Cationic mCS / Lys hydrogel and application thereof

Abstract

The invention belongs to the technical field of medicine research, and particularly relates to cationic mCS / Lys hydrogel and application thereof. The cationic mCS / Lys hydrogel is prepared according to the following steps: carrying out reductive amination reaction by taking chitosan and an aldehyde compound as raw materials to obtain dodecyl modified chitosan; mixing the modified chitosan with lysine, and then carrying out covalent cross-linking with genipin, thereby obtaining the product. The hydrogel provided by the invention shows excellent injectability and biocompatibility, and can significantly slow down cartilage degeneration and rebuild joint lubrication. Meanwhile, a good long-term analgesic effect is achieved, and the progress of osteoarthritis is delayed.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pharmaceutical research, and particularly relates to a cationic mCS / Lys hydrogel and application thereof. BACKGROUND

[0002] Osteoarthritis (OA) is a disabling joint disease with rapidly increasing global incidence, affecting more than 595 million people by 2021, and is expected to reach 1,101.6 million people by 2050. Its main features include cartilage degradation and calcification, synovial inflammation, etc. The key but often overlooked factor in the progression of OA is the imbalance of lubrication-calcification coupling. In the physiological state, the joint cartilage and lubricating factors such as phosphatidylcholine (PC) maintain the superlubrication of the joint. During the OA process, the loss of lubricating factors and cartilage damage lead to increased friction, which increases the friction coefficient from 0.001~0.05 to 0.3~0.5. The nucleic acids released by the apoptosis of chondrocytes recruit calcium and phosphorus ions, promote mineral deposition, and induce pathological calcification to occur, further damaging the articular cartilage. Ultimately, the lubrication failure and calcification form a vicious cycle.

[0003] Current treatment methods for OA focus on symptomatic rather than etiological treatment, and have many shortcomings. For example, hyaluronic acid (HA), the most commonly used in clinical, has a short duration in the body and needs to be administered frequently. At the same time, it cannot inhibit calcification. Although the latest research such as liposomes and self-lubricating hydrogels have improved lubrication performance, similar to HA, these lubricants have a short duration and need to be administered frequently. Due to the complex preparation process, the promotion is limited and the biocompatibility is difficult to guarantee. More importantly, the above research has not solved the interaction between the decrease in lubrication and pathological calcification. Therefore, there is an urgent need for therapeutic drugs that can target the lubrication-calcification coupling for a long time to break the OA disease cycle. SUMMARY

[0004] Based on the above technical problems, the present application provides an injectable cationic mCS / Lys hydrogel with dual functions of lubrication and anti-calcification. The hydrogel exhibits good injectability and biocompatibility, can significantly slow down cartilage degradation, and relieve pain and delay the progression of OA.

[0005] The specific technical scheme of the present application is: In the first aspect of the present application, a cationic mCS / Lys hydrogel is provided, which is prepared according to the following steps: Reducing amination reaction is carried out with chitosan and aldehyde compounds as raw materials to obtain alkylated modified chitosan; The modified chitosan is mixed with lysine, and then covalently crosslinked with genipin, and then dialyzed and purified to obtain the cationic mCS / Lys hydrogel.

[0006] As an embodiment of the present application, the aldehyde compound is selected from lauryl aldehyde (dodecanal).

[0007] As an embodiment of the present application, the molar ratio of the chitosan to the lauryl aldehyde is 10:1~3.

[0008] As an embodiment of the present application, the molar ratio of the modified chitosan to the lysine is 1:1.

[0009] As an embodiment of the present application, the molar ratio of the total amino groups of the modified chitosan and lysine mixture to the genipin is 1:1~1.5.

[0010] As an embodiment of the present application, the reductive amination reaction is stirred at 37℃ for 8~12h.

[0011] As an embodiment of the present application, the mixing of the modified chitosan and lysine is stirred at 24~37℃ for 4~6h.

[0012] As an embodiment of the present application, the covalent crosslinking of the mixed product and genipin is ultrasonically treated at 40kHz for 30~40min.

[0013] As an embodiment of the present application, the cationic mCS / Lys hydrogel can adsorb extracellular nucleic acids and endogenous lubricating factors.

[0014] As an embodiment of the present application, the cationic mCS / Lys hydrogel has a three-dimensional porous structure.

[0015] In the second aspect of the present application, the cationic mCS / Lys hydrogel is used for preparing a drug for treating OA.

[0016] As a preferred embodiment of the present application, the cationic mCS / Lys hydrogel is used for preparing a drug for treating temporomandibular joint osteoarthritis.

[0017] In the third aspect of the present application, the cationic mCS / Lys hydrogel is used for preparing a drug for promoting cartilage reconstruction.

[0018] In the fourth aspect of the present application, the cationic mCS / Lys hydrogel is used for preparing an analgesic drug.

[0019] Compared with the prior art, the present application has the following beneficial effects: 1、The present application integrates the two functions of "restoring cartilage lubrication" and "inhibiting pathological calcification" which are crucial to OA in a single hydrogel system, directly attacking the pathological cycle of "lubrication-calcification coupling disorder". Among them, the hydrogel can efficiently recruit PC in the joint synovial fluid through electrostatic and hydrophobic interactions by the bottlebrush-like structure and cationic properties, forming a stable hydrated lubricating layer on the surface of the hydrogel. In addition, the bottlebrush-like structure can further enhance boundary lubrication through entropy effect and reduce friction under physiological load. In addition, the cationic properties of mCS and Lys can capture negatively charged extracellular nucleic acids. Experiments have proved that the combination of mCS / Lys and PC is comparable to HA, and the binding capacity of nucleic acids is stronger than HA. The calcified nodule area of the hydrogel treatment group is reduced by 48.8% compared with the OA group, and the calcified cartilage is reduced by 78.4%, effectively inhibiting the formation of nucleic acid-amorphous calcium phosphate calcification precursor.

[0020] 2、The hydrogel of the present application benefits from the stable three-dimensional network constructed by covalent cross-linking of alkyl branches and genipin, and can be stable in the joint cavity for more than 35 days (still retaining 20.3% on the 35th day), far exceeding the commonly used HA, achieving long-acting treatment, and reducing the burden and risk of frequent injections.

[0021] 3、The hydrogel prepared by the present application has good biological safety. In vitro cell experiments show that the survival rate of chondrocytes is more than 95%, and the hemolysis rate is only 2.8% (below the safety threshold of 5%); no toxic reaction is found in the main organs of in vivo experiments, confirming its reliable safety as an implant material.

[0022] 4、The hydrogel provided by the present application has a three-dimensional porous structure, which is beneficial to cell ingrowth. Its rheological properties and compression modulus match the mechanical environment of cartilage, and can be smoothly injected through a 26G needle, having good injectability.

[0023] 5、In the mouse temporomandibular joint osteoarthritis model, the hydrogel provided by the present application can promote cartilage regeneration and repair on the one hand: significantly up-regulating the key gene SOX9 of cartilage synthesis and down-regulating the osteogenesis gene RUNX2. Histology shows that the OARSI score is reduced, the content of proteoglycan is significantly higher than that of the OA group, and the synthesis of cartilage matrix is enhanced. On the other hand, the hydrogel can effectively relieve pain: behavioral tests show that the exploration behavior of treated mice increases, and anxiety-like behavior caused by pain decreases. Mechanical hyperalgesia test shows that the analgesic effect is durable and superior to HA, and can effectively relieve pain at 6 weeks.

[0024] In summary, this invention successfully developed an injectable, biocompatible cationic hydrogel with both long-lasting lubrication and highly effective anti-calcification functions. It demonstrates superior efficacy compared to the clinical gold standard HA in multiple dimensions, including lubrication durability, anti-calcification ability, and long-lasting analgesia. This provides a novel, multifunctional, synergistic, and innovative solution for OA treatment, with broad prospects for clinical translation and application. Attached Figure Description

[0025] Figure 1 This study focuses on the synthesis of injectable cationic mCS / Lys hydrogels and their dual role in the treatment of osteoarthritis: long-lasting lubrication and inhibition of pathological calcification. Ingredients include: chitosan (CS); alkylated chitosan (mCS); lysine (Lys); temporomandibular joint (TMJ); phosphatidylcholine (PC); and extracellular nucleic acid (exNA).

[0026] Figure 2 This document describes the synthesis and physicochemical characterization of mCS / Lys hydrogels. A) Schematic diagram of mCS / Lys hydrogel synthesis. B) Hydrogel formation process. C-D) Thermodynamic changes during crosslinking: TGA and DSC. E-F) Chemical structure: ATR-FTIR and XRD.

[0027] Figure 3 The physicochemical characterization of the mCS / Lys hydrogel is as follows: A-C) SEM images and quantitative analysis of CS / Lys and mCS / Lys hydrogels (scale bar = 200 μm); D-E) Surface wettability assessment of CS / Lys and mCS / Lys hydrogels; F) Characterization of the swelling property of mCS / Lys; G-H) Particle size and Zeta potential; I) Characterization of the injectability of the mCS / Lys hydrogel.

[0028] Figure 4 The following are the biocompatibility assessments of the hydrogels: A-B) live / dead cell staining and corresponding quantitative data (scale bar = 100 micrometers); C) cell proliferation assay; D-E) hemolysis test; F) tissue toxicity H&E staining (scale bar = 100 micrometers).

[0029] Figure 5 This section describes the mechanical characterization of the mCS / Lys hydrogel. A) Schematic diagram of the AFM testing process; B-C) ​​Thermal and statistical graphs of Young's modulus; D-E) Thermal and statistical graphs of adhesion force; F) Rheological frequency scanning; G) In vitro degradability; H) Characterization of compressive modulus.

[0030] Figure 6 Characterization of the lubrication properties of mCS / Lys hydrogel. A) Schematic diagram of lubrication mechanism. B~C) Changes in surface roughness over time and quantitative analysis. D~E) Friction coefficient-time curves; F~I) Molecular dynamics simulation of the adsorption process of mCS and HA phosphatidylcholine.

[0031] Figure 7 This describes the therapeutic effect of mCS / Lys hydrogel on pathological calcification. A) Schematic diagram of animal model experimental protocol. B) Alizarin Red staining image of cells (scale bar = 200 μm). C) Immunofluorescence imaging of Alizarin Red staining and calcification-related proteins in vivo (scale bar = 30 μm). D) Semi-quantitative analysis of in vitro mineralization. E~G) Semi-quantitative analysis of calcification markers in vivo.

[0032] Figure 8 This describes the therapeutic effect of mCS / Lys hydrogel. A) H&E and SF staining (scale bar = 50 μm); B) Micro-CT images (scale bar = 250 μm); C) In vivo retention of mCS / Lys hydrogel; D-F) OARSI histopathological scores and semi-quantitative staining analysis; G-I) Quantitative assessment of bone volume / total volume (BV / TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N); J) Quantitative analysis of in vivo fluorescence intensity.

[0033] Figure 9 This is a pain-related behavioral assessment using mCS / Lys hydrogel. A~B) Movement trajectories in the open field and elevated cross maze. C) Schematic diagram of the behavioral test. D~G) Quantitative analysis of the open field and elevated cross maze. H) Von Frey.

[0034] Figure 10 This illustrates the mechanism by which mCS / Lys hydrogel inhibits pathological calcification. A) Schematic diagram of mCS / Lys inhibiting calcification. B) Transcriptomics results. C-G) Kinetic simulation of the adsorption process of mCS and HA phosphatidylcholine.

[0035] Statistical indicators: ns: not significant, *: p <0.05, **: p <0.01, ***: p <0.001; mean ± SD, n =5. Detailed Implementation

[0036] The present invention will now be described in further detail with reference to specific embodiments and accompanying drawings.

[0037] This invention provides a cationic mCS / Lys hydrogel, which is prepared according to the following steps: Chitosan and aldehyde compounds were used as raw materials for a reductive amination reaction to obtain alkylated chitosan; Modified chitosan was mixed with lysine and then covalently crosslinked with genipin. After dialysis and purification, cationic mCS / Lys hydrogel was obtained.

[0038] Chitosan is a natural cationic polysaccharide that has been approved by the FDA for human use due to its excellent biocompatibility. Alkylated chitosan (mCS) retains the cationic properties of chitosan while enhancing its bioactivity and structural stability. Lysine (Lys) is an essential cationic amino acid that can inhibit pathological calcification. mCS and Lys, through electrostatic and hydrophobic interactions, can firmly target the cartilage matrix while dynamically capturing lubricating factors such as PC and calcification precursors such as extracellular nucleic acids, thereby achieving the goal of treating osteoarthritis.

[0039] The materials used in the embodiments of this invention are as follows: Chitosan, acetic acid, L-lysine, genipin, sodium hydroxide, 1,2-dimyristoyl-sn-glycerol-3-phosphocholine, and dimethyl sulfoxide were purchased from Maclean's (China). Lauraldehyde, sodium cyanoborohydride, and fluorescein isothiocyanate were purchased from Aladdin (China). Dexamethasone, L-ascorbic acid, disodium β-glycerophosphate, Alizarin Red S, and Triton X-100 were purchased from MilliporeSigma (USA). Penicillin-streptomycin solution and fetal bovine serum (FBS) were purchased from Pronossa (China). Hyaluronidase, Safranin O / Fix Green staining solution, H&E staining solution, and Alizarin Red S solution (1%, pH=4.2) were purchased from Solarbio (China). Runx2 and SOX9 antibodies were purchased from Santa Cruz (USA), catalog numbers: sc-390351, sc-166505. α-Minimum essential medium and phosphate-buffered saline were purchased from Carlsbad (USA). The LIVE / DEAD™ cell staining kit was purchased from Invitrogen (USA). Hyaluronic acid and Cell Counting Kit-8 were purchased from GLPBIO Technology LLC (USA). All chemicals were commercially available and used as is.

[0040] Example 1 Synthesis of mCS / Lys hydrogels, such as Figure 1 As shown: CS (4 g) was dissolved in 200 mL of 2% acetic acid. After complete dissolution, lauraldehyde was added at a molar ratio of 10:1, and the mixture was stirred at 37 °C for 12 hours. The pH was adjusted to 5.0 with 1 M NaOH, and then excess sodium cyanoborohydride was added. After 3 hours, the pH was neutralized to 7.0 with sodium hydroxide. The precipitate was washed with ethanol and freeze-dried to obtain alkyl chitosan (mCS).

[0041] 2 g of mCS was dissolved in 200 mL of 2% acetic acid. Lysine was added at a molar ratio of 1:1 to mCS, and the mixture was stirred at 37 °C for four hours. Genipin was added for crosslinking (the molar ratio of genipin to the total primary amines of the alkylated chitosan-lysine mixture was maintained at 1:1), followed by sonication at 40 kHz for 30 minutes and dialyzing with deionized water. The sample was then treated with 0.1 M sodium hydroxide for two hours, washed until neutral, lyophilized, and stored at 4 °C.

[0042] Example 2 Synthesis of mCS / Lys hydrogel CS (4 g) was dissolved in 200 mL of 2% acetic acid. After complete dissolution, lauraldehyde was added at a molar ratio of 10:3, and the mixture was stirred at 37 °C for 12 hours. The pH was adjusted to 5.0 with 1 M NaOH, and then excess sodium cyanoborohydride was added. After 3 hours, the pH was neutralized to 7.0 with NaOH. The precipitate was washed with ethanol and freeze-dried to obtain alkyl chitosan (mCS).

[0043] 2 g of mCS was dissolved in 200 mL of 2% acetic acid. Lysine was added at a molar ratio of 1:1 to mCS, and the mixture was stirred at 37 °C for four hours. Genipin was added for crosslinking (the molar ratio of genipin to the total primary amines of the alkylated chitosan-lysine mixture was maintained at 1:1), followed by sonication at 40 kHz for 30 minutes and dialyzing with deionized water. The sample was then treated with 0.1 M NaOH for two hours, washed until neutral, lyophilized, and stored at 4 °C.

[0044] Example 3 Synthesis of mCS / Lys hydrogel CS (4 g) was dissolved in 200 mL of 2% acetic acid. After complete dissolution, lauraldehyde was added at a molar ratio of 10:1, and the mixture was stirred at 37 °C for 12 hours. The pH was adjusted to 5.0 with 1 M NaOH, and then excess sodium cyanoborohydride was added. After 3 hours, the pH was neutralized to 7.0 with NaOH. The precipitate was washed with ethanol and freeze-dried to obtain alkyl chitosan (mCS).

[0045] 2 g of mCS was dissolved in 200 mL of 2% acetic acid. Lysine was added at a molar ratio of 1:1 to mCS, and the mixture was stirred at 37 °C for four hours. Genipin was added for crosslinking, maintaining a molar ratio of genipin to total primary amines in the alkylated chitosan-lysine mixture at 1:1. The mixture was then sonicated at 40 kHz for 30 minutes and dialyzed with deionized water. The sample was then treated with 0.1 M NaOH for two hours, washed until neutral, lyophilized, and stored at 4 °C.

[0046] Example 4 Synthesis of mCS / Lys hydrogel CS (4 g) was dissolved in 200 mL of 2% acetic acid. After complete dissolution, lauraldehyde was added at a molar ratio of 10:1, and the mixture was stirred at 37 °C for 12 hours. The pH was adjusted to 5.0 with 1 M NaOH, and then excess sodium cyanoborohydride was added. After 3 hours, the pH was neutralized to 7.0 with NaOH. The precipitate was washed with ethanol and freeze-dried to obtain alkyl chitosan (mCS).

[0047] 2 g of mCS was dissolved in 200 mL of 2% acetic acid. Lysine was added at a molar ratio of 1:1 to mCS, and the mixture was stirred at 37 °C for four hours. Genipin was added for crosslinking, maintaining the molar ratio of genipin to the total primary amines in the alkylated chitosan-lysine mixture at 1.5:1. The mixture was then sonicated at 40 kHz for 30 minutes and dialyzed with deionized water. The sample was then treated with 0.1 M NaOH for two hours, washed until neutral, lyophilized, and stored at 4 °C.

[0048] Example 5 Synthesis of mCS / Lys hydrogel CS (4 g) was dissolved in 200 mL of 2% acetic acid. After complete dissolution, lauraldehyde was added at a molar ratio of 10:1, and the mixture was stirred at 37 °C for 8 hours. The pH was adjusted to 5.0 with 1 M NaOH, and then excess sodium cyanoborohydride was added. After 3 hours, the pH was neutralized to 7.0 with NaOH. The precipitate was washed with ethanol and freeze-dried to obtain alkyl chitosan (mCS).

[0049] 2 g of mCS was dissolved in 200 mL of 2% acetic acid. Lysine was added at a molar ratio of 1:1 to mCS, and the mixture was stirred at 37 °C for four hours. Genipin was added for crosslinking (the molar ratio of genipin to the total primary amines of the alkylated chitosan-lysine mixture was maintained between 1:1 and 1.5:1), followed by sonication at 40 kHz for 30 minutes and dialyzing with deionized water. The sample was then treated with 0.1 M NaOH for two hours, washed until neutral, lyophilized, and stored at 4 °C.

[0050] Example 6 Synthesis of mCS / Lys hydrogel CS (4 g) was dissolved in 200 mL of 2% acetic acid. After complete dissolution, lauraldehyde was added at a molar ratio of 10:1, and the mixture was stirred at 37 °C for 12 hours. The pH was adjusted to 5.0 with 1 M NaOH, and then excess sodium cyanoborohydride was added. After 3 hours, the pH was neutralized to 7.0 with NaOH. The precipitate was washed with ethanol and freeze-dried to obtain alkyl chitosan (mCS).

[0051] 2 g of mCS was dissolved in 200 mL of 2% acetic acid. Lysine was added at a molar ratio of 1:1 to mCS, and the mixture was stirred at 24 °C for 6 hours. Genipin was added for crosslinking (the molar ratio of genipin to the total primary amines of the alkylated chitosan-lysine mixture was maintained between 1:1 and 1.5:1), followed by sonication at 40 kHz for 30 minutes and dialyzing with deionized water. The sample was then treated with 0.1 M NaOH for two hours, washed until neutral, lyophilized, and stored at 4 °C.

[0052] Example 7 Synthesis of mCS / Lys hydrogel CS (4 g) was dissolved in 200 mL of 2% acetic acid. After complete dissolution, lauraldehyde was added at a molar ratio of 10:1, and the mixture was stirred at 37 °C for 12 hours. The pH was adjusted to 5.0 with 1 M NaOH, and then excess sodium cyanoborohydride was added. After 3 hours, the pH was neutralized to 7.0 with NaOH. The precipitate was washed with ethanol and freeze-dried to obtain alkyl chitosan (mCS).

[0053] 2 g of mCS was dissolved in 200 mL of 2% acetic acid. Lysine was added at a molar ratio of 1:1 to mCS, and the mixture was stirred at 37 °C for four hours. Genipin was added for crosslinking (the molar ratio of genipin to the total primary amines of the alkylated chitosan-lysine mixture was maintained between 1:1 and 1.5:1), followed by sonication at 40 kHz for 40 minutes and dialyzing with deionized water. The sample was then treated with 0.1 M NaOH for two hours, washed until neutral, lyophilized, and stored at 4 °C.

[0054] Hydrogels with the expected effects of the present invention were prepared in Examples 1 to 7. Therefore, the hydrogel in Example 1 is used as an example to describe its performance and effects below.

[0055] Experimental Example 1 Physicochemical properties characterization of mCS / Lys hydrogel 1. Experimental Methods Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR; model FTIR-8400S, Shimadzu, Japan) was used in the range of 4000~4000 cm⁻¹. -1 The chemical structure was characterized within the spectral range using 32 scans at a resolution of 4 cm⁻¹. -1 The analysis was performed using IRsolution software version 1.40 (Shimadzu).

[0056] Thermal analysis (TGA) and differential scanning calorimetry (DSC) were performed using a STA 8000 instrument (PerkinElmer, USA) under a nitrogen atmosphere at a heating rate of 10 °C / min, with a temperature range of 35–800 °C.

[0057] Crystallinity was assessed by X-ray diffraction (XRD) using an Empyrean diffractometer (Panalytical, Netherlands) with Cu Kα radiation (λ=1.5406Å), a scan range of 5°–50°, a step size of 0.02°, and a scan rate of 2° / min. Data analysis was performed using Jade 9.0 software (MDI, USA) in conjunction with the ICDD PDF-4+ database.

[0058] Morphology was observed using a scanning electron microscope (SEM; S-4800, Hitachi, Japan) at 5 kV. The lyophilized hydrogel was treated with gold sputtering coating (5 nm). The swelling properties of the hydrogel were evaluated by immersion in phosphate buffer at 37 °C. After reaching swelling equilibrium, the hydrogel was lyophilized, and the water content percentage was calculated using the formula [(M1-M2) / M1]×100, where M1 represents the swollen mass and M2 represents the dry mass. The residual mass percentage was calculated as (M1-M2) / M1. t / M0)×100, where M0 represents the initial mass, M t This represents the mass at a given time t.

[0059] The degradation properties of the hydrogel were assessed by immersing it in phosphate buffer containing 0.1 U / mL hyaluronidase at 37 °C. The hydrogel was weighed periodically, and its mass change over time was plotted.

[0060] Surface wettability was assessed using an Easy Drop K100 instrument (Krüss, Germany), with 2 μL water droplets applied to a hydrogel surface measuring 10 mm × 2 mm (n=5). Furthermore, the zeta potential and hydrodynamic diameter were determined by dynamic light scattering at 25 °C using a Litesizer 500 (Anton Paar, Austria), ensuring transmittance greater than 80%.

[0061] The compressive modulus was measured using a bio-nanoindenter (Piuma, Optics11, Netherlands) with a spherical tip (10 μm radius, 5 μm displacement), and the force-indentation curve was fitted to a Hertzian model (n=5 samples, 80~100 indentation points per sample).

[0062] An atomic force microscope (AFM; Keysight 5500, USA) with a tapered tip (force constant 13 N / m) was used for nanomechanical indentation testing (sample thickness 50 μm). Rheological properties (viscoelasticity) were measured on a rheometer (MCR102e, Anton Paar, Austria) at 25 °C: frequency scan (0.1~1000 Hz, constant strain 2%).

[0063] Friction performance was evaluated using a ball-disc friction testing machine (UMT-2MT, CETR, USA). Silicon nitride balls (8 mm in diameter) and poly(2-hydroxyethyl methacrylate) were used for testing. The amplitude was 4 mm, the speed was 6 mm / s, the temperature was 37℃, and the relative humidity was 40–50%. The lubricant used was pure water or 1 mg / mL polyacrylic acid. Surface roughness was analyzed using a 3D optical profilometer (ST400, Nanovea, USA) with Nanovea Software v7.455. Simulated synovial fluid consisted of PBS + 1% bovine serum albumin + 0.1% PC.

[0064] The structures of mCS, HA, Lys, and PC were modeled using Charmm-Gui, while extracellular nucleic acids were modeled using Discovery Studio. FF19SB, OL24, GAFF2, and lipids21 force fields were applied to Lys, DNA, CS, HA, and PC, respectively. The solvent system underwent energy minimization using a 5000-step steepest descent method and a conjugate gradient algorithm, followed by heating from 0 K to 300 K over 500 ps. Constrained MD simulations were then performed for 500 ps each in the NVT and NPT ensembles until equilibrium was reached. A 100 ns MD simulation was performed in the NPT ensemble, and the binding free energy was calculated using the MM-GBSA method.

[0065] 2. Results 2.1 Preparation and Characterization of mCS / Lys Hydrogel To prepare bifunctional cationic hydrogels, this invention employs a two-step synthesis method. Figure 2 (A) The first step in the synthesis involves linking the dodecyl side to the chitosan backbone via reductive amination to generate mCS. Subsequently, a doubly crosslinked hydrogel network is constructed by crosslinking mCS and Lys with genipin, transforming the fluid mCS / Lys precursor into a solid hydrogel. Figure 2 (B). Thermodynamic analysis showed new alkyl-related decomposition peaks in the final product, indicating improved overall thermal stability compared to chitosan. Figure 2 (C~D). FTIR spectroscopy further supports the occurrence of the amidation reaction. Primary amine (approximately 1590 cm⁻¹). -1 The strength is significantly reduced, and the height is between 1540 and 1560 cm. -1 The presence of a secondary amine signal in the region indicates that the free amino group has been converted into an amide bond. Figure 2 XRD analysis showed a significant decrease in crystallinity and the appearance of new diffraction peaks characteristic of alkyl chain incorporation, thus confirming the structural modification caused by alkylation and crosslinking. Figure 2 (Middle F).

[0066] SEM showed that the hydrogel has a three-dimensional porous structure similar to the fibrous network structure of natural articular cartilage. Figure 3 Alkylation modification slightly increased the porosity of the hydrogel from 82.3% to 90.1%, and the average pore size increased from 87.5±8.9 μm to 103.2±7.7 μm. Figure 3 (B~C). The relatively mild changes may be attributed to the reduction of intermolecular hydrogen bonding and partial phase separation during gelation, which alters the packing density of the polymer chains. The retained high porosity and interconnected pore network ensure sufficient space for cell infiltration and nutrient diffusion, while mitigating the excessive mechanical brittleness typically associated with high-density cross-linked hydrogels, highlighting its potential for cartilage tissue engineering applications.

[0067] Contact angle measurements showed that alkylation enhanced surface hydrophobicity, with the water contact angle increasing from 34.9° to 44.9°. Figure 3 While the hydrogels exhibit moderate hydrophilicity (D~E), they still retain a degree of hydrophilicity, a characteristic similar to that of natural cartilage tissue within the 30~50° range. Swelling tests confirmed that both alkylated hydrogels (93.7%) and non-alkylated hydrogels (94.3%) possessed comparable swelling capacity. Figure 3 The presence of dodecyl side chains (F) indicates that the introduction of dodecyl side chains did not alter the water retention capacity. Microstructural and physicochemical analyses show that the alkylated hydrogel retains key structural and surface features similar to natural cartilage, including high porosity, interconnected pores, moderate hydrophilicity, and good water retention. These properties make this hydrogel a promising candidate for cartilage regeneration scaffolds, providing a biomimetic microenvironment conducive to cell viability, migration, and extracellular matrix generation.

[0068] Dynamic light scattering measurements confirmed that the modified hydrodynamic diameter decreased significantly from 3842.47±26.28 nm to 2855.67±20.11 nm. Figure 3 (G). Due to alkylation modification, the number of free amino groups is reduced, resulting in a lower charge in the mCS / Lys group compared to the unmodified chitosan group, but it still maintains strong cationic properties. Figure 3 (H). Through electrostatic interactions, the hydrogel effectively binds to anionic cartilage and captures anionic nucleic acids, disrupting the metastable system of nucleic acid-associated amorphous calcium phosphate, thereby inhibiting pathological calcification. Injectability assessment showed that it could be smoothly extruded through a 26G needle, and the "L"-shaped structure remained intact. Figure 3 (I). This property is attributed to the good viscoelasticity and controllable gelation time of genipin hydrogel under weakly acidic conditions and in appropriate proportions, which is beneficial for intra-articular local injection and minimally invasive treatment applications.

[0069] 2.2 Mechanical characterization of cationic mCS / Lys hydrogels Complex and dynamic joint loading environments place stringent demands on the mechanical properties of biomaterials. To systematically evaluate the suitability of the cationic mCS / Lys hydrogel of this invention, a series of comprehensive mechanical and stability assessments were conducted. Nanoindentation revealed that alkylation significantly enhanced local mechanical stiffness, increasing Young's modulus from 23.1 ± 5.4 kPa to 42.1 ± 5.8 kPa, and adhesion force from 41.4 ± 3.5 nN to 79.1 ± 8.1 nN. Figure 5 (A~E). This invention attributes this improvement to three synergistic mechanisms: (i) the physical interlocking of the hydrophobic dodecyl chains, which increases cohesive energy and enhances interfacial interactions; (ii) the enhanced entropic elasticity due to restricted polymer chain movement, thereby increasing resistance to deformation; and (iii) additional covalent crosslinking generated through genipin-mediated bridging, enhancing overall network integrity. Rheological analysis further confirms the strong gel-like properties of the alkylated hydrogel. The storage modulus (G′) reaches 475.7±16.2 Pa, almost twenty times the loss modulus (G′′) (24.2±0.6 Pa), while the loss factor (tan δ) remains below 0.1 over a wide frequency range of 0.1~100 Hz. Figure 5 (F). This characteristic indicates that it primarily exhibits an elastic solid response, which is crucial for withstanding repetitive joint loads without irreversible deformation.

[0070] Resistance to degradation under physiological conditions is another key parameter for evaluating hydrogel performance. In an enzymatic degradation test conducted at 37°C in phosphate buffer containing hyaluronidase, the alkylated hydrogel retained 53.2% of its initial mass after 35 days. Figure 5 (G). This extended stability indicates that hydrophobic aggregation and enhanced cross-linking synergistically mitigate hydrolysis and enzymatic attack, thereby prolonging the survival time. Compression mechanical tests using a bio-nanoindenter showed that the modified hydrogel had a compressive modulus of 38.7 ± 2.6 kPa, more than twice that of the control group (19.3 ± 3.3 kPa). Figure 5 (H). This data matches the mechanical environment of natural articular cartilage. Overall, these data demonstrate that alkylation imparts substantial improvements in stiffness, adhesive interactions, elastic response, and environmental stability to the hydrogel while preserving its water-rich, biomimetic properties. This makes mCS / Lys hydrogels a promising candidate for cartilage tissue engineering.

[0071] 2.3. Lubricating properties and mechanism of cationic mCS / Lys hydrogel The cationic mCS / Lys hydrogel of the present invention can provide sustained low frictional properties through two complementary mechanisms. Figure 6(A) First, the hydrogel, acting as a supramolecular reservoir, captures and retains PC from the synovial fluid through electrostatic attraction and intermolecular interactions, thereby forming a stable boundary lubrication layer rich in hydration interfaces. Second, the grafted dodecyl side chains adopt a bottle-brush conformation, minimizing the interpenetration of polymer chains and suppressing entropic friction during sliding, effectively compensating for joint lubricant depletion.

[0072] Stability is crucial for long-term lubrication. The mCS / Lys hydrogel maintained a low surface roughness (Ra = 453.5 ± 4.9 nm) in artificial lubricants for over 35 days. Figure 6 (B and C). Under physiological contact pressure (maximum 25.68 MPa, normal load 1 N), the mCS / Lys friction coefficient decreased to 0.125 ± 0.006, while the non-alkyl modified group was 0.176 ± 0.008. Figure 6 (D and E). When 1 mM of exogenous PC was added, the mCS / Lys friction coefficient further decreased to 0.036 ± 0.003, while the non-alkyl modified group only reached 0.095 ± 0.020. This significant enhancement highlights the crucial role of the phosphatidylcholine hydrated lubricating film.

[0073] MD simulations model the molecular interactions between mCS and PC at the atomic level. The calculated non-covalent binding energy is –97.3 kcal·mol⁻¹. −1 Furthermore, in the 100 ns trajectory, the equilibrium adsorption amount for each mCS monomer is 0.46 PC molecules ( Figure 6 (F~I). Notably, these values ​​are comparable to those of HA (0.41 ± 0.02 PCs per disaccharide unit), confirming that the mCS / Lys system possesses PC binding affinity comparable to that of natural synovial lubricants. By integrating supramolecular PC capture with entropy-driven friction reduction via alkyl side chains, the mCS / Lys hydrogel provides durable low-friction protection, reduces cartilage wear, and prolongs intra-articular retention time, thereby reducing the frequency of clinical interventions. This bifunctional design not only addresses the biomechanical deficiencies of current lubricants but also harmonizes with the biochemical environment of degenerated joints, offering a promising translational pathway for OA treatment.

[0074] Experimental Example 2 Biocompatibility 1. Experimental Methods In vitro cell compatibility: Cytotoxicity was assessed using the CCK-8 assay with ATDC5 chondrocytes (5 × 10⁻⁶ cells). 3Cells / wells were incubated with the hydrogel extract for 12 / 24 hours; absorbance was measured at 450 nm. Cell viability was observed by live / dead staining (calcein-AM / propidium iodide) and using confocal microscopy (A1R, Nikon, Japan). Hemolysis rate was quantified by incubating fresh mouse blood with the hydrogel extract (1 h, 37 °C) and calculating the hemolysis rate (%) based on absorbance (540 nm) relative to Triton X-100 (positive control) and PBS (negative control).

[0075] In vivo biocompatibility: The hydrogel (10 μL) was injected intraperitoneally into C57BL / 6J mice (n=5). Eight weeks later, major organs such as the heart, liver, spleen, lung, and kidney were harvested, fixed in formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Histological images were then obtained using an optical microscope (Olympus, Japan).

[0076] 2. Results Live / dead cell staining assays showed that chondrocyte survival exceeded 95% after three days of exposure to 500 μg / mL hydrogel extract. Figure 4 (A~B)

[0077] CCK-8 assays showed no statistically significant difference in cell viability between the treatment and control groups within 120 hours. Figure 4 (C)

[0078] Hemolysis assays showed a hemolysis rate of 2.8 ± 0.4%, far below the acceptable 5% threshold for biological materials. Figure 4 (D~E). In vivo safety assessment showed no signs of inflammation, necrosis, or structural damage in major organs after eight weeks. Figure 4 (Middle F).

[0079] Experimental Example 3 Therapeutic effect of cationic mCS / Lys hydrogel mouse model 1. Experimental Methods Cell culture and calcification induction: ATDC5 cells (CL-0856, Wuhan Pronosai Life Science Technology Co., Ltd.) were cultured in αMEM medium containing 10% FBS and 1% penicillin / streptomycin at 37°C and 5% CO2. The calcification medium contained 10 nM dexamethasone, 100 μM ascorbic acid, 1.1 mM CaCl2, and 10 mM β-glycerophosphate.

[0080] Alizarin Red S staining: After fixing cells with 10% formaldehyde, they were stained with Alizarin Red S (40mM, pH 4.2) for 30 minutes and then rinsed. After washing away excess staining solution, mineralized nodules were quantified using ImageJ (NIH, USA).

[0081] OA Mouse Model and Hydrogel Administration: Temporomandibular joint osteoarthritis was induced in 8-week-old C57BL / 6J mice via unilateral anterior crossbite. Hydrogel (10 µL), HA, or PBS was injected into the temporomandibular joint cavity. The animal experimental protocol was approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University (IACUC-20250164).

[0082] In vivo retention: Isothiocyanate-labeled mCS / Lys hydrogel was injected into the temporomandibular joint cavity, and fluorescence retention was monitored weekly using IVIS (Lumina XRMS, PerkinElmer, USA; excitation / emission: 465 / 520nm) for 5 weeks.

[0083] Behavioral assessment: The Von Frey test was used to assess the mechanical abnormal pain threshold, the open field test was used to assess motor activity, and the elevated cross maze was used to assess anxiety-like behaviors to evaluate pain behavior. Data were analyzed using VisuTrack 8.0 from Softmaze, China.

[0084] Imaging (Micro-CT) evaluation: The mandible was scanned using a Quantum GX2 (PerkinElmer; 16µm resolution) and reconstructed using a Mimics 21.0 (Materialise, Belgium).

[0085] Histological characterization: Decalcified tissue sections (7µm) were stained with H&E or Safranin O / Fix Green and observed using an optical microscope (DM4, Leica, Germany). Immunofluorescence was quantified using ImageJ.

[0086] 2. Results To evaluate the therapeutic effect of cationic mCS / Lys hydrogel, this invention employed a mouse unilateral anterior reverse bite (UAC) model. Mice received intra-articular injections of mCS / Lys, HA, or PBS every four weeks, and tissue samples were collected at eight weeks. Figure 7 (A)

[0087] In vitro alizarin red staining showed that the area of ​​calcified nodules in the OA+mCS / Lys group was reduced by 48.8±8.1% compared with the OA control group, which was superior to the OA+HA group (reduction of 8.7±3.7%). Figure 7 (B, D). In vivo results showed that mCS / Lys reduced the ratio of calcified cartilage to total cartilage thickness by 78.4±4.3%, significantly higher than HA's 18.5±5.3%. Figure 7(C, E). These findings indicate that mCS / Lys is a more effective inhibitor of pathological calcification than current clinical standards. Immunofluorescence analysis further elucidated the mechanism promoting cartilage regeneration. The expression of SOX9, a key gene in cartilage matrix synthesis, was upregulated 40.1-fold in the OA+mCS / Lys group, more than twice that in the HA group (C, E). Figure 7 (C, F). The key gene RUNX2, which promotes bone differentiation, was downregulated by 95.5% in the OA+mCS / Lys group, while it was downregulated by 67.4% in the HA group. Figure 7 (C, G).

[0088] Safranin-Fix-Green and H&E staining showed that the cartilage layer in the mCS / Lys group was intact and rich in glycosaminoglycans, which contrasted sharply with the severe matrix loss and structural disintegration in the OA group. Figure 8 (Middle A). Quantitative analysis showed that the OARSI scores of the mCS / Lys group and the HA group were significantly lower than those of the control group. Simultaneously, the cartilage layer was thicker, and the proteoglycan content was also higher. Figure 8 (D~F). MicroCT analysis showed that mCS / Lys not only repaired the articular cartilage but also restored the subchondral bone structure (D~F). Figure 8 (B, G~I). Compared with OA+PBS, the mCS / Lys group and HA group had significantly higher bone volume fraction (BV / TV), and also increased trabecular bone number (Tb.N) and thickness (Tb.Th). These results indicate that mCS / Lys can promote cartilage and subchondral bone remodeling. Joint cavity retention time is crucial for treatment efficacy. In vivo imaging showed that 20.3% of mCS / Lys remained in the joint cavity on day 35, which was 18.5 times that of the blank control group and far exceeded the 14-day retention time of HA. Figure 8 (C, J). This ensures sustained lubrication and anti-calcification activity in degenerative joint environments.

[0089] Experiment Example 4 Study on the analgesic effect of cationic mCS / Lys hydrogel Pain is a major symptom of osteoarthritis (OA) and severely impairs patients' quality of life. Therefore, assessing the analgesic potential of therapeutic hydrogels is crucial for OA treatment. To comprehensively evaluate pain-related behaviors, this invention employs multidimensional behavioral tests, including assessments of anxiety-like responses and mechanical hyperalgesia. Figure 9 (C)

[0090] Anxiety levels were quantified using the elevated cross maze (EPM) and open field test (OFT). Compared to the OA+PBS group, both the mCS / Lys and HA groups showed lower anxiety-related indices, with the mCS / Lys group exhibiting better anti-anxiety effects: in the OFT, the total distance traveled and the average movement speed of the mCS / Lys group were significantly higher than those of the HA and PBS treatment groups. Figure 9(A, D~E). Similarly, EPM analysis showed that compared with the HA and OA+PBS groups, the mCS / Lys group had a significantly higher percentage of time spent in open arms and a significantly higher frequency of entering open arms. Figure 9 (B, F~G). These findings suggest enhanced exploratory behavior and reduced anxiety-like phenotypes, implying that mCS / Lys is more effective than HA in alleviating anxiety induced by OA-related spontaneous pain.

[0091] Assess mechanical hyperalgesia using the Von-Frey test. Figure 9 (H). The mCS / Lys group showed long-term pain threshold recovery, reaching 61.7±18.1% at 3 weeks and 51.2±9.8% at 6 weeks, both exceeding the 50% threshold. In contrast, the HA group achieved a recovery of 55.3±13.9% at 3 weeks, but decreased to 28.9±11.8% at 6 weeks. This time difference highlights the long-acting analgesic effect of mCS / Lys, which is comparable to HA in the short term but superior to HA in the long term, possibly due to the long-term retention time of the hydrogel (H). Figure 8 (C)

[0092] Experimental Example 5 Therapeutic mechanism of cationic mCS / Lys hydrogel Extracellular nucleic acids are becoming a key mediator of pathological calcification in osteoarthritis (OA). Their polyanionic properties stabilize amorphous calcium phosphate (ACP) precursors, forming nucleic acid-ACP complexes. These complexes are adsorbed onto collagen fibers via hydrogen bonds and van der Waals forces, promoting ACP penetration into the fiber core and facilitating intrafiber mineralization. Nucleic acid deposition spatially co-localizes with calcification foci, and targeted degradation of these substances can reverse the calcification process. Based on this pathological framework, cationic mCS / Lys hydrogels combat calcification through multiple mechanisms. Figure 10 (A). RNA sequencing confirmed 286 differentially expressed genes associated with OA (SOX, MMP, COX, HSPA, BMP, CILP, etc.). Figure 10 Both B and C have changed.

[0093] MD results confirmed that the binding energies of alkylated chitosan and lysine to extracellular nucleic acids were comparable to those of HA, and even exceeded those of HA in the cross-linked case. Figure 10 (C~G). In terms of binding number, each monomer of mCS and Lys bound 1.95 and 2.49 nucleic acid molecules, respectively, higher than HA (1.86). Figure 10 (C~G). In summary, mCS / Lys chelates extracellular nucleic acids, inhibits hydroxyapatite crystallization, and simultaneously regulates cartilage matrix gene expression. This dual-action strategy not only inhibits calcification nucleation but also promotes cartilage repair, providing a durable anti-calcification modality different from existing single-function approaches.

[0094] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A cationic mCS / Lys hydrogel, characterized in that, It is prepared according to the following steps: Alkylated chitosan was obtained by reductive amination reaction of chitosan and aldehyde compounds as raw materials. The modified chitosan was mixed with lysine, and the resulting mixture was then covalently crosslinked with genipin. After dialysis and purification, the cationic mCS / Lys hydrogel was obtained.

2. The cationic mCS / Lys hydrogel according to claim 1, characterized in that, The aldehyde compound is selected from lauraldehyde.

3. The cationic mCS / Lys hydrogel according to claim 2, characterized in that, The molar ratio of chitosan to lauraldehyde is 10:1~3; The molar ratio of modified chitosan to lysine is 1:1; The total amino group ratio of the modified chitosan and lysine mixture to genipin is 1:1~1.

5.

4. The cationic mCS / Lys hydrogel according to claim 1, characterized in that, The reductive amination reaction was carried out by stirring at 37°C for 8-12 hours. The modified chitosan and lysine were mixed at 24-37°C for 4-6 hours. The covalent cross-linking of the mixed product with genipin was performed by ultrasonic treatment at 40 kHz for 30-40 min.

5. The cationic mCS / Lys hydrogel according to claim 1, characterized in that, The cationic mCS / Lys hydrogel has a three-dimensional porous structure.

6. The use of the cationic mCS / Lys hydrogel according to any one of claims 1 to 5 in the preparation of a medicament for treating osteoarthritis.

7. The application according to claim 6, characterized in that, The cationic mCS / Lys hydrogel is used to prepare a drug for treating temporomandibular joint osteoarthritis.

8. The use of the cationic mCS / Lys hydrogel according to any one of claims 1 to 5 in the preparation of a drug that promotes cartilage regeneration.

9. The use of the cationic mCS / Lys hydrogel according to any one of claims 1 to 5 in the preparation of analgesic drugs.

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