.

[0058] 1.4 Statistical Analysis of Data

[0059] All statistical data in this invention were calculated using the statistical analysis software Graphpad Prism. The data are expressed as mean ± standard deviation (Means ± SD). The comparison between multiple groups of data was performed using the one-way ANOVA test. ns, no significant difference, *, P < 0.05, **, P < 0.01, ***, P < 0.001, P < 0.0001, ****.

[0060] 1.5 Experimental Results

[0061] 1.5.1 Synthesis and Characterization of HA-TK-BETA

[0062] Compared to the infrared spectrum of HA, the spectrum of HA-TK is significantly different in the wavenumber range of 1570–1515 cm⁻¹. -1 A distinct -NH bending vibration absorption peak and a CN stretching vibration absorption peak were observed at the location. Figure 2 This indicates that an amide bond exists in HA-TK, and TK was successfully modified onto HA via an amide reaction.

[0063] Comparison of BETA and BETA-1 1 The 1H NMR spectrum showed three new absorption peaks at δ = 12.13, 2.57, and 2.45 ppm, corresponding to the hydrogen atoms of the terminal carboxyl group (a), and sequentially from the terminal carboxyl group to the two tertiary carbon hydrogens on the bulk (b, c). Figure 3 A). Comparison between BETA and BETA-1 13The C NMR spectrum showed four new peaks at δ = 173.61, 171.61, 28.49, and 27.61 ppm, corresponding to the four carbon atoms (a, b, c, d) from the main structure to the end. Figure 3 B). HPLC-MS / MS spectra showed that the molecular weight of the synthesized BETA-1 was 491.1, which was basically consistent with the molecular weight of 492.5 obtained by Chemdraw software simulation. Figure 3 C). The above results prove that BETA was successfully modified with a carboxyl group.

[0064] The HPLC chromatogram shows that BETA-1 eluted at around 5 min, while HA-TK-BETA eluted at around 3 min. Figure 4 A), therefore, it can be inferred that no free BETA-1 exists in the synthesized and purified HA-TK-BETA. The fluorine spectra of both BETA and HA-TK-BETA show the same characteristic absorption peak at δ = -165.19. Figure 4 B) This suggests the presence of a BETA structure in the HA-TK-BETA product. 1 The 1H NMR spectrum shows an absorption peak of BETA-1 at δ = 8.5–4.5, while the characteristic peak of BETA-1 corresponding to the hydrogen in the carboxyl group at δ = 12.28 is not observed. Figure 4 C).

[0065] The infrared spectrum of HA35-TK-BETA shows wavenumbers in the range of 1570–1515 cm⁻¹. -1 The absorption peaks of the -NH bending vibration and CN stretching vibration disappear at wavenumbers of 1680–1610 cm⁻¹. -1 An absorption peak for a double bond appeared at that location. Figure 4 D). The above results indicate that BETA-1 is linked to HA-TK via an amide reaction to obtain the HA-TK-BETA polymer.

[0066] 1.5.2 Characterization of particle size and morphology of synthesized particles

[0067] Polymers HA5-TK-BETA and HA10-TK-BETA were synthesized using HA molecules with molecular weights of 5 kDa and 10 kDa, respectively. These polymers self-assembled in PBS solution to form drug-loaded particles H5TB and H10TB. In PBS solution containing ROPI, the polymers self-assembled into ROPI-encapsulated HA5k-TK-BETA / ROPI particles (H5TB / R) and HA10k-TK-BETA / ROPI particles (H10TB / R). DLS data showed that the particle sizes of H5TB and H5TB / R were approximately 280 nm and 620 nm, respectively. Figure 5 A to Figure 5 B), while the particle sizes of H10TB and H10TB / R are approximately 350nm and 800nm, respectively. Figure 5 D to Figure 5 E), all are nanoparticles (NP). The ZETA potentiogram shows that the potentials of H5TB and H5TB / R are -23.2±1.8 mV and -21.7±1.3 mV, respectively. Figure 5 C), the potentials of H10TB and H10TB / R are -28.3±1.9mV and -23.0±2.5mV, respectively. Figure 5 F). Polymers HA35-TK-BETA and HA100-TK-BETA were synthesized using HA at 35k and 100k. The self-assembled particles formed by these polymers can be abbreviated as H35TB and H100TB, and the ROPI-loaded particles can be abbreviated as H35TB / R and H100TB / R. SEM (scanning electron microscopy) showed that H35TB particles had a diameter of approximately 2 μm, while H35TB / R particles had a diameter of approximately 5 μm and were spherical. Figure 5 G to Figure 5 H). The ZETA potentials are -34.5±1.0mV and -30.5±1.3mV, respectively. Figure 5 I). The particle size of H100TKB is around 5μm, and the particle size of H100TB / R is around 10μm. Figure 5 J and Figure 5 K), the ZETA potential is approximately -44mV. Figure 5 Therefore, drug-loaded particles prepared from high molecular weight HA are micron-sized particles (MP).

[0068] As seen in the UV spectrum, HA and HA-TK in formamide show only trace absorption at 280 nm, while BETA and HA-TK-BETA have essentially the same peak shape and strong absorption at 280 nm. Figure 6 A). A standard curve was plotted based on the absorbance of BETA at 280 nm. Figure 6 B) provides a basis for determining the BETA loading and encapsulation efficiency of particles.

[0069] In methanol, HA, HA-TK, and HA-TK-BETA polymers showed only trace absorption at 220 nm, while ROPI exhibited strong absorption at 220 nm. Figure 6 C). A standard curve was plotted based on the ROPI absorbance at 220 nm. Figure 6 D), and accordingly calculate the drug loading and encapsulation efficiency of ROPI in the particles.

[0070] The drug loading of HTB / R particles synthesized from HA of different molecular weights was characterized using standard curves. The results showed that the BETA loading of drug-loaded particles prepared from HA of molecular weights of 5k, 10k, 35k, and 100k was 15.5±0.8, 11.4±0.4, 8.6±0.6, and 2.6±0.3 mg / mg particle, respectively, while the ROPI loading was 23.8±1.0, 31.1±1.0, 35.7±0.9, and 15.3±0.6 mg / mg particle, respectively. Considering both particle size and drug loading, H10TB / R and H35TB / R will be selected for further investigation.

[0071] 1.5.3 Release kinetics of HTB / R

[0072] H10TB / R and H35TB / R release slowly in PBS, but in a 50mM H2O2 environment, approximately 50% of BETA and ROPI are released within 24 hours, and after 48 hours, both drugs release more than 60% of their contents. Figure 6 E to Figure 6 F) demonstrates that the delivery system has good responsive release capability in a high ROS environment.

[0073] 1.5.4 Characterization of Thermosensitive Hydrogels

[0074] The solution of PLGA-PEG-PLGA polymer undergoes a phase transition upon temperature increase, forming a non-flowing colloid. Further heating results in the precipitation of a white compound. Figure 7 A). PLGA-PEG-PLGA polymers can form gels at 37°C within a mass-to-volume ratio range of 18-24 (m / v%). Figure 7 B). To ensure rapid gelation of the polymer solution in the human body and within the normal body temperature range of rats (38.5℃~39.5℃), a 20m / v% hydrogel was subsequently used in the experiments. SEM images showed that the freeze-dried thermosensitive hydrogel structure was porous, with a pore size of approximately 2μm. Figure 7 C).

[0075] UV spectra of different concentrations of H35TB / R (particle size in the micrometer range, hereinafter referred to as MP) in PBS are as follows: Figure 7 As shown in Figure D, 240 nm was selected as the characteristic absorption wavelength of the particles in PBS, and a standard curve was constructed. Figure 7 E). The experiment revealed that the solubility of 12 mg MP in 200 μL PBS was approaching saturation, making it impossible to completely dissolve more MP. MP solutions were prepared at this concentration and added to different masses of temperature-sensitive polymers. After gelation, the MP treatments without gel loading were tested, and it was found that the gels could achieve an encapsulation rate of over 90% for the added MP. Figure 7 F).

[0076] 1.5.5 In vitro degradation and drug release kinetics of drug-loaded thermosensitive hydrogels

[0077] H10TB / R (abbreviated as NP) and H35TB / R (abbreviated as MP) were prepared using Cy5-labeled HA-TK-BETA polymers. NP or MP was added to a temperature-sensitive polymer solution, and the mixture was used to write "spinal" and "LDH" respectively. After gelation at elevated temperature, PBS was added, and the mixture was incubated at 37°C. The blue color of the English words gradually faded, indicating slow gel degradation and drug release. The blue text in the NP@Gel group disappeared around day 8, while the blue text in the MP@Gel group did not disappear until day 12. Figure 8 A to Figure 8 B).

[0078] In PBS solution, the drug was released in trace amounts from the thermosensitive gel; NP@Gel released approximately 20% of the drug by day 7, and MP@Gel by day 12. In 50 mM H2O2, the NP@Gel group achieved over 60% drug release by day 2, and approximately 80% by day 7. Figure 8 C). The MP@Gel group showed a good sustained-release effect, with continuous slow release of the drug over 12 days. By day 7, the drug release had reached approximately 60%, and by day 11, the drug release had exceeded 80%. Figure 8 D).

[0079] 1.5.6 Evaluation of the in vitro anti-inflammatory efficacy of drug-loaded thermosensitive hydrogels

[0080] Adding LPS to BV2, NPC, or PM cells stimulates the secretion of pro-inflammatory cytokines, followed by drug treatment. Compared to free drugs and MP, MP@Gel can achieve a longer-lasting inhibitory effect on inflammation. On day 4, the MP@Gel group reduced the secretion of IL-6 and IL-1β in BV2 cells by 29% and 56%, respectively, compared to the free drug group. Figure 9 A); inhibited NPC secretion by 34% IL-6 and 14% IL-1β (A). Figure 9 B); reduced PM secretion by 16% IL-6 and 49% IL-1β. Figure 9 C).

[0081] 1.5.7 Alleviation of Cellular Inflammatory Damage by Drug-Loaded Thermosensitive Hydrogels

[0082] Following LDH, TNF-α secreted by immune cells infiltrating the IVD site can induce NPC apoptosis by activating the NF-κB pathway. Excessive NPC apoptosis accelerates the LDH process; therefore, inhibiting TNF-α-induced NPC apoptosis is of great significance. GC-based drugs are potent NF-κB pathway inhibitors and can effectively suppress NF-κB pathway activation by binding to intracellular GC receptors, thereby inhibiting in vivo inflammation levels. This invention will verify the ability of drug-loaded thermosensitive hydrogels to inhibit TNF-α-induced NPC apoptosis.

[0083] CCK8 results showed that co-incubation of the pro-inflammatory factor TNF-α with NPC induced NPC apoptosis. The IC50 values ​​of TNF-α after 24 h and 96 h of co-incubation with NPC were 446.1 ng / mL and 63.37 ng / mL, respectively. Figure 10 A to Figure 10 B). To ensure a fairer assessment, we incorporated the corresponding IC (Intensive Care Unit) values ​​for efficacy evaluation at different time points. 50 Concentrations of TNF-α induced inflammation. After co-incubation with TNF-α (446.1 ng / mL), the drug, and NPC for 24 h, the free drug, drug microparticles, and drug-loaded gel increased the activity of NPC by 49.4%, 45.1%, and 27.3%, respectively. Figure 10 C). The poor efficacy of the gel was due to insufficient drug release at the detection time point caused by sustained release. However, after co-incubation for 96 hours, the efficacy of the drug-loaded gel group was comparable to that of the free drug and microparticle groups. Figure 10 D). Unfortunately, due to the limitation of NPC cell in vitro culture time, it was not possible to compare the efficacy of each group at a longer time point that would better reflect the advantages of the gel.

[0084] Studies have shown that after BV2 cells are activated by LPS, the released IL-1β can induce neuronal necrosis through Toll-like receptor 4 (TLR4), while GC can inhibit LPS-induced BV2 cell activation through the NF-κB pathway, significantly reducing the production of pro-inflammatory cytokines. Therefore, drug-loaded thermosensitive gels can inhibit LPS-induced BV2 activation, thereby alleviating inflammation-induced damage to PC12 neurons. CCK8 assay results show... Figure 11As shown, cell culture media (CM) obtained after co-incubating different concentrations of LPS with BV2 for 24 h significantly inhibited PC12 activity. When CM obtained from co-incubating BV2 with LPS and the drug for 24 h or 96 h was added to PC12 cells, it was found that at 24 h, both the free drug and MP groups significantly improved PC12 cell viability, but the MP@Gel group did not show significant efficacy. This is presumably because the released drug concentration was not as high as the free drug concentration due to the sustained-release effect of the gel. After adding CM obtained from 96 h of co-incubation to PC12 cells, the free drug, MP, and MP@Gel groups all significantly increased PC12 cell activity by approximately 20% compared to the LPS group, showing comparable efficacy and indicating that the drug-loaded gel showed improved efficacy at longer time points. Unfortunately, due to the growth conditions of PC12 cells, it was not possible to obtain CM at longer time points to demonstrate the advantages of the drug-loaded gel.

[0085] 1.5.8 Safety evaluation of gel drug delivery system

[0086] Empty gels, free drugs, drug-loaded microparticles, and thermosensitive gels encapsulating microparticles were co-incubated with four cell types: PM, NPC, BV2, and PC12, respectively, and cell viability was assessed. Results showed that none of the in vitro delivery systems exhibited significant toxicity to the test cells. Figure 12 ).

[0087] 1.5.9 Behavioral and physiological changes in rats after modeling

[0088] The model used in this invention is a non-compression rat autologous NPP (nucleus pulposus) transplantation model. Using bone forceps, the inferior articular process of L5, the superior articular process of L6, and the L5 hemilamine were removed to expose the nerve roots of L5 and L6. The removed rat caudal NPP was placed at the exposed nerve roots. During suturing, gelatin sponge was placed on top to prevent the NPP from compressing the nerve roots and causing mechanical compression. Figure 13 A)

[0089] Behavioral data showed that both the Sham group and the LDH modeling group experienced pain after modeling, but the Sham group recovered to the same level as the healthy group by day 7, while the LDH modeling group maintained a state of pain hypersensitivity for 28 days. Figure 13 B to Figure 13 C). ELISA data showed that on day 7, both IL-6 (interleukin-6) and IL-1β (interleukin-1β) levels were significantly higher in the LDH modeling group compared to the Sham group. IL-6 levels in blood, IVD and surrounding tissues, and DRG increased by 5-fold, 5-fold, and 10-fold, respectively. Figure 13 D), while IL-1β increased by 8-fold, 13-fold, and 56-fold, respectively. Figure 13E). Q-PCR results showed that the mRNA level of the pro-inflammatory cytokine IFN-γ (interferon-γ) in the DRG of LDH-modeling rats was 81% higher than that in the Sham group. Simultaneously, the mRNA expression levels of pain-related molecules PGE-2 (prostaglandin E2) and NGF (nerve growth factor) were increased by 68% and 79%, respectively, compared to the Sham group. Figure 13 F). The above results indicate that this model can be used to simulate the inflammatory and pain state following the occurrence of LDH.

[0090] 1.5.10 Comparison of in vivo therapeutic effects of NP@Gel and MP@Gel

[0091] Seven days after modeling, a gel solution was injected paravertebrally using the touch method. X-ray images confirmed that the needle was indeed near the rat vertebral modeling site during this injection. Figure 14 A). Mechanical pain behavioral data showed that the NP@Gel group provided sustained pain relief in rats for 7 days, while the MP@Gel group provided pain relief for rats for 12 days. Figure 14 B) MP@Gel's better sustained-release effect makes it more effective than NP@Gel, so MP@Gel will be used in subsequent experiments.

[0092] 1.5.11 In vivo efficacy comparison and behavioral verification of thermosensitive hydrogels

[0093] Seven days after modeling, paravertebral injections of PBS, free drug, drug microparticles (MP), and drug-loaded hydrogel (MP@Gel) were administered. Free drug provided analgesia for only 4 hours, MP provided effective analgesia for 3 days, while MP@Gel showed a durable analgesic effect, significantly inhibiting pain for 12 days. Figure 14 C to Figure 14 E).

[0094] 1.5.12 In vivo release of drug-loaded thermosensitive hydrogel

[0095] To investigate the in vivo release kinetics of MP@Gel, we established an LDH model in rats and injected the fluorescently labeled drug paravertebrally. Fluorescence at the administration site in rats was detected at different time points using a small animal in vivo imaging system. Figure 15 A). The fluorescence intensity of free Cy5 decreased most rapidly in rats, decreasing by 52% 4 hours after administration, and was close to that of the PBS group after 1 day. Figure 15 B). The fluorescence intensity of the MP-Cy5 group decreased to 32% within 3 days, while the fluorescence intensity of the MP-Cy5@Gel group decreased more slowly, with clearly visible fluorescence throughout 12 days. On day 12, it still had about 40% fluorescence, which is basically consistent with the in vitro release kinetics trend.

[0096] 1.5.13 In vivo efficacy validation of thermosensitive hydrogel

[0097] In the mechanistic experiment, serum, IVD (IVD and surrounding tissues), and DRG (dorsal root ganglion) were collected on day 7, and the expression of pro-inflammatory cytokines or pain-related molecules was quantitatively analyzed. Figure 16 A). Compared with the free drug group, the MP@Gel group reduced IL-6 protein expression in serum, IVD, and DRG by 30%, 65%, and 40%, respectively, and reduced IL-1β protein expression by 74%, 41%, and 46%, respectively. Figure 16 B). Compared with the free drug group, the MP@Gel group inhibited 46% of IFN-γ mRNA expression in the DRG ( Figure 16 C). Simultaneously, the MP@Gel group significantly downregulated the expression of pain-related molecules PGE-2 and NGF in DRG; compared with the free drug group, the mRNA expression levels of PGE-2 and NGF decreased by 56% and 37%, respectively. Figure 16 D).

[0098] Results 11 days after treatment showed that ( Figure 17 A) Due to the near-complete release of the drug, the efficacy of the MP@Gel group had decreased, and the expression levels of IL-6 in serum and IVD were no longer statistically different from those in the PBS group. However, it still had some effect on IL-1β in DRG, with an inhibition rate of 29%. Figure 17 B). The inhibitory trend on IL-6 protein expression is similar to that of IL-1β. Figure 17 C). The MP@Gel group showed no statistically significant difference in IFN-γ mRNA expression compared to PBS, while the inhibition rates of pain-related molecules PGE-2 and NGF mRNA expression remained at 51% and 63%, respectively. Figure 17 D). Therefore, by day 11, the anti-inflammatory effect of MP@Gel had largely disappeared, but the analgesic effect remained.

[0099] In summary, this invention modifies hyaluronic acid (HA) with the ROS-sensitive crosslinking agent TK and the drug BETA to synthesize a HA-TK-BETA polymer. The HA-TK-BETA polymer can self-assemble in physiological saline, and the hydrophobic cavities formed internally can encapsulate ROPI in the solution, forming drug-loaded particles. The particle solution is mixed with a PLGA-PEG-PLGA thermosensitive polymer to form a thermosensitive polymer particle solution with body temperature gelation capability. After paravertebral injection of the mixture of microparticles and the thermosensitive polymer, a gel can form around the protruding IVD.

[0100] The drug-loaded thermosensitive gel responds to ROS in the inflammatory environment, causing TK bond breakage and releasing the drug loaded in the hydrogel. This provides long-lasting relief of the inflammatory environment around the incisional ulcer (IVD) for up to 12 days and effectively alleviates pain. This system significantly reduces the expression levels of IL-6 and IL-1β in vivo. On day 7, it inhibited the expression levels of IL-6, IL-1β, and IFN-γ in the dorsal root ganglion (DRG), and reduced the expression of the pain mediator prostaglandin E2 (PGE-2) and the pain signaling pathway activating molecule nerve growth factor (NGF) in the DRG by 52% and 37%, respectively.

[0101] The treatment system designed in this invention can not only effectively relieve patients' pain symptoms, but also alleviate LDH from the perspective of inhibiting inflammation, improve efficacy, solve the shortcomings of traditional drug administration methods such as multiple administrations and large side effects, improve patients' quality of life, and have better patient acceptance.

[0102] The above description is only a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions that fall within the scope of the present invention are within the scope of protection of the present invention.

Claims

1. A method for preparing a drug-loaded thermosensitive hydrogel, characterized in that, The drug betamethasone was modified onto hyaluronic acid using the reactive oxygen species-sensitive crosslinking agent ketithiolide to synthesize a hyaluronic acid-ketithiolide-betamethasone polymer. This polymer can self-assemble in physiological saline, and the hydrophobic cavities formed inside can encapsulate ropivacaine in the solution to form drug-loaded particles. The particle solution is then mixed with a PLGA-PEG-PLGA thermosensitive polymer to form a drug-loaded thermosensitive polymer particle solution with body temperature gelation capability. The steps for synthesizing the hyaluronic acid-ketothiolate-betamethasone polymer include modifying hyaluronic acid with the reactive oxygen species-sensitive crosslinking agent ketothiolate. Synthesis of hyaluronic acid-ketothiolate: Hyaluronic acid was dissolved in deionized water, and dichloroethane and N-hydroxysuccinimide were added. The mixture was activated in an oil bath at 45°C for 1 hour. Ketothiolate was then added, and the mixture was stirred at 45°C for 24 hours. The sample was then taken out and added to a 3500 Da dialysis bag for dialysis. The dialysis medium was deionized water, and the water was changed every 12 hours. Dialysis was performed for three days, and the resulting dialysate was then freeze-dried under vacuum. Synthesis of betamethasone-1: Betamethasone, dodecyl succinic anhydride and 4-dimethylaminopyridine were dissolved in a 1:1 mixture of dichloromethane and acetonitrile. Triethylamine was added and the mixture was stirred overnight. After the reaction was completed, the reaction solution was separated and purified by column chromatography. The collected sample was evaporated under reduced pressure using a rotary evaporator to obtain a white solid powder. Synthesis of hyaluronic acid-ketothiolate-betamethasone: Hyaluronic acid-ketothiolate was dissolved in formamide, and betamethasone-1,O-benzotriazole-tetramethylurea hexafluorophosphate was dissolved in dimethyl sulfoxide. The dimethyl sulfoxide solution was added to the formamide, and N,N-diisopropylethylamine at room temperature was added. The mixture was stirred at 45°C for 24 h. The reaction solution was added to a 3500 Da dialysis bag for dialyzing. The solution was dialyzed with dimethyl sulfoxide, anhydrous ethanol, 50% ethanol and deionized water for 24 h each. The dialysate was changed every 12 h. The dialyzed sample was freeze-dried. The drug-loaded particles are hyaluronic acid-ketothiol-betamethasone / ropivacaine particles, and their synthesis steps include: Hyaluronic acid-ketothiolated betamethasone was dissolved in deionized water, and ropivacaine was dissolved in dichloromethane. The hyaluronic acid-ketothiolated betamethasone solution was placed on a magnetic stirrer and stirred at 100 rpm / min and 25°C. The dichloromethane solution was slowly added dropwise to the hyaluronic acid-ketothiolated betamethasone solution, resulting in a two-layer solution. The upper layer was a deionized aqueous solution of hyaluronic acid-ketothiolated betamethasone, and the lower layer was a dichloromethane solution of ropivacaine. The two-layer solution was sonicated at 40 kHz for 30 min and 4°C to obtain a milky white solution. The sonicated solution was placed in a fume hood at 25°C and stirred overnight to allow the dichloromethane to evaporate. Dichloromethane was added and shaken to dissolve the unreacted ropivacaine. The lower layer of dichloromethane solution was collected using a separatory funnel, and the upper layer was collected and freeze-dried under vacuum to obtain the dried product. The drug-loaded thermosensitive hydrogel is used to prepare a long-acting drug for treating intervertebral disc herniation, and the administration method is paravertebral injection.

2. The method for preparing the drug-loaded thermosensitive hydrogel according to claim 1, characterized in that, The steps of mixing the particle solution with the PLGA-PEG-PLGA thermosensitive polymer to form a drug-loaded thermosensitive polymer particle solution with body temperature gelation capability include: Take hyaluronic acid-ketothiolate-betamethasone / ropivacaine granules, dissolve them in phosphate buffer, then take an appropriate amount of the temperature-sensitive polymer PLGA-PEG-PLGA, vortex thoroughly to prepare a polymer solution, add an appropriate amount of water to the metal bath heating module, set the temperature to 25℃, place the vial containing the polymer solution below the liquid surface, increase the temperature by 1℃ each time after the temperature stabilizes for 5 minutes, place the polymer that has formed a gel in liquid nitrogen to freeze into a solid, and freeze-dry the solid to obtain the final product.

3. The method for preparing the drug-loaded thermosensitive hydrogel according to claim 2, characterized in that, After adding different masses of temperature-sensitive polymers to hyaluronic acid-ketothiol-betamethasone / ropivacaine particles to form a gel, the gel's encapsulation rate of the hyaluronic acid-ketothiol-betamethasone / ropivacaine particles was tested and found to be no less than 90%.

4. A drug-loaded thermosensitive hydrogel prepared by the method according to any one of claims 1-3.

5. The use of the drug-loaded thermosensitive hydrogel according to claim 4 in the preparation of a long-acting drug for the treatment of intervertebral disc herniation.

6. The use according to claim 5, characterized in that, The drug-loaded thermosensitive hydrogel can respond to reactive oxygen species in the inflammatory environment, causing the ketothiol bonds to break and releasing the drug loaded in the hydrogel. This can provide long-lasting relief from the inflammatory environment around the herniated disc and effectively alleviate pain.

7. The use according to claim 5, characterized in that, The drug-loaded thermosensitive hydrogel can reduce the expression of pro-inflammatory factors and pain-related genes in vivo, either as protein or mRNA.

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