Viscoelastic hydrogel microspheres with adjustable stress relaxation performance as well as preparation method and application of viscoelastic hydrogel microspheres

Abstract

The invention provides viscoelastic hydrogel microspheres with adjustable stress relaxation performance as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. According to the invention, the idea of a'cell shock absorption 'strategy is realized, and the viscoelastic hydrogel microspheres with adjustable stress relaxation performance are successfully constructed. Chemical bonds in a hydrogel network are regulated and controlled through oxidation and hydrazine coupling reaction, and the hydrogel microspheres capable of absorbing impact and relieving cartilage cell ER stress induced by mechanical stress are prepared. The preparation method comprises the following steps: encapsulating cationic liposome modified with cartilage targeting peptide Wyrgrl and tauroursodeoxycholic acid (TUDCA) in a microsphere; the microsphere system not only can obviously dissipate impact force in a joint cavity and reduce vibration and pressure conduction, but also can realize continuous release of TUDCA, so that ER stress is relieved, and OA progress is delayed. According to the microsphere strategy, dual protection on cartilage injury can be realized by reducing mechanical stress and relieving ER stress, and the microsphere shows a good application prospect in the aspect of delaying osteoarthritis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a viscoelastic hydrogel microsphere with adjustable stress relaxation performance, a preparation method and an application thereof. Background Art

[0002] Cell motility is fundamental to the function of living systems. Cells interact with their mechanical microenvironment through mechanotransduction and matrix-dependent motility. Mechanical stresses of varying intensities induce distinct patterns of cell motility. Moderate mechanical stress promotes directional cell movement and distribution, enhancing migration efficiency and promoting tissue repair. Excessive mechanical stress, however, activates endoplasmic reticulum stress, oxidative stress, and a series of key signaling pathways (such as p38 MAPK, JNK, and NF-κB), inhibiting cell migration and leading to apoptosis or necrosis. Furthermore, cell motility can in turn influence the mechanical microenvironment. For example, during wound healing, fibroblasts migrate to the site of injury and release matrix metalloproteinases to degrade the extracellular matrix, thereby enhancing their migratory capacity. However, excessive fibroblast activity can also induce pathological changes, such as abnormal tissue remodeling during liver or lung tissue repair, leading to fibrotic diseases. Therefore, providing materials with appropriate mechanical stress is crucial for tissue repair.

[0003] In osteoarthritis, chondrocytes are highly sensitive to mechanical stress, making cartilage more susceptible to mechanical damage. Excessive mechanical stress can induce chondrocyte dysfunction, disrupt their normal function, accelerate apoptosis, and exacerbate the degradation of the extracellular matrix, ultimately driving cartilage degeneration and osteoarthritis progression. Therefore, maintaining cell survival in a stable mechanical environment is crucial for the repair and functional homeostasis of cartilage tissue.

[0004] Current treatments for osteoarthritis, such as hyaluronic acid injections, mainly relieve symptoms by lubricating joints and promoting smooth sliding between tissues. As drug carriers, hydrogel microspheres have good injectability and sustained release properties, which can achieve precise drug delivery and long-term release. Studies have shown that the structural parameters of microspheres, such as particle size and porosity, have a significant effect on cartilage regeneration. Particle size affects the drug release rate and cell uptake efficiency, while porosity affects cell growth and nutrient exchange capacity. However, these strategies have failed to fundamentally address the mechanism of cartilage degeneration, namely the abnormal mechanical sensitivity of chondrocytes in high-stress environments and their adverse microenvironmental interactions.

[0005] To combat mechanical stress-induced cell damage, new therapeutic strategies are urgently needed to weaken the transmission of mechanical vibration and pressure, thereby reducing the mechanical sensitivity of chondrocytes. This strategy is expected to provide a "cell shock-absorbing" effect, thereby slowing the progression of osteoarthritis. Although preliminary studies have shown that hydrogel microspheres have a certain effect in cushioning cells, their inherent elasticity may cause additional elastic stress on cartilage in the complex mechanical environment of joints. Therefore, the development of hydrogel microspheres with stress relaxation properties that can mimic the viscoelasticity of natural cartilage is of great significance for alleviating mechanical vibrations and maintaining cell movement and function.

[0006] The endoplasmic reticulum (ER) is a key organelle for sensing mechanical signals such as stress, tension, and matrix stiffness. Distributed throughout the cytoplasm, the ER rapidly reshapes its structure during cell deformation and mediates force transmission through interactions with cytoskeletal components such as actin and microtubules. Studies have shown that the actin-binding protein Filamin A interacts with PERK, a key ER kinase, and IRE1, a key sensor of the unfolded protein response (UPR). In addition to its role in the stress response, the ER is also responsible for the synthesis and folding of extracellular matrix proteins in chondrocytes. Excessive mechanical stress activates ER stress, leading to the accumulation of unfolded or misfolded proteins within the lumen, which in turn triggers the unfolded protein response (UPR) through three canonical pathways: IRE1, ATF6, and PERK. Although the UPR initially helps restore ER homeostasis, its sustained activation induces chondrocyte apoptosis, further exacerbating mechanical stress-induced damage. Therefore, therapeutic strategies targeting mechanical stress relief and ER stress regulation may provide new breakthroughs in the effective treatment of osteoarthritis.

[0007] However, targeted hydrogel microspheres with the above-mentioned therapeutic strategies have not yet been successfully developed. Even if there are reports of stress-relaxation hydrogels, they exhibit poor stress relaxation properties. In the stress relaxation modulus test, the stress recovery is too fast and the external mechanical energy cannot be effectively dissipated.

[0008] For example, patent document CN 116444819 A discloses a hyaluronic acid-based composite hydrogel, which is prepared by light-induced free radical polymerization and contains both static covalent crosslinks and phenylboronic acid ester dynamic crosslinks. The hydrogel exhibits good biocompatibility and stress relaxation properties, which is beneficial for maintaining the morphology and function of chondrocytes. However, the hydrogel does not have a significant shock-absorbing effect and has difficulty relieving the rapid transmission of external mechanical stress, which cannot meet the expected function of relieving mechanical loads. On the other hand, the hydrogel is a block hydrogel, which requires a large traumatic implantation operation, and there is a disadvantage of compressing or restricting the movement of the joint cavity.

[0009] Therefore, how to provide a hydrogel microsphere that is injectable, has adjustable stress relaxation properties, has a significant shock-absorbing effect, can effectively alleviate the rapid conduction of external mechanical stress, is used to relieve endoplasmic reticulum stress, and achieves a significant effect of promoting cartilage regeneration has become a technical problem that needs to be solved urgently. Summary of the Invention

[0010] The present invention aims to address the aforementioned technical problems and provides viscoelastic hydrogel microspheres with adjustable stress relaxation properties, as well as a preparation method and application thereof. The technical objective of the present invention is to provide hydrogel microspheres with excellent stress relaxation properties that can be used to buffer short-term mechanical impacts, exhibit significant shock absorption, effectively alleviate endoplasmic reticulum stress, and significantly promote cartilage regeneration.

[0011] In order to achieve the above technical objectives, the technical solutions adopted by the present invention are as follows:

[0012] The present invention first provides a method for preparing viscoelastic hydrogel microspheres with adjustable stress relaxation properties, comprising the following steps:

[0013] (1) WYRGRL peptide was reacted with DSPE-PEG2000-NHS via a Mannich reaction to synthesize DSPE-PEG2000-WYRGRL;

[0014] (2) Phospholipids, cholesterol, octadecylamine, DSPE-PEG2000-WYRGRL, and DSPE-PEG2000-NH2 were mixed in a weight ratio of 5:1:3:1.2:1.5 to prepare Lipo-WYRGRL@TUDCA cationic liposomes;

[0015] (3) Hydrogel microspheres were prepared by microfluidics using aldehyde-modified methacrylated hyaluronic acid and a photoinitiator as the inner aqueous phase, Lipo-WYRGRL@TUDCA cationic liposomes, amino-modified methacrylated hyaluronic acid and a photoinitiator as the outer aqueous phase, and Span 80 and paraffin oil as the oil phase.

[0016] Existing studies have shown that chronic mechanical vibration and endoplasmic reticulum (ER) stress are the main driving factors for the progression of osteoarthritis (OA). The present invention proposes a novel "cell shock absorption" strategy, successfully constructing viscoelastic hydrogel microspheres with adjustable stress relaxation properties, which well implements the above-mentioned envisioned strategy. The method of the present invention regulates the chemical bonds in the hydrogel network through oxidation and hydrazine coupling reactions to prepare hydrogel microspheres that can absorb shock and alleviate mechanical stress-induced chondrocyte ER stress. By encapsulating cationic liposomes modified with the cartilage-targeting peptide Wyrgrl in the microspheres (the liposomes can also be loaded with tauroursodeoxycholic acid (TUDCA)). This microsphere system can not only significantly dissipate the impact force in the joint cavity, reduce vibration and pressure transmission, but also achieve the sustained release of TUDCA, thereby alleviating ER stress and delaying the progression of OA. In vitro studies have shown that the stress relaxation time constant (τ) of the microspheres can be adjusted to 23.81 seconds, which is close to the mechanical properties of natural cartilage matrix. These mechanical properties, combined with the targeted release of TUDCA, significantly reduced Grp78 and CHOP expression, alleviated ER stress, and inhibited chondrocyte apoptosis. In in vivo experiments, the microspheres effectively maintained articular cartilage structure, inhibited ER stress responses, and significantly delayed OA progression. Overall, the microsphere strategy of the present invention achieves dual protection against cartilage damage by reducing mechanical stress and alleviating ER stress, showing promising application prospects in delaying osteoarthritis.

[0017] As shown in the comparative examples of the present invention, when the inventors attempted to use a variety of common single-component hydrogel systems, including HAMA, GelMA, HAMA-CHO, and HAMA-NH2, to prepare hydrogel microspheres, they found that although these materials had good gelation and biocompatibility, the hydrogel microspheres formed from these materials generally did not have good stress relaxation properties. In the stress relaxation modulus test, the stress recovery was too fast, and the external mechanical energy could not be effectively dissipated. Therefore, it could not meet the significant shock absorption effect sought by the present invention. Under simulated mechanical impact conditions, these microsphere structures did not have a significant shock absorption effect, and it was difficult to alleviate the rapid transmission of external mechanical stress, and could not meet the expected function of relieving mechanical loads.

[0018] On the other hand, the stress-relaxing hydrogel microsphere system constructed in this invention demonstrated unexpected synergistic effects during experiments. The therapeutic effect demonstrated by the system is not the sum of the material components or the drug release behavior itself, but rather the carrier material achieves a significant therapeutic effect. Specifically, the following points are included:

[0019] (1) The stress relaxation property itself has a greater effect on alleviating ER stress than expected

[0020] Initially, the inventors hypothesized that TUDCA, as a drug component, played a dominant role in alleviating ER stress. However, experiments revealed that even without TUDCA loading, using only hydrogel microspheres with stress-relaxing properties reduced the expression of ER stress-related markers such as GRP78 and CHOP, while also protecting the structural integrity of articular cartilage. This suggests that the mechanical behavior of the material itself has the potential to intervene in cellular states and protect physiological structures. This result goes beyond the previous treatment approach, which focused solely on drug delivery.

[0021] (2) Synergistic effect of mechanical shock absorption and drug delivery

[0022] In the stress-relaxation hydrogel microsphere system constructed in this scheme, the inventors observed that its effect in alleviating chondrocyte endoplasmic reticulum stress was significantly better than that of the control group with a traditional non-stress relaxation structure. In particular, after loading with TUDCA, the microsphere system can significantly downregulate the expression of ER stress-related markers such as GRP78 and CHOP, and maintain the morphology and matrix production capacity of chondrocytes. The experimental results show that the effect of hydrogel microspheres with stress relaxation properties when used in conjunction with drugs is significantly better than that of non-relaxation microsphere drug delivery systems. This observation suggests that the mechanical response behavior of the material may play an important synergistic role in the process of cellular stress relief, significantly improving the therapeutic effect.

[0023] (3) The excellence of microsphere structure

[0024] The stress-relaxing hydrogel microspheres constructed in this protocol exhibit excellent injectability and can be smoothly delivered through a fine-caliber needle into the complex spatial structure of the joint cavity, eliminating the need for invasive implantation and significantly improving clinical operability. Furthermore, after injection into the joint cavity, the microspheres exhibit excellent dispersibility and fluid adaptability, preventing mechanical obstruction or foreign body sensation during joint movement. They maintain the joint's original range of motion and functional state, avoiding the drawbacks of traditional bulky hydrogels or blocky materials that compress or restrict joint cavity movement.

[0025] Furthermore, in step (1), DSPE-PEG2000-NHS is dissolved in DMF and reacted with WYRGRL peptide and triethylamine in a molar ratio of 1:1.1:3 to obtain DSPE-PEG2000-WYRGRL.

[0026] Furthermore, the solvent used in step (2) is a mixed solution of chloroform and water in a volume ratio of 3:1.

[0027] Furthermore, the aldehyde-modified methacrylated hyaluronic acid in step (3) is prepared by reacting hyaluronic acid and methacrylic acid under alkaline conditions to obtain methacrylated hyaluronic acid, which is then reacted with sodium periodate.

[0028] Furthermore, the amino-methacrylated hyaluronic acid in step (3) is prepared by activating the methacrylated hyaluronic acid with EDC and HOBt, and then reacting it with oxalyl dihydrazide.

[0029] Furthermore, the photoinitiator in step (3) is LAP.

[0030] Furthermore, in step (3), the weight ratio of the aldehyde-modified methacrylated hyaluronic acid, the amino-modified methacrylated hyaluronic acid and the cationic liposome is 4:2:1.

[0031] Furthermore, a drug is loaded into the Lipo-WYRGRL@TUDCA cationic liposomes in step (2), wherein the drug includes tauroursodeoxycholic acid, wherein the weight ratio of phospholipid, cholesterol, octadecylamine, DSPE-PEG2000-WYRGRL, DSPE-PEG2000-NH2 and tauroursodeoxycholic acid is 5:1:3:1.2:1.5:1.

[0032] A second object of the present invention is to provide viscoelastic hydrogel microspheres with adjustable stress relaxation properties prepared by the above method.

[0033] A third object of the present invention is to provide the use of the above-mentioned viscoelastic hydrogel microspheres with adjustable stress relaxation properties in the preparation of drugs for treating osteoarthritis.

[0034] The beneficial effects of the present invention are as follows:

[0035] (1) The present invention proposes a novel "cell shock-absorbing hydrogel microsphere" strategy. Aldehyde-modified HAMA (HAMA-CHO) and amino-modified HAMA (HAMA-NH2) were prepared by coupling oxidized hyaluronic acid with hydrazine groups. Cationic liposomes loaded with TUDCA and Wyrgrl peptide (Lipo-Wyrgrl@TUDCA) were prepared by thin film dispersion method, and encapsulated in stress-relaxing hydrogel microspheres through imine bond action and microfluidic technology to obtain hydrogel microspheres with viscoelastic adjustability (Stress-relaxed HAMA@Lip). The microspheres can buffer the mechanical stress in cartilage tissue and reduce ER stress-induced chondrocyte apoptosis, thereby delaying the progression of osteoarthritis. Stress-relaxed HAMA@Lip can simulate the viscoelasticity of natural cartilage tissue and has good molecular chain mobility and deformation ability. At the same time, TUDCA-loaded Lipo-Wyrgrl@TUDCA is anchored to chondrocytes through electrostatic interaction and the targeting function of Wyrgrl peptide, assisting the processing of unfolded proteins, alleviating ER stress, and reducing cell apoptosis rate.

[0036] (2) The stress relaxation properties and ER stress regulation effects of the microspheres were evaluated in vitro using rheometers, immunofluorescence, qPCR, and Western blot. The results showed that stress-relaxed HAMA@Lip microspheres can effectively alleviate mechanical stress damage and ER stress response in chondrocytes, showing the potential to delay the progression of osteoarthritis. The therapeutic strategy constructed by alleviating excessive mechanical stress, restoring cell motility, and maintaining cell function not only has important clinical significance, but also provides new treatment ideas for diseases related to cell-mechanical environment interactions, such as muscle atrophy and cardiovascular disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The construction and characterization of cationic liposomes and stress-relaxing HAMA@Lip are presented; (A) Transmission electron microscopy (TEM) images of liposomes; (BC) Dynamic light scattering (DLS) analysis of the particle size and zeta potential of different liposome formulations, including empty liposomes (Lipo), WYRGRL-modified liposomes (Lipo-WYRGRL), TUDCA-loaded liposomes (Lipo-WYRGRL@TUDCA), and DiR dye-labeled liposomes (Lipo-WYRGRL@DiR); (DF) Optical microscopy images, phosphorus elemental mapping analysis, and scanning electron microscopy (SEM) images compare the microspheres of different groups (HAMA microspheres). (G) 1H nuclear magnetic resonance (1HNMR) spectra and substitution rates of various chemically modified hyaluronic acid products (HAMA, HAMA-CHO, HAMA-NH2); (H) Confocal microscopy images showing the uniform loading of Lipo-WYRGRL@DiR in stress-relaxation HAMA microspheres; (IK) Particle size distribution of microspheres in different groups (HAMA microspheres, stress-relaxation HAMA, stress-relaxation HAMA@Lip); (L) Cumulative drug release curves of Lipo-WYRGRL@TUDCA and stress-relaxation HAMA@Lip.

[0038] Figure 2Figure 3 Mechanical properties of stress relaxation hydrogels with different compositions; (A) Frequency sweep curves of hydrogels with different compositions at 37°C, fixed strain of 0.1%, and frequency range of 0.1-10 Hz (n=3); the shaded area indicates the standard deviation of each data set; (B) Amplitude sweep curves of hydrogels with different compositions at 37°C, fixed frequency of 1 Hz, and strain range of 1%-100% (n=3); the shaded area indicates the standard deviation of each data set; (C) The generalized ternary Maxwell-Wiechert model was used to fit the experimental data; (D) The Maxwell-Wiechert model fits the experimental data well, and the goodness of fit is determined by the coefficient of determination R 2 . Open circles represent individual data points for each group (n = 3), and gray triangles represent the mean of each data set. (E) Stress relaxation curves of hydrogels with different compositions at 37°C. Each set of fitting data is from independently prepared hydrogel samples (n = 3). (F) Stress relaxation constants of hydrogels with different compositions over a 600-second observation period (n = 3). Error bars are not plotted because the relaxation time of the HAMA group exceeded 600 seconds.

[0039] Figure 3 Stress-relaxing HAMA@Lip has good biocompatibility and can alleviate ER stress and osteoarthritis phenotypes in chondrocytes. (A) CCK-8 assay results comparing the effects of the blank group, Lipo-Wyrgrl@TUDCA, stress-relaxing HAMA, and stress-relaxing HAMA@Lip loaded with hydrogel-encapsulated Lipo on chondrocyte proliferation on days 1, 2, and 3. (B) Growth curves of chondrocytes treated with different concentrations of tunicamycin.

[0040] (CD) Thioflavin T staining results and quantitative analysis of fluorescence intensity of each treatment group (n=3); (EF) Type II collagen (Col II) immunofluorescence staining results and quantitative analysis of fluorescence intensity of each treatment group (n=3); (GH) Aggrecan staining results and quantitative analysis of fluorescence intensity of each treatment group (n=3); (IJ) MMP13 staining results and quantitative analysis of fluorescence intensity of each treatment group (n=3); Statistical analysis method was one-way analysis of variance (ANOVA) with Tukey's post hoc test; ns: no significant difference, *p<0.05, **p<0.01.

[0041] Figure 4Stress-relaxing HAMA@Lip promotes protein folding, alleviates endoplasmic reticulum stress, and reduces cell apoptosis; (A) Western blot results of CHOP, GRP78, ATF6, phosphorylated eIF2α (p-eIF2α), and Actin protein expression in each treatment group; (BE) Quantitative analysis of grayscale values ​​of CHOP, GRP78, ATF6, and p-eIF2α, all results were normalized to the internal reference Actin (n=3); (F) Flow cytometric analysis of early and late apoptosis in chondrocytes in each treatment group; (G I) Quantitative statistical analysis of total, early, and late apoptosis rates in each treatment group (n = 3); (J) Schematic diagram of co-culture of stress-relaxation HAMA@Lip and chondrocytes; (KN) Expression levels of osteoarthritis-related genes in chondrocytes after co-culture (n = 3); Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test; ns: no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Figure 5 Figure 3 Imaging evaluation of the in vivo therapeutic effect of stress-relaxation HAMA@Lip; (A) Flowchart and timeline of the animal experiment; (B) X-ray images showing the knee joint space and osteophyte formation, shown in the anteroposterior (AP) and lateral (LAT) views; (C) Microcomputed tomography (Micro-CT) images showing two-dimensional and three-dimensional imaging results of the knee joint and its subchondral bone structure; (DG) Quantitative analysis of subchondral bone, including bone volume fraction (BV / TV), trabecular thickness (Tb.Th), trabecular space (Tb.Sp), and trabecular structure index (Tb.Pf) (n=3); One-way analysis of variance (ANOVA) with Tukey's post hoc test; *p<0.05, **p<0.01.

[0042] Figure 6Stress-relaxing HAMA@Lip alleviates endoplasmic reticulum stress and protects cartilage structure in osteoarthritis; (A) Representative H&E staining images of the knee joints of rats in different groups; (B) Representative Safranin-O / FastGreen staining images of the knee joints of rats in different groups; (C) Representative immunofluorescence images of CHOP and GRP78 in the joints of rats in different groups; (D) OARSI scores of osteoarthritis progression in rats in different groups (n=3), the scoring criteria are detailed in the supplementary materials; (E) Quantitative analysis of articular cartilage thickness in rats in different groups (n=3); (FG) Quantitative analysis of CHOP and GRP78 fluorescence intensity in the joints of rats in different groups (n=3); Statistical method was one-way analysis of variance (ANOVA) with Tukey's post hoc test; *p<0.05, **p<0.01, ***p<0.001.

[0043] Figure 7 Stress-relaxing HAMA@Lip inhibits the progression of osteoarthritis in rats; (A) Representative immunohistochemical staining images of Aggrecan in the joints of rats in different groups; (B) Representative immunohistochemical staining images of MMP13 in the joints of rats in different groups; (C) Representative immunohistochemical staining images of type II collagen (Col II) in the joints of rats in different groups; (D) Quantitative analysis of Aggrecan expression in rat joints, with the normal group as the normal reference (n=3); (E) Quantitative analysis of MMP13 expression in rat joints, with the normal group as the normal reference (n=3); (F) Quantitative analysis of Col II expression in rat joints, with the normal group as the normal reference (n=3); One-way analysis of variance (ANOVA) was used for statistical analysis, followed by Tukey's post hoc test; *p<0.05, **p<0.01.

[0044] Figure 8 Schematic diagram and experimental flow chart of the present invention; (A) Preparation of cationic liposomes targeting cartilage; (B) Chemical modification of MA-grafted HAMA via oxidation and OPA / N-nucleophile condensation reaction; (C) Construction of stress-relaxing HAMA@Lip microspheres using multiphase microfluidics and UV cross-linking; (D) Targeting mechanism of stress-relaxing HAMA@Lip and its mechanical stress release mechanism; (E) TUDCA alleviates cell apoptosis by assisting protein folding and inhibiting the UPR pathway.

[0045] Figure 9 This is the NMR detection spectrum of WYRGRL peptide.

[0046] Figure 10 This is the mass spectrum of the WYRGRL peptide.

[0047] Figure 11HPLC results of WYRGRL peptide.

[0048] Figure 12 is the liposome encapsulation efficiency.

[0049] Figure 13 Live-dead staining results of Stress-relaxed HAMA@Lip.

[0050] Figure 14 This is a physical picture of the formation of hydrogel.

[0051] Figure 15 The degradation performance of stress-relaxedHAMA@Lipo-Wyrgrl@TUDCA microspheres.

[0052] Figure 16 Diagram of the animal experiment steps for Stress-relaxedHAMA@Lip.

[0053] Figure 17 In the figure, a, b, and c are the modulus test results (frequency sweep and strain sweep), stress relaxation test results, and fitting curves of HAMA, respectively.

[0054] Figure 18 In the figure, a, b, and c are the modulus test results (frequency sweep and strain sweep), stress relaxation test results, and fitting curves of HAMA-CHO, respectively.

[0055] Figure 19 In the figure, a, b, and c are the modulus test results (frequency sweep and strain sweep), stress relaxation test results, and fitting curves of HAMA-NH2, respectively. DETAILED DESCRIPTION

[0056] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is described in detail below with reference to the following embodiments. It is necessary to point out that the following embodiments are only used to explain and illustrate the present invention and are not intended to limit the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above invention content still fall within the scope of protection of the present invention.

[0057] Example 1

[0058] 1. Experimental Materials and Methods

[0059] 1. Experimental materials and sources

[0060] Cholesterol, octadecylamine, phosphatidylcholine, thiazolin T, sodium periodate, oxalyl dihydrazide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and 1-hydroxybenzotriazole (HOBt) were purchased from Aladdin (Shanghai). WYRGRL (Trp-Tyr-Arg-Gly-Arg-Leu), DSPE-PEG2000-NH2, and DSPE-PEG2000-WYRGRL were purchased from Apeptide (Shanghai). Tauroursodeoxycholic acid (TUDCA) was purchased from Yesen Biotechnology (Shanghai). Hyaluronic acid (HA, molecular weight = 168 kDa), methacrylic anhydride (MA, ≥94%), and lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP, ≥95%) were purchased from Sigma-Aldrich. DMEM / F12 medium, 0.25% trypsin-EDTA, fetal bovine serum (FBS), and antibiotics were purchased from Invitrogen (California, USA). Tunicamycin (SC0393) was purchased from Beyotime. Rhodamine phalloidin and DAPI were purchased from MedChemExpress.

[0061] Antibodies were used at a dilution of 1:1000 for western blotting and 1:200 for immunofluorescence, unless otherwise stated. Antibodies against Col II (AF0135), MMP13 (AF5355), Aggrecan (DF7561), ADAMTS5 (DF13268), and β-actin (AF7018) were purchased from Affinity Biosciences. Antibodies against ATF-6 (D4Z8V) (#65880), CHOP (L63F7) (#2895), BiP (Grp78) (#3183), and phosphorylated eIF2α (Ser51) (#9721) were purchased from CST. RNA sequences used for qPCR are provided in the supplementary material. All RNA was purchased from Sangon Biotech (Shanghai).

[0062] 2. Synthesis and characterization of cationic liposomes

[0063] (1) WYRGRL was linked to DSPE-PEG2000-NHS by Mannich reaction to synthesize DSPE-PEG2000-WYRGRL. The specific steps were as follows: 100 mg of DSPE-PEG2000-NHS was dissolved in DMF, 1.1 equivalents of WYRGRL peptide and 3 equivalents of triethylamine were added, and the reaction was continued for 12 hours. The product was purified by dialysis bag with MWCO of 1000 Da for 24 hours, lyophilized and stored. 1 HNMR ( Figure 9 ) and mass spectrometry verification ( Figure 10), the synthesis rate was calculated by HPLC ( Figure 11 ).

[0064] (2) Lipo-WYRGRL@TUDCA cationic liposomes were prepared by reverse evaporation method. The specific steps were as follows: phospholipids, cholesterol, octadecylamine, DSPE-PEG2000-WYRGRL, DSPE-PEG2000-NH2 and TUDCA were dissolved in chloroform and water (volume ratio 3:1) at a mass ratio of 5:1:3:1.2:1.5:1, and then ultrasonic emulsification was performed to form an oil-in-water emulsion. The organic solvent was removed by rotary evaporation at 60°C to form a lipid membrane, which was then hydrated with PBS (pH = 7.4). Subsequently, probe ultrasonication (30% amplitude, 5 min) and filtration were performed to form liposomes ( Figure 8 The particle size and Zeta potential of Lipo-WYRGRL@TUDCA were measured using Malvern laser particle size analyzer (DLS). Figure 1 B, C); the morphology was observed using a transmission electron microscope (TEM) and stained with phosphotungstic acid ( Figure 1 Middle A).

[0065] 3. Synthesis and characterization of stress relaxation hydrogel microspheres

[0066] (1) Synthesis of HAMA-CHO:

[0067] First, HA (10 g) and MA (2 ml) were reacted under alkaline conditions overnight, dialyzed for 4 days, and lyophilized to obtain HAMA; then, 0.8 g of HAMA was dissolved in 50 mL of water, 450 mg of sodium periodate was added, and the reaction was continued for 4 h (final concentration 40 mM), and HAMA-CHO ( Figure 8 Middle B, Figure 1 Middle G).

[0068] (2) Synthesis of HAMA-NH2:

[0069] Synthesis of HAMA-NH2: 2 g HAMA was dissolved in 0.1 M MES buffer (pH = 6.0), pre-activated with EDC (1.2 g) and HOBt (0.8 g) for 30 min, and then oxalyl dihydrazide (4.58 g, 260 mM) was added. The mixture was reacted for 12 h, purified by dialysis, and lyophilized to obtain HAMA-NH2 ( Figure 8 Middle B, Figure 1 Middle G).

[0070] Rheological tests were performed using a Discovery Hybrid Rheometer (TA) with a 20 mm plate and a 500 μm gap. The material was prepared at 2 wt%, mixed, and then 0.5% LAP was added. The material was cross-linked using 405 nm blue light for 300 s. Frequency sweeps (5% strain, 0.1-10 Hz), strain sweeps (1 Hz, 1%-200%), and stress relaxation tests (5% strain, 300 s) were performed. All experiments were repeated three times ( Figure 2 A, B, E in the figure). The stress relaxation test data was fitted using the Maxwell-Wiechert three-element model to fit the decay curve, R 2 Between 0.95-0.99, the fitting accuracy is high ( Figure 2 (C, D).

[0071] (3) Preparation of stress-relaxed HAMA@Lip by three-phase microfluidic method:

[0072] The inner aqueous phase is 2wt% HAMA-CHO + 0.5wt% LAP, the outer aqueous phase is Lipo-WYRGRL@TUDCA + 2wt% HAMA-NH2 + 0.5wt% LAP, and the oil phase is paraffin oil containing 8wt% Span 80. After the droplets are generated, they are cooled to -40°C, cross-linked by blue light, and the oil phase is removed to obtain microspheres ( Figure 8 Middle C).

[0073] Microsphere characterization includes: bright field microscopy observation of synthesis and particle size ( Figure 1 I, J, K), laser confocal microscopy observation of the distribution of DIR dye-labeled liposomes ( Figure 1 TUDCA encapsulation efficiency and release (in PBS for 25 days) were measured by UV spectrophotometer. Encapsulation efficiency = W / W0 × 100% ( Figure 1 Middle L, Figure 12 ); SEM observation of microsphere morphology, pores and particle size after freeze drying ( Figure 1 (D, E, F).

[0074] 4. Isolation and culture of primary chondrocytes

[0075] Articular cartilage tissue of one-week-old rats was obtained, digested with 0.2% type II collagenase at 37°C overnight, filtered, washed and inoculated into F12 / DMEM containing 10% FBS, cultured at 37°C and 5% CO2, and used for experiments for 3-5 generations.

[0076] 5. Biocompatibility and in vitro model establishment

[0077] Live / dead staining and CCK-8 assay were used to evaluate the effect of stress-relaxed HAMA@Lip (2.5 mg / mL) on chondrocyte proliferation. Figure 13 The microspheres were placed in the upper chamber of the Transwell and the cells were placed in the lower chamber. Calcein-AM / PI staining was used for observation on the 1st, 2nd and 3rd day, and CCK-8 was used to detect the activity ( Figure 3 In addition, CCK-8 was used to evaluate the toxicity of different concentrations of marine mycin and to establish an ER stress model ( Figure 3 Middle B).

[0078] 6. Western blot analysis

[0079] The cells were lysed with RIPA, transferred to a membrane after SDS-PAGE electrophoresis, blocked and treated with primary antibody (in 3% BSA), followed by HRP-labeled secondary antibody, developed with ECL, and analyzed with ImageJ for grayscale values, which were normalized to β-actin. Figure 4 (A, B)

[0080] 7. Immunofluorescence: Chondrocytes were seeded on confocal microplates, fixed with 4% paraformaldehyde, permeabilized with TritonX-100, and blocked with 5% BSA. Incubated with primary antibody overnight, the next day with fluorescent secondary antibody for 30 minutes, stained with rhodamine phalloidin for cytoskeleton, and DAPI for nuclei. Observed under a confocal microscope. Quantitative analysis was performed using image analysis with mean fluorescence intensity. Figure 3 C, D, E, F, G, H, I, J)

[0081] 8. qPCR: Chondrocytes were pretreated with actinomycin for 24 hours and then co-cultured with stress-relaxed HAMA@Lip at different TUDCA concentrations for 24 hours. RNA was extracted and reverse transcribed into cDNA using the RevertAid kit. qPCR was performed using the SYBR Green method. -ΔΔCT Calculate by this method and repeat 3 times ( Figure 4 (K, L, M, N).

[0082] 9. Flow cytometry: Annexin V-FITC / PI double staining was used to detect cell apoptosis. The treated cells were collected, washed with PBS, and resuspended in binding buffer. Annexin V-FITC and PI were added and incubated in the dark for 10-15 minutes. After staining, binding buffer was added and the cells were immediately tested. Data were collected by flow cytometry and analyzed using FlowJo software. A two-parameter scatter plot was drawn. According to the signal intensity of Annexin V and PI, the cells were divided into viable cells (Annexin V- / PI-), early apoptotic cells (Annexin V- / PI-), and thrombocytopenic cells (Annexin V- / PI-). + / PI-), late apoptotic cells (Annexin V + / PI + ) and necrotic cells (Annexin V- / PI+ The proportion of cells in each quadrant was counted, and a bar graph was drawn to show the percentage changes of each type of cells in different treatment groups to evaluate the differences in apoptosis levels ( Figure 4 (F, G, H, I).

[0083] 10. OA animal models

[0084] Animal experiments were conducted with the ethics approval of Ruijin Hospital (IACUC-20240301-01). Thirty male Sprague-Dawley rats (~200 g) aged 6-8 weeks were used. The modeling group underwent resection of the MCL, ACL, and medial meniscus, while the sham group underwent only skin incision. The model was confirmed to be successful 4 weeks after modeling. From weeks 5 to 8, 100 μL of PBS, HAMA, stress-relaxed HAMA, or stress-relaxed HAMA@Lip ( Figure 5 Middle A).

[0085] 11. X-ray and Micro-CT

[0086] At 5 weeks after surgery, X-rays (lateral and anteroposterior) were taken using the Faxitron system. The animals were then sacrificed and joint samples were taken. Micro-CT scanning and three-dimensional reconstruction were performed using SkyScan1172 to analyze osteophyte formation and subchondral bone conditions. Figure 5 (B, C, D, E, F, G).

[0087] 12. Histological Analysis

[0088] The joints were fixed and decalcified 45 days later, and the sections were embedded in paraffin. The sections were stained with HE and Safo green, and the OARSI scoring system was used to evaluate the pathology ( Figure 6 Immunohistochemistry was performed using Col II, AGGRECAN, and MMP13 antibodies, and DAB staining was performed followed by analysis of expression area and intensity using ImageJ ( Figure 7 Immunofluorescence staining was performed using CHOP and GRP78 antibodies, labeled with FITC or Cy5, and the nuclei were stained with DAPI and then scanned and imaged to analyze the fluorescence intensity ( Figure 6 C, F, G).

[0089] 13. Statistical analysis

[0090] GraphPad Prism 9 was used for analysis, and Student's t-test was used for comparison between two groups. One-way or two-way ANOVA was used for comparison between multiple groups. P < 0.05 was considered significant.

[0091] 2. Experimental Results and Discussion

[0092] 1. Preparation and characterization of microspheres

[0093] We first constructed cartilage-targeted cationic liposomes to deliver TUDCA, aiming to alleviate ER stress. DSPE-PEG2000-Wyrgrl was synthesized by reacting DSPE-PEG2000-NHS with the Wyrgrl peptide in a triethylamine environment and purified by dialysis and freeze-drying. HPLC, high-resolution mass spectrometry, and H-NMR analyses confirmed its structural correctness ( Figure 9-11 ).

[0094] Then, DSPE-PEG2000-NH2, DSPE-PEG2000-Wyrgrl, phosphatidylcholine, cholesterol, octadecylamine and TUDCA were used as raw materials to prepare the targeted liposome Lipo-Wyrgrl@TUDCA ( Figure 8 The encapsulation efficiency was determined to be approximately 84.65% by UV-visible spectroscopy ( Figure 12 Transmission electron microscopy revealed that the liposomes had a multilayer structure and a particle size of approximately 150 nm ( Figure 1 Middle A).

[0095] A composite system of liposomes and hydrogel microspheres was further constructed. DIR-labeled liposomes were introduced, and four types of liposomes were prepared: Lipo, Lipo-Wyrgrl, Lipo-Wyrgrl@TUDCA, and Lipo-Wyrgrl@DIR. Dynamic light scattering tests showed that because they all contained the cationic component octadecylamine, the Zeta potential of all liposomes was about +12mV, reflecting their cationic properties. Particle size analysis showed that Lipo was about 90nm, Lipo-Wyrgrl was about 110nm, and Lipo-Wyrgrl@TUDCA and Lipo-Wyrgrl@DIR were both about 160nm, with a PDI of 0.24, indicating a good particle size distribution ( Figure 1 (B, C).

[0096] The size and surface charge of liposomes significantly influence their function. Positive charge helps overcome the cartilage barrier and achieve effective targeting. Combined with Wyrgrl modification, liposomes enhance their ability to penetrate the cartilage matrix and target chondrocytes, while also achieving sustained release of TUDCA.

[0097] 2. Modulus and viscoelasticity of hydrogel microspheres

[0098] To mimic the cushioning and shock-absorbing properties of cartilage matrix, we chemically modified hyaluronic acid (HA, molecular weight 168kDa) to manipulate the influence of material composition on modulus and viscoelastic properties. First, HA was reacted with methacrylic anhydride (MA) in an ice bath overnight to introduce photocrosslinking groups, resulting in methacryloylated hyaluronic acid (HAMA). Its synthesis was confirmed by H NMR spectroscopy, and the degree of substitution was approximately 31% ( Figure 1Middle G).

[0099] Subsequently, HAMA was oxidized with periodate to generate aldehyde-modified HAMA (HAMA-CHO) with a degree of substitution of approximately 27%. HAMA then reacted with hydrazine hydrochloride to generate amino-modified HAMA (HAMA-NH2) with a degree of substitution of 28.67%, both of which were verified by NMR ( Figure 1 Middle G).

[0100] Aldehyde groups and amino groups can form imine bonds (C=N), with a bond energy of 100-150 kJ / mol, significantly lower than conventional covalent bonds (300-400 kJ / mol). This weak crosslinked structure dissociates under stress, imparting fluidity to the hydrogel. By adjusting the material ratio, the initial swelling and degradability of the gel can be adjusted, thereby achieving stress relaxation properties, simulating the viscoelasticity of cartilage tissue, and effectively alleviating damage to articular cartilage caused by elastic stress.

[0101] The synthesized HAMA, HAMA-CHO, and HAMA-NH2 were dissolved in deionized water at a mass concentration of 2%, and 0.5% of the photoinitiator NAP (6-methoxy-2-naphthaldehyde) was added. After dissolution, blue light cross-linking was performed to form a hydrogel (see Figure 14 ). The following groups were set up in the experiment: a) HAMA group; b) HAMA: HAMA-CHO (1:1) group; c) HAMA: HAMA-NH2 (1:1) group; d) HAMA-NH2: HAMA-CHO (1:1) group; e) HAMA-CHO: HAMA-NH2: Lipo-Wyrgrl@TUDCA groups with different ratios (1:1:1, 1:2:1, 1:4:1, 2:1:1, 4:1:1, 2:4:1, 4:2:1, 1:1:2). All groups were added with 0.5% photoinitiator NAP. The mechanical properties of each group were tested using a rheometer, including frequency sweep (5% strain) and strain sweep (1Hz). The results showed that the storage modulus of HAMA after cross-linking alone was approximately 5000Pa, which decreased to about 600Pa after adding equal proportions of HAMA-CHO and HAMA-NH2, indicating that the imine bond significantly affects the stiffness of the material. After further addition of Lipo-Wyrgrl@TUDCA, the modulus dropped to about 400 Pa, because the liposomes could not form effective covalent bonds, affecting the overall cross-linking density ( Figure 2 (A, B).

[0102] The difference in modulus among each group reflects the change in internal bond strength: when the reaction ratio of aldehyde group to amino group is optimal, the imine bond density is the largest, corresponding to the maximum storage modulus; while liposomes have difficulty forming effective bonds, and increasing the ratio further reduces the modulus.

[0103] The viscoelastic properties of the material were evaluated by stress relaxation experiments, and the stress decay process with time was monitored under constant strain conditions. The generalized Maxwell-Wiechert ternary model was used for fitting. 2 Between 0.95 and 0.99, the fitting accuracy is high, indicating that the model is reliable ( Figure 2 (C, D).

[0104] The stress relaxation time constant (τ) of normal cartilage tissue is about 20 seconds. Among the tested formulas, the τ of the HAMA-CHO:HAMA-NH2:Lipo-Wyrgrl@TUDCA 4:2:1 group was 23.81 seconds, which is closest to the intrinsic viscoelasticity of cartilage ( Figure 2 The modulus of this group is approximately 400 Pa, which can both fit the joint surface and relieve the mechanical impact generated during activities. It is defined as stress-relaxed HAMA.

[0105] Stress-relaxed HAMA microspheres were prepared by microfluidic device. The obtained microspheres were uniformly dispersed under optical microscope, with a diameter of 232.16±88.98μm ( Figure 1 Due to the electrostatic dipole interaction between liposomes and hyaluronic acid, and the formation of an imine bond between DSPE-PEG2000-NH2 and HAMA-CHO in the liposomes, the liposomes are effectively anchored in the hydrogel matrix, thereby improving the stability of the system. Using DIR-labeled liposomes (instead of TUDCA), it was observed under a confocal microscope that they were evenly distributed in the microspheres and maintained good stability ( Figure 1 (D, E, F).

[0106] Further scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) analysis of the freeze-dried microspheres revealed that as the complexity of the microsphere components increased, their porosity gradually decreased. The porosities of the HAMA, Stress-relaxedHAMA, and Stress-relaxedHAMA@Lipo-Wyrgrl@TUDCA groups were 41.16%, 31.40%, and 26.10%, respectively. The distribution of C, N, O, and P in the elemental distribution map further confirmed the presence of liposomes in the hydrogel microspheres. Figure 1 (D, E, F).

[0107] In terms of drug encapsulation efficiency, the TUDCA encapsulation efficiency of Stress-relaxedHAMA@Lipo-Wyrgrl@TUDCA was 55.95% ( Figure 12In vitro release experiments showed that free Lipo-Wyrgrl@TUDCA released 80% of TUDCA within 4.5 days at 37°C, while the TUDCA release time of stress-relaxed HAMA@Lipo-Wyrgrl@TUDCA was extended to 25 days, with a cumulative release of 72.15% ( Figure 1 Middle L).

[0108] In addition, in PBS containing hyaluronidase at 37°C to simulate the in vivo enzymatic degradation environment, the mass of the stress-relaxed HAMA@Lipo-Wyrgrl@TUDCA microspheres gradually decreased, and the residual mass was only 11.05% after 10 days, indicating that the system has good biodegradability ( Figure 15 ).

[0109] 3. Biocompatibility

[0110] To evaluate the biocompatibility of the materials, stress-relaxed HAMA, Lipo-Wyrgrl@TUDCA (hereafter referred to as Lip), and stress-relaxed HAMA@Lip were co-cultured with rat primary chondrocytes. Live / dead cell counts were determined using Calcein-AM / PI double staining, and cell viability was analyzed using CCK-8 assays at 24, 48, and 72 hours.

[0111] The results showed that at any time point, there was no significant difference in cell density and the proportion of dead cells among the groups ( Figure 13 In the CCK-8 test results, there was no statistical difference in the absorbance values ​​at 450nm among the groups ( Figure 3 (A) in the middle, indicating that Stress-relaxedHAMA@Lipo-Wyrgrl@TUDCA has good cell compatibility.

[0112] 4. Alleviate endoplasmic reticulum stress

[0113] Considering that the endoplasmic reticulum (ER) is a key organelle for protein synthesis in chondrocytes, the abundant extracellular matrix (ECM) in articular cartilage requires a large amount of protein synthesis to maintain stability. In osteoarthritis (OA), ER stress is common in chondrocytes. To establish an in vitro ER stress model, chondrocytes were treated with Tunicamycin (which inhibits glycosylation and leads to the accumulation of misfolded proteins). Based on the results of the CCK-8 experiment, the final concentration was selected as 2 μg / ml ( Figure 3(B) TUDCA (taurodeoxyursodeoxycholic acid), a chemical chaperone, can assist in the refolding of misfolded proteins and prevent their aggregation, thereby alleviating ER stress. This experiment used TUDCA at concentrations of 100, 300, and 500 μmol / L.

[0114] ThT staining (binding to protein aggregates) showed that Tunicamycin significantly induced the aggregation of misfolded proteins, and TUDCA could reduce their aggregation in a dose-dependent manner. The 500 μmol / L group was even lower than the blank control group ( Figure 3 C, D). Immunofluorescence staining showed that the expression of type II collagen and Aggrecan decreased and the expression of MMP13 increased in the Tunicamycin-treated group; while the stress-relaxed HAMA@Lip-treated group significantly restored the levels of type II collagen and Aggrecan and reduced the expression of MMP13, suggesting that it has an alleviating effect on the OA phenotype ( Figure 3 Chinese EJ).

[0115] Western blot results showed that TUDCA could dose-dependently downregulate the expression of ER stress markers GRP78, hydrolyzed phosphorylated eIF2α (p-eIF2α) and CHOP, and inhibit the expression of ATF6 in the UPR pathway ( Figure 4 Flow cytometry further verified that TUDCA significantly reduced the proportion of late apoptosis under ER stress conditions, while there was no significant change in early apoptosis. The late apoptosis rate in the model group was 7.54% ± 0.89%, in the blank control group it was 2.4% ± 0.21%, and in the high-dose TUDCA group it dropped to 3.87% ± 1.01% ( Figure 4 Chinese FI).

[0116] In a Tunicamycin-induced chondrocyte OA model, stress-relaxed HAMA@Lip was released at different TUDCA concentrations (100, 300, and 500 μmol / L). qPCR showed that the expression of type II collagen, Aggrecan, MMP13, and ADAMTS5 were all regulated. MMP13 expression steadily decreased with increasing concentration, while ADAMTS5 expression increased briefly at medium doses and then decreased at high doses, suggesting that their response mechanisms have certain differences ( Figure 4 Chinese JN).

[0117] The above results indicate that Stress-relaxedHAMA@Lip can effectively alleviate ER stress, inhibit cell apoptosis, and reduce chondrocyte loss, providing a potential therapeutic strategy for delaying the progression of OA.

[0118] 5. Animal experiments

[0119] Further animal experiments verified the therapeutic effect of stress-relaxedHAMA@Lip, which is called a "cell shock absorber" for the treatment of OA. A rat osteoarthritis model was established by a modified Hulth method, including anterior cruciate ligament transection, medial collateral ligament transection and medial meniscus resection ( Figure 5 Middle A, Figure 16 The rats were randomly divided into five groups: sham operation group, PBS control group, HAMA group, Stress-relaxed HAMA group and Stress-relaxedHAMA@Lip group.

[0120] The intra-articular injection treatment was started on the 14th day after surgery. At this time, all rats showed no abnormalities such as infection, ulceration or necrosis at the surgical site. Each group was injected once a week for four consecutive weeks. The rats were killed on the 6th week after surgery, and the knee joints were collected for X-ray imaging, Micro-CT scanning, histological staining, immunohistochemistry and immunofluorescence analysis ( Figure 5 (B, C).

[0121] X-ray results showed that the Stress-relaxed HAMA@Lip group had significantly less osteophyte formation than the other surgical groups. Although the joint space of all surgical groups was wider than that of the blank group, this was believed to be due to the destruction of the joint capsule structure and short-term effusion caused by the surgery, and not a true manifestation of cartilage degeneration. In addition, the shorter modeling period (6 weeks) more reflects the early OA phenotype, and the typical characteristics of late-stage OA, such as narrowing of the joint space or the formation of a large number of osteophytes, have not yet appeared. This phenomenon is consistent with the short-term observation results of the modified Hulth model or partial meniscectomy model in existing studies. Therefore, the present invention does not use the width of the joint space as a quantitative indicator of OA progression.

[0122] Micro-CT scanning obtained high-resolution three-dimensional images, which accurately evaluated the structure and density of the subchondral bone. The bone volume fraction (BV / TV) of the Stress-relaxedHAMA@Lip group was significantly higher than that of the other surgical groups, although slightly lower than that of the blank group, indicating that the treatment effectively inhibited subchondral bone loss in the early and middle stages of OA. Further analysis showed that the trabecular thickness (Tb.Th) of the Stress-relaxedHAMA@Lip group increased, the trabecular spacing (Tb.Sp) decreased, and the trabecular structural factor (Tb.Pf) decreased, indicating that the quality of bone structure improved and bone resorption was delayed ( Figure 5 (in DG).

[0123] 6. Histological staining

[0124] Histological staining (HE and safranin fast green staining) showed that the cartilage surface was obviously eroded and the matrix was severely degraded in the PBS and HAMA groups ( Figure 6In the stress-relaxed HAMA@Lip group, the cartilage structure was intact, with neatly arranged chondrocytes and minimal matrix loss. Semi-quantitative OARSI scores showed that the model group scored 16.67±1.50, while the stress-relaxed HAMA@Lip group scored 8.33±0.25 (p<0.001), significantly better than the HAMA group (14±0.58) and the stress-relaxed HAMA group (11±1). Cartilage thickness was 173.16±18.58μm in the model group and 267.11±31.71μm in the stress-relaxed HAMA@Lip group (p<0.05), significantly better than the HAMA group (216.16±54.13μm) and the stress-relaxed HAMA group (245.00±25.68μm). These results indicate that the stress-relaxed HAMA@Lip microsphere system can significantly reduce cartilage damage by continuously releasing TUDCA and providing mechanical support, demonstrating superior OA treatment efficacy ( Figure 6 (D, E).

[0125] Immunohistochemistry results showed that the expression levels of Col II and Aggrecan in the stress-relaxed HAMA@Lip group were significantly higher than those in the PBS and HAMA groups, while the expression of MMP13 was the lowest ( Figure 7 A, B, C in the middle). Compared with the blank group, the expression of Aggrecan and type II collagen in the OA model group decreased by 55% and 32% respectively (p<0.05), and the expression of MMP13 increased by 3.3 times (p<0.01), indicating that the cartilage matrix was severely degraded. In the HAMA group, the levels of Aggrecan and Col II did not change much, and the decrease in MMP13 expression was not statistically significant. In contrast, the cartilage protection effect of the stress-relaxedHAMA@Lip group was the most obvious, with Aggrecan and type II collagen restored to 82% and 88% of the blank group (p<0.05), and MMP13 decreased to 18% of the model group (p<0.01), even lower than the blank group ( Figure 7 (D, E, F).

[0126] Immunofluorescence staining results showed that the expression of GRP78 and CHOP in the cartilage tissue of the Stress-relaxed HAMA@Lip group was significantly reduced, indicating that it effectively inhibited ER stress. The HAMA microsphere group had no obvious ER stress relief effect, while the Stress-relaxed HAMA@Lip group had the strongest regulatory effect. Fluorescence quantitative analysis showed that compared with the PBS group, the proportion of CHOP-positive cells in the Stress-relaxed HAMA@Lip group decreased by 46.86% (p<0.05), and the proportion of GRP78-positive cells decreased by 42.29% (p<0.01), indicating that it significantly relieved cartilage ER stress through mechanical buffering and drug synergistic release, delaying the progression of OA ( Figure 6 C, F, G).

[0127] In summary, this study successfully constructed hydrogel microspheres with stress relaxation properties that mimic the cushioning effect of cartilage matrix, acting as "cellular shock absorbers" to slow the progression of OA. These microspheres combine the stress relaxation properties of articular cartilage with the sustained release mechanism of TUDCA, significantly reducing ER stress in chondrocytes and alleviating mechanical loads.

[0128] IV. Conclusion

[0129] The present invention successfully constructed a hydrogel microsphere with stress relaxation properties, named "Stress-relaxedHAMA@Lip", which aims to relieve mechanical stress, improve the mechanical environment of joints, and thus delay the progression of osteoarthritis (OA). By integrating multi-channel microfluidics technology with cationic liposome preparation technology, efficient encapsulation and sustained release of TUDCA were achieved. The hydrogel microspheres effectively relieved the mechanical stress on cartilage during joint movement, reduced the endoplasmic reticulum (ER) stress level of chondrocytes, and reduced cell apoptosis ( Figure 8 Experimental results demonstrated that Stress-relaxed HAMA@Lip significantly reduced cartilage degeneration, maintained the integrity of cartilage tissue structure, and effectively delayed disease progression in a rat OA model. Its sustained release of TUDCA inhibited the expression of ER stress markers and enhanced chondrocyte activity. In summary, this study proposed a novel and effective OA treatment strategy, highlighting the potential of hydrogel microspheres in reducing mechanical stress and alleviating ER stress. This study lays a theoretical foundation for the development of biomaterial-based OA interventions and provides innovative ideas for clinical treatment.

[0130] Comparative Example 1

[0131] Only HAMA, HAMA-CHO or HAMA-NH2 was used as the hydrogel material, and hydrogel microspheres were prepared according to the scheme of Example 1. The hydrogel microspheres were subjected to the stress test and stress relaxation test. The results are shown in FIG. Figure 9-11 shown.

[0132] Modulus and stress relaxation tests were conducted using a Discovery Hybrid Rheometer (TA) with a 20 mm plate and an 800 μm gap setting. The materials were mixed at a 2% wt ratio, and 0.5% LAP was added. The materials were cross-linked using 405 nm blue light for 300 seconds. Frequency sweeps (5% strain, 0.1-10 Hz), strain sweeps (1 Hz, 1%-200%), and stress relaxation tests (5% strain, 300 seconds) were performed.

[0133] Figure 17 In the figure, a, b, and c are the modulus test results (frequency sweep and strain sweep), stress relaxation test results, and fitting curves of HAMA, respectively. Figure 18 In the figure, a, b, and c are the modulus test results (frequency sweep and strain sweep), stress relaxation test results, and fitting curves of HAMA-CHO, respectively. Figure 19 In the figure, a, b, and c are the modulus test results (frequency sweep and strain sweep), stress relaxation test results, and fitting curves of HAMA-NH2, respectively.

[0134] The above results demonstrate that the stress-relaxing hydrogel microspheres formed from aldehyde-modified hyaluronic acid (HAMA-CHO) and hydrazine-modified components exhibit superior stress relaxation properties in both frequency and strain sweeps, significantly buffering short-term mechanical impacts and achieving significant shock absorption. In contrast, the three hydrogel microsphere materials used in the comparative examples generally lack excellent stress relaxation properties, exhibiting excessively rapid stress recovery in stress relaxation modulus tests and failing to effectively dissipate external mechanical energy.

[0135] In addition, the inventors also used GelMA as the hydrogel material to prepare hydrogel microspheres, and found that they also had the problem of too fast stress recovery.

Claims

1. A method for preparing viscoelastic hydrogel microspheres with adjustable stress relaxation properties, characterized in that: The following steps are involved: (1) WYRGRL peptide was reacted with DSPE-PEG2000-NHS via a Mannich reaction to synthesize DSPE-PEG2000-WYRGRL; (2) Phospholipids, cholesterol, octadecylamine, DSPE-PEG2000-WYRGRL, and DSPE-PEG2000-NH2 were mixed in a weight ratio of 5:1:3:1.2:1.5 to prepare Lipo-WYRGRL@TUDCA cationic liposomes; (3) Hydrogel microspheres were prepared by microfluidics using aldehyde-modified methacrylated hyaluronic acid and a photoinitiator as the inner aqueous phase, Lipo-WYRGRL@TUDCA cationic liposomes, amino-modified methacrylated hyaluronic acid and a photoinitiator as the outer aqueous phase, and Span 80 and paraffin oil as the oil phase.

2. The method according to claim 1, characterized in that In step (1), DSPE-PEG2000-NHS is dissolved in DMF and reacted with WYRGRL peptide and triethylamine at a molar ratio of 1:1.1:3 to obtain DSPE-PEG2000-WYRGRL.

3. The method according to claim 1, characterized in that The solvent used in step (2) is a mixed solution of chloroform and water in a volume ratio of 3:

1.

4. The method according to claim 1, wherein The aldehyde-modified methacrylated hyaluronic acid in step (3) is prepared by reacting hyaluronic acid and methacrylic acid under alkaline conditions to obtain methacrylated hyaluronic acid, which is then reacted with sodium periodate.

5. The method according to claim 1, wherein The amino-methacrylated hyaluronic acid in step (3) is prepared by activating the methacrylated hyaluronic acid with EDC and HOBt, and then reacting it with oxalyl dihydrazide.

6. The method according to claim 1, characterized in that The photoinitiator in step (3) is LAP.

7. The method according to claim 1, characterized in that In step (3), the weight ratio of the aldehyde-modified methacrylated hyaluronic acid, the amino-modified methacrylated hyaluronic acid and the cationic liposome solution is 4:2:

1.

8. The method according to claim 1, characterized in that The drug is loaded into the Lipo-WYRGRL@TUDCA cationic liposomes in step (2), wherein the drug includes tauroursodeoxycholic acid, wherein the weight ratio of phospholipid, cholesterol, octadecylamine, DSPE-PEG2000-WYRGRL, DSPE-PEG2000-NH2 and tauroursodeoxycholic acid is 5:1:3:1.2:1.5:

1.

9. Viscoelastic hydrogel microspheres with adjustable stress relaxation properties prepared by the method according to any one of claims 1 to 8.

10. Use of the viscoelastic hydrogel microspheres with adjustable stress relaxation properties according to claim 9 in the preparation of a drug for treating osteoarthritis.

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