A stress-triggered selectively targeted hydrogel microsphere, its preparation method and application

Abstract

This invention provides a stress-triggered selective targeting hydrogel microsphere, its preparation method, and its application, belonging to the field of biomedical technology. The core of this invention is the "stress-triggered charge reversal" mechanism. It utilizes the interaction between weakly cross-linked disulfide bonds broken under stress and "charge reversal" messenger molecules to achieve "charge reversal" of nanoparticles under stress. A dual-network hydrogel microsphere system is prepared using microfluidic technology to achieve bearing lubrication and stress relaxation within the joint cavity. Stress-triggered charge reversal is achieved at stress-overloaded cartilage lesions, imparting a positive charge to the drug-loaded nanoparticles within the hydrogel microspheres. This significantly enhances the ability of the drug-loaded nanoparticles to penetrate deep into the cartilage matrix and target chondrocytes for drug release under the guidance of the charge. This enables the selective and targeted release of appropriate drug concentrations to deep-seated chondrocytes in pathological microenvironments induced by different stress intensities, achieving highly effective and low-toxicity treatment of osteoarthritis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a stress-triggered selective targeting hydrogel microsphere, its preparation method, and its application. Background Technology

[0002] Mechanical stress plays a crucial role in biological research, particularly in cellular function and disease progression. Recent studies have shown that cellular responses to mechanical stress not only affect physiological function but may also play a key role in the occurrence and development of diseases. For example, in the skeletal system, appropriate mechanical loads promote bone remodeling through a balance between osteoblasts and osteoclasts. When mechanical stress is abnormal and this balance is disrupted, it can lead to osteoporosis or other skeletal diseases. Simultaneously, abnormal mechanical stress can activate the immune system, triggering inflammatory responses. For instance, in the cardiovascular system, abnormal shear stress on the arterial wall activates endothelial cells to release pro-inflammatory factors, attracting leukocyte infiltration and thus promoting the formation and development of atherosclerosis. Therefore, improving the biomechanical microenvironment of lesion tissues and mitigating the negative impact of abnormal mechanical stress on cellular physiological function represents a novel therapeutic target.

[0003] To mitigate the negative effects of abnormal mechanical stress on cells, researchers have developed numerous biomaterial systems with stress-triggered functions. While these stress-triggered systems can effectively alleviate cell damage and apoptosis caused by stress overload, the pathological changes in cells caused by long-term, continuous abnormal mechanical stress still require further correction.

[0004] In the pathological microenvironment of osteoarthritis (OA), persistent abnormal mechanical stress causes chondrocytes to regulate their metabolic activities and cytokine expression in response to changes in the extracellular mechanical stress environment. Among these changes, abnormally synthesized transforming growth factor-β1 (TGF-β1) induces cellular redox imbalance by increasing the production of reactive oxygen species (ROS) in the mitochondrial respiratory chain of various cell populations, including chondrocytes, leading to apoptosis, senescence, and the expression of fibrosis genes. More importantly, after abnormal synthesis and secretion by chondrocytes, TGF-β1 is deposited in the articular cartilage matrix. Under stress, the deposited TGF-β1 is induced to transform into its active form, and the magnitude of mechanical stress is positively correlated with the degree of TGF-β1 activation. Subsequently, activated TGF-β1 binds to the AKL-1 receptor on the chondrocyte membrane, triggering downstream signaling pathways, inducing terminal differentiation of chondrocytes, and increasing the expression of matrix metalloproteinase-13 (MMP-13), thereby leading to the degradation of the chondrocyte extracellular matrix.

[0005] To address this, researchers have developed multifunctional hydrogel microsphere systems for releasing drugs deep into cartilage tissue, thereby alleviating pathological changes in chondrocytes under abnormal mechanical stress. For example, Lin et al. developed a charge-guided hydrogel microsphere system that not only effectively alleviates stress overload but also guides positively charged nanoparticles to penetrate deep into the cartilage matrix through positive and negative charge interactions, targeting chondrocytes for drug release and effectively inhibiting the negative effects of TGF-β1 activation.

[0006] While charge-guided hydrogel microsphere systems can effectively alleviate pathological changes in chondrocytes caused by abnormal TGF-β1 synthesis by targeting deep chondrocytes with positively charged drug-loaded nanoparticles, the curved surface of articular cartilage and the misalignment of the lower limb's force lines often lead to uneven stress distribution on the cartilage surface. This results in varying stress-induced TGF-β1 concentrations in different parts of the cartilage tissue. Therefore, indiscriminate drug release will inhibit physiologically functional TGF-β1, causing significant side effects. To address these challenges, it is necessary to construct a hydrogel microsphere system with selective targeting of deep chondrocytes for drug release. This system would selectively target deep chondrocytes with appropriate drug concentrations based on the TGF-β1 concentration in the pathological microenvironment of the cartilage, achieving highly effective and low-toxicity treatment of osteoarthritis (OA).

[0007] In recent years, selective drug delivery technology has experienced rapid development. This technology refers to the precise delivery of drugs to target sites within the body using specific biomaterial carriers. Through a special mechanism, these biomaterials are endowed with the ability to selectively release drugs at lesion sites while selectively not releasing drugs at healthy sites, thus achieving selective drug release, improving efficacy, and reducing side effects. Currently, selective drug delivery biomaterials based on the "stimulus-response-charge reversal" concept are increasingly being applied to the selective release of drugs under specific pathological conditions. For example, researchers have developed charge-reversal nanomicelles loaded with Nap-N3 using the specific reaction between N-(2-hydroxyethyl)-4-azido-1,8-naphthalimide (Nap-N3) and hydrogen sulfide (H2S). Triggered by H2S within the tumor lesion, Nap-N3 is converted to Nap-NH3. +This process transforms the nanomicelles into positively charged particles, increasing uptake at the lesion site. Ultimately, this allows the biomaterial system to release high concentrations of drug in the central region of the lesion, while releasing low concentrations or no drug at all in the surrounding area, significantly reducing drug side effects and improving efficacy. Therefore, introducing the concept of charge reversal into stress-triggered hydrogel microsphere systems holds promise for imparting a positive charge to drug delivery nanoparticles within the hydrogel microspheres under the triggering of characteristic factors (i.e., stress) in cartilage lesions. This, guided by the charge, significantly enhances the ability of drug-loaded nanoparticles to penetrate deep into the cartilage matrix and target deep chondrocytes for drug release.

[0008] However, in healthy cartilage, the drug-loaded nanoparticles within the hydrogel microsphere system are electrically neutral, preventing them from penetrating deep into the cartilage matrix. Furthermore, nanoparticles free within the joint cavity are quickly cleared by the synovium, thus failing to release drugs to chondrocytes. Therefore, how to effectively combine stress stimulation with charge reversal in hydrogel systems to provide a selectively targeted hydrogel system based on stress-triggered charge reversal, where the hydrogel system can target and release more drugs into deeper tissues at sites of higher stress, while releasing no drugs in healthy areas, thereby endowing the hydrogel with the function of selectively targeting deep diseased chondrocytes to release drugs, achieving highly effective and low-toxicity treatment of osteoarthritis, has become an urgent technical problem to be solved. Summary of the Invention

[0009] This invention aims to solve the aforementioned technical problems by providing stress-triggered selective targeting hydrogel microspheres, their preparation method, and applications. The technical objective of this invention is to reduce side effects on healthy tissues while ensuring drug efficacy. Through a "stress-triggered charge reversal" mechanism, sufficient positively charged nanoparticles are successfully generated in diseased tissues to deliver more drug. Simultaneously, in healthy tissues, due to insufficient "stress triggering," charge reversal does not occur, significantly reducing the side effects of nanoparticles on healthy tissues, thereby achieving more efficient and less toxic treatment for osteoarthritis.

[0010] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: This invention first provides a method for preparing stress-triggered selectively targeted hydrogel microspheres, comprising the following steps: (1) Nanoliposomes were prepared by thin film hydration using lecithin, cholesterol, LSKL peptide, N-(benzoylthio)benzamide and DSPE-PEG-Nap-N3 as raw materials; (2) Methacrylated hyaluronic acid (HAMA) and thiolized hyaluronic acid (HASH) were constructed by esterification reaction as the basic framework of the dual network hydrogel system. Combined with microfluidic technology, the double bond free radical polymerization reaction of HAMA polymer network and the thiol polymerization reaction of HASH polymer network were initiated by ultraviolet light to construct dual network micro-nano hydrogel microspheres loaded with "charge reversal" secondary nanostructure.

[0011] The preparation method provided by this invention is based on the "stress-triggered charge reversal" mechanism. It utilizes the interaction between weakly cross-linked disulfide bonds broken under stress and "charge reversal" messenger molecules to achieve "charge reversal" of nanoparticles under stress. A dual-network hydrogel microsphere system is then prepared using microfluidic technology to achieve bearing lubrication and stress relaxation within the joint cavity. Stress-triggered charge reversal is achieved at stress-overloaded cartilage lesions, imparting a positive charge to the drug-loaded nanoparticles within the hydrogel microspheres. This significantly enhances the ability of the drug-loaded nanoparticles to penetrate deep into the cartilage matrix and target chondrocytes for drug release under the guidance of the charge. This enables the selective and targeted release of appropriate drug concentrations to deep-seated lesions in the pathological microenvironment induced by different stress intensities, achieving highly effective and low-toxicity treatment of osteoarthritis (OA).

[0012] The hydrogel microspheres of this invention can still generate sufficient positively charged nanoparticles in diseased tissues, thereby delivering adequate drug delivery. In healthy tissues, however, insufficient "stress triggering" prevents "charge reversal" from occurring. Therefore, healthy tissues contain very few positively charged nanoparticles, significantly reducing drug release and side effects.

[0013] Furthermore, the weight ratio of lecithin, cholesterol and DSPE-PEG-Nap-N3 in step (1) is 4:1:0.1-10, the concentration of LSKL peptide is 0.1-100 mg / mL, and the concentration of N-(benzoylthio)benzamide is 0.1-100 mg / mL.

[0014] Furthermore, the reaction conditions for the thin film hydration method described in step (1) are hydration at 37°C for 0.5-2.0 h, followed by ultrasonic power of the probe at 5-80% and ultrasonic time of 5-30 min.

[0015] Furthermore, the preparation process of thiolated hyaluronic acid in step (2) is as follows: HOBt and mercaptoethylamine are added to sodium hyaluronate solution, the pH is adjusted to 3.0-6.5, then EDC is added, and the reaction is carried out at room temperature for 24 h. After the reaction is completed, the pH is adjusted to 7.5-11.0, then dithiothreitol is added, and the reaction is continued for 24 h.

[0016] Furthermore, the weight ratio of methacrylated hyaluronic acid and thiolized hyaluronic acid in step (2) is 1:0.1-10.

[0017] Furthermore, the microfluidic reaction conditions in step (2) are controlled by a flow rate ratio of water phase: oil phase = 1:1-10.

[0018] Furthermore, in step (2), the photoinitiator is LAP, and the conditions for UV photoinitiation are curing by irradiation with 365 nm UV light for 5-30 min.

[0019] Furthermore, the concentration of the dual-network hydrogel system described in step (2) is 3.0-5.0 wt%.

[0020] A second objective of this invention is to provide stress-triggered selective targeting hydrogel microspheres prepared by the method described above.

[0021] A third objective of this invention is to provide the application of the stress-triggered selective targeting hydrogel microspheres described above in the preparation of drugs for treating osteoarthritis.

[0022] The beneficial effects of this invention are as follows: This invention first prepares "charge-reversed" nanoliposomes with a Nap-N3 structure and N-(benzoylthio)benzamide as a messenger molecule via thin-film hydration. Subsequently, methacrylated hyaluronic acid (HAMA) and thiolized hyaluronic acid (HASH) are constructed as the basic framework of a dual-network hydrogel system through esterification reactions. Combining microfluidic technology, a dual-network micro-nano hydrogel microsphere system loaded with "charge-reversed" secondary nanostructures is constructed through UV-initiated double-bond free radical polymerization of the HAMA polymer network and thiol polymerization of the HASH polymer network. This hydrogel microsphere system can utilize its unique spherical structure to achieve bearing lubrication of articular cartilage, reducing frictional stress damage. Furthermore, in "stress overload" regions, it absorbs elastic energy through the breaking of weakly cross-linked disulfide bonds, thereby achieving "stress relaxation" of the articular cartilage and effectively alleviating stress damage. Furthermore, under stress triggering, the free thiol groups generated after the weak cross-linked disulfide bonds break will trigger the interaction between "Nap-N3" and "messenger molecules" on the surface of the "charge reversal" liposomes. This reversal of charge from neutral to positive secondary nanostructures simultaneously rebuilds the disulfide bonds in the hydrogel microsphere network. Thus, the hydrogel microsphere system achieves "charge reversal" under stress triggering, transforming from electrically neutral hydrogel microspheres in the resting state into a micro-nano hydrogel microsphere system with "charge-guided" function, loaded with positively charged secondary nanostructures. The released positively charged nanoparticles will penetrate deep into the cartilage matrix and deliver medication close to chondrocytes via "charge guidance," thereby inhibiting the negative effects of abnormal TGF-β1 synthesis and activation. Ultimately, this effectively inhibits chondrocyte apoptosis, maintains the homeostasis of extracellular matrix synthesis and degradation, and significantly improves the efficacy of OA treatment. The construction of this intelligent hydrogel microsphere system, as well as the study of the mechanism linking the differences in the spatiotemporal distribution of stress and TGF-β1 with the pathological progression of OA to date, provides a solid theoretical basis and experimental foundation for the design of future biomaterials, and also provides a brand-new diagnostic and treatment strategy for the prevention and treatment of OA. Attached Figure Description

[0023] Figure 1 Construction and functional verification of stress-triggered charge reversal hydrogel systems; (A) HAMA, HASH, DSPE-PEG-Nap-N3 polymers 1(A) 1H NMR detection results; (B) Strain curves of hydrogel networks with different concentrations under pressure; (C) Morphology of charge-reversed nanoliposomes under transmission electron microscopy; (D) Particle size distribution of charge-reversed nanoliposomes; (E) Schematic diagram of stress relaxation performance of dual-network hydrogel system; (F) Stress relaxation curve of dual-network hydrogel system; (G) Schematic diagram of charge reversal experiment of nanoliposomes; (H) Proportion of charge reversal achieved by nanoliposomes under stress triggering of different intensities (by fluorescence intensity reaction); (I) Schematic diagram of experiment to detect Zeta potential of charge-reversed nanoparticles; (J) Zeta potential of two groups of nanoliposomes in resting state and stress-triggered state.

[0024] Figure 2 The following are the results of the construction, characterization, and biocompatibility testing of the hydrogel microsphere system: (A) Morphology of the hydrogel microsphere system under an optical microscope; (B) Statistical graph of particle size of the hydrogel microsphere system; (C) Morphology of the hydrogel microsphere system under a scanning electron microscope; (D) Elemental energy dispersive spectroscopy (EDS) detection of the hydrogel microsphere system; (E) Effect of different concentrations of nanoparticles on chondrocyte proliferation; (F) Effect of different concentrations of hydrogel microsphere system on chondrocyte proliferation; (G) Effect of the highest concentration of nanoparticles and hydrogel microsphere system on the live / dead state of chondrocytes; (ns represents no significant difference).

[0025] Figure 3 To alleviate TGF-β1-induced chondrocyte dysfunction using a hydrogel microsphere system; (A) Oxygen consumption rate (OCR) of chondrocytes in different groups was detected using a Seahorse energy metabolism analyzer to assess mitochondrial respiratory chain function; (B) Mitochondrial respiratory chain function curves of chondrocytes in different experimental groups (n = 3); (C) Quantitative analysis of basal respiratory function; (D) Quantitative analysis of ATP production; (E) Quantitative analysis of maximum respiratory function; (F) Quantitative analysis of reserve respiratory function; (G) Flow cytometry apoptosis maps of chondrocytes in different experimental groups; (H) Quantitative analysis of chondrocyte flow cytometry apoptosis results (n = 3); (ns: no significant difference, * P < 0.05, ** P < 0.01, *** P < 0.001). Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is described in detail below with reference to embodiments. It should be noted that the following embodiments are for explanation and illustration only and are not intended to limit the invention. Non-essential improvements and adjustments made by those skilled in the art based on the above description are still within the scope of protection of this invention. Example

[0027] I. Experimental Materials and Methods 1. Synthesis of Methacrylamide Hyaluronic Acid (HAMA) 1000 mg of sodium hyaluronate was weighed and dissolved in 50 mL of deionized water, and mechanically stirred until completely dissolved. 2 mL of methacrylic anhydride was added dropwise, followed by 2 mL of 5 M NaOH solution, with a dropping rate controlled at 0.1 mL / min. The reaction system was placed in an ice-water bath and stirred continuously for 12 h. After the reaction was complete, the mixture was centrifuged at 7000 rpm for 15 min, and the supernatant was collected and placed in a dialysis bag with a molecular weight cutoff of 8000 Da. Dialysis was performed in deionized water for 3 days, changing the solution every 8 h. After dialysis, the solution was freeze-dried to obtain a white, spongy solid. The degree of substitution of the methacryloyl group was calculated by ¹H NMR (D₂O₃ as solvent).

[0028] 2. Synthesis of Thiolized Hyaluronic Acid (HASH) Weigh 500 mg of sodium hyaluronate and dissolve it in 150 mL of deionized water. Add 100-500 mg of HOBt and 100-500 mg of mercaptoethylamine, and adjust the pH to 3.0-6.5. Add 50-200 mg of EDC and react at room temperature for 24 h. After the reaction is complete, adjust the pH to 7.5-11.0, add 100-500 mg of dithiothreitol (DTT), and continue the reaction for 24 h. Dialyze the reaction solution in a buffer solution at pH 4.0 for 3 days, and then dialyze it in deionized water for 2 days. Freeze-dry to obtain HASH solid. Calculate the degree of thiol substitution by ¹H NMR.

[0029] 3. Synthesis of DSPE-PEG-Nap-N3 Under argon protection, 10-100 mg of DSPE-PEG-COOH, 214 mg of DMAP, and 1.45 g of DCC were dissolved in 100 mL of anhydrous tetrahydrofuran and stirred in an ice bath. 0.5-10 g of Nap-N3 was dissolved in 10 mL of anhydrous tetrahydrofuran and slowly added dropwise to the above system. After reacting in an ice bath for 4 h, the reaction was allowed to proceed at room temperature for 48 h. After the reaction was complete, a small amount of deionized water was added dropwise to quench the DCC, the precipitate was removed by filtration, and the solvent was removed by rotary evaporation. The resulting solid was reconstituted with dichloromethane, and excess diethyl ether was added to precipitate the solid. The mixture was then allowed to stand overnight at -20°C. The precipitate was collected and dried under vacuum to obtain DSPE-PEG-Nap-N3.

[0030] 4. Preparation of charge-reversed nanoliposomes The liposomes were prepared using a thin-film hydration method: lecithin, cholesterol, DSPE-PEG-Nap-N3 (mass ratio 4:1:0.1-10), and 0.1-100 mg / mL N-(benzoylthio)benzamide were dissolved in 1.5 mL of chloroform, and a homogeneous lipid film was formed by rotary evaporation. PBS buffer (pH 7.4) containing 10.1-100 mg / mL LSKL peptide was added, and the mixture was hydrated at 37°C for 0.5-2.0 h. Subsequently, the mixture was sonicated with a probe (power 5-80%, time 5-30 min) to obtain a nanoliposome suspension. Particle size and zeta potential were determined by DLS.

[0031] 5. Preparation of dual-network hydrogel microspheres HAMA and HASH were dissolved in deionized water at a mass ratio of 1:0.1-10. Charge-reversal nanoliposome suspension (final concentration 2.5 wt%) and photoinitiator LAP (0.5 wt%) were added to form the aqueous phase. The oil phase was paraffin oil containing 5 wt% Span 80. The aqueous and oil phases were injected separately into a capillary microfluidic device using a syringe pump, controlling the flow rate ratio (aqueous phase:oil phase = 1:1-10) to form emulsion droplets. The emulsion was cured by irradiation with 365 nm UV light for 5-30 min. The microspheres were collected, washed with isopropanol, and centrifuged to obtain hydrogel microspheres.

[0032] 6. Material characterization and performance testing (1) Mechanical property test: The hydrogel was compressed to 15% strain at a rate of 1 mm / min using a universal mechanical testing instrument. The stress-strain curve was recorded and the elastic modulus was calculated. (2) Stress relaxation test: The strain was fixed at 15%, and the stress change over time was continuously recorded. (3) Tribological test: The lubrication performance of the hydrogel microsphere suspension (1 mg / mL) was tested using a UMT-3 tribological testing machine with a load of 3.0 N, a frequency of 1 Hz, and an amplitude of 4 mm. (4) Charge reversal performance verification: Different stresses (1.0–4.0 N) were applied to the hydrogel. After the nanoparticles were released, the change in Zeta potential was detected, and the reversal efficiency was quantitatively analyzed by fluorescence intensity (408 nm excitation). (5) Cell compatibility test: C28 / I2 chondrocytes were co-cultured with hydrogel microspheres. Cell proliferation was detected by CCK-8 assay, and cell viability was assessed by Calcein-AM / PI staining. (6) Mitochondrial function detection: Oxygen consumption rate (OCR) of chondrocytes was detected using a Seahorse XFe24 analyzer to assess ATP production and maximum respiratory capacity. (7) Animal experiments: SD rats were modeled by ACL transection and medial meniscus resection. Hydrogel microspheres were injected into the joint cavity at 2 and 5 weeks postoperatively. Samples were taken at 8 weeks for Micro-CT and histological (H&E, Safranin-Fixed Green, TUNEL, immunofluorescence) analysis.

[0033] II. Experimental Results and Analysis 1. Construction and functional verification of the "stress-triggered charge reversal" hydrogel system First, the applicant successfully synthesized HAMA and HASH polymers that constitute the hydrogel network, as well as the key polymer DSPE-PEG-Nap-N3 for constructing charge-reversed nanoparticles. 1 ¹H NMR detection and integral calculation showed that the grafting rate of HAMA was about 34%, and the grafting rate of HASH was about 18%. Figure 1 (A). Subsequently, the mechanical properties of the dual-network hydrogel system at different concentrations were tested. The results showed that when the concentration of the dual-network hydrogel system reached 3.0-5.0 wt%, the mechanical properties of the system were significantly improved. Figure 1 (B). Therefore, 5.0 wt% is a relatively ideal candidate concentration for the hydrogel system.

[0034] Secondly, the applicant successfully prepared nanoliposomes with charge reversal function, and observed their morphology using transmission electron microscopy. The liposomes showed uniform size distribution and the characteristic shell-core structure of liposomes. Figure 1(C). Simultaneously, the particle size distribution of the liposomes was detected using dynamic light scattering (DLS), and the Gaussian curve fitting yielded a liposome particle size of 116.2 ± 49.01 nm (C). Figure 1 (D).

[0035] Next, the applicant conducted preliminary verification of the function of the "stress-triggered charge reversal" hydrogel system. First, to verify the stress relaxation performance of the dual-network hydrogel system, based on a "stress overload" pressure of approximately 150 kPa for the weight-bearing cartilage, the applicant continuously monitored the stress curves of the dual-network hydrogel system at 10% fixed strain (pressure approximately 102.23 kPa) and 15% fixed strain (pressure approximately 295.84 kPa), respectively. The results showed that the breakage of weakly cross-linked disulfide bonds was only triggered when the "stress overload" pressure was reached. Figure 1 (E). Meanwhile, the stress in the dual-network hydrogel system decreases slowly, exhibiting a good stress relaxation effect. Figure 1 (Middle F).

[0036] Furthermore, the applicant utilized the Nap-N3 functional group, which exhibits fluorescence properties during charge reversal, to conduct preliminary verification of the charge reversal performance of nanoliposomes. Figure 1 (G). By applying stresses of varying magnitudes to the "stress-triggered charge reversal" hydrogel system and observing the green fluorescence emitted by a 408 nm laser, it can be seen that under "stress overload" conditions, the nanoliposomes successfully trigger charge reversal, and the proportion of triggered charge reversal increases with increasing stress. Figure 1 (H). Finally, the nanoliposomes within the hydrogel system were released, and their zeta potentials were measured (H). Figure 1 (I). It can be seen that under stress triggering, nanoliposomes with charge reversal function successfully changed from negative to positive charge (I). Figure 1 (J).

[0037] In conclusion, the "stress-triggered charge reversal" hydrogel system constructed by the applicant in the preliminary experiments is successful and effective.

[0038] 2. Construction and characterization of hydrogel microsphere system A stress-triggered charge reversal (STCR) hydrogel microsphere system was successfully prepared using a microfluidic device, and the prepared system was characterized. Optical microscopy revealed that the hydrogel microsphere system exhibited near-perfect sphericity. Figure 2 (A). Simultaneously, the particle size of the hydrogel microsphere system was measured and statistically analyzed, revealing a uniform particle size distribution, concentrated around 80 μm. Figure 2(B) Subsequently, the hydrogel microsphere system was observed using a scanning electron microscope, revealing that the hydrogel microsphere system possesses a relatively uniform pore structure. Figure 2 (C). Simultaneously, energy dispersive spectroscopy (EDS) analysis of the hydrogel microsphere system revealed that, in addition to the conventional carbon (C) and oxygen (O) elements, it is also rich in phosphorus (P), thus proving that the hydrogel microsphere system loaded with nanoparticles was successfully constructed. Figure 2 (D).

[0039] 3. Biocompatibility testing of the hydrogel microsphere system The biocompatibility of the hydrogel microsphere system was tested. This was achieved by combining chondrocytes with different concentrations of nanoparticles (…). Figure 2 (E) and hydrogel microsphere system ( Figure 2 The cells were co-cultured with chondrocytes (F1) for 48 h, and the proliferation of chondrocytes was detected using a CCK-8 assay kit. The results showed that the hydrogel microsphere system had no negative impact on chondrocyte proliferation and exhibited good biocompatibility. Finally, the highest concentration of nanoparticles and the hydrogel microsphere system involved in the above experiments were co-cultured with chondrocytes for 48 h, and the toxicity of the hydrogel microsphere system was verified by live / dead cell staining. Figure 2 (G). Fluorescence microscopy revealed that most chondrocytes were live cells (exhibiting green fluorescence), with very few dead cells (exhibiting red fluorescence). Therefore, our constructed hydrogel microsphere system exhibits almost no cytotoxicity and is a biocompatible medical biomaterial.

[0040] 4. Study on the relief of TGF-β1-induced chondrocyte dysfunction by hydrogel microsphere system The efficacy of the constructed hydrogel microsphere system in alleviating TGF-β1-induced mitochondrial respiratory chain dysfunction and chondrocyte apoptosis in chondrocytes was validated. First, the oxygen consumption rate (OCR) of chondrocytes was measured using a Seahorse energy metabolism analyzer to assess the mitochondrial respiratory chain function of chondrocytes. Figure 3 (A). By plotting the mitochondrial respiratory chain functional curves of chondrocytes in each experimental group ( Figure 3 (B), and quantitative analysis of important indicators related to respiratory chain function reflected by the curve ( Figure 3 In our study (CF), we found that chondrocytes in the disease-simulated group with added active TGF-β1 experienced dysfunction of the mitochondrial respiratory chain due to the abnormal increase in active TGF-β1, resulting in significant decreases in key indicators such as ATP production and maximum respiratory function compared to the control group. Meanwhile, chondrocytes in the treatment-simulated group co-cultured with a hydrogel microsphere system showed significant improvement in mitochondrial respiratory chain function, with all key indicators significantly higher than those in the disease-simulated group.

[0041] Subsequently, flow cytometry was used to preliminarily examine the efficacy of the hydrogel microsphere system in inhibiting TGF-β1-induced chondrocyte apoptosis. Figure 3 (G). Quantitative analysis of flow cytometry apoptosis results ( Figure 3 (H) found that the hydrogel microsphere system can effectively inhibit TGF-β1-induced chondrocyte apoptosis, thus promoting the repair and regeneration of cartilage tissue.

[0042] III. Conclusion This invention centers on the "stress-triggered charge reversal" mechanism. It utilizes the interaction between weakly cross-linked disulfide bonds broken under stress and "charge reversal" messenger molecules to achieve stress-triggered charge reversal in nanoparticles. A dual-network hydrogel microsphere system is then prepared using microfluidic technology to achieve intra-articular bearing lubrication and stress relaxation. Stress-triggered charge reversal is achieved at stress-overloaded cartilage lesions, imparting a positive charge to the drug-loaded nanoparticles within the hydrogel microspheres. This significantly enhances the ability of the drug-loaded nanoparticles to penetrate deep into the cartilage matrix and target chondrocytes for drug release under the guidance of this charge. This enables the selective and targeted release of appropriate drug concentrations to deep-seated lesions in pathological microenvironments induced by different stress intensities, achieving highly effective and low-toxicity treatment of osteoarthritis (OA).

[0043] IV. Creative Description of the Invention In this invention, the "stress-triggered charge reversal" mechanism is difficult to achieve. Previous literature contains many examples of stress-triggered hydrogels and nanoparticles capable of charge changes. However, linking these two processes through a single mechanism is challenging. The inventors explored numerous stress-triggered and charge-change schemes, but all failed to achieve the goal of inducing further charge changes in the hydrogel after stress triggering; realizing this linked reaction is extremely difficult.

[0044] Existing stress-relaxation hydrogels generally lack the potential for further chemical reactions after stress triggering, making it impossible to connect to the subsequent charge reversal. Furthermore, existing charge reversal nanoparticles, primarily based on reactions with substances like ROS or pH, cannot react with the components of the hydrogel, thus failing to generate a linkage with the stress-triggered hydrogel. Therefore, finding a mechanism that enables the stress-triggered charge reversal phenomenon is extremely difficult. This invention, through extensive exploration and innovation, ultimately achieves this effect.

[0045] The greatest advantage of this invention is that it significantly reduces side effects on healthy tissues while ensuring therapeutic efficacy. In existing technologies, positively charged drug delivery materials deliver drugs to deeper tissues by utilizing positive charges. However, this delivery lacks controllability, resulting in the delivery of large amounts of drug to both diseased and healthy tissues, leading to significant side effects in healthy tissues. In contrast, this invention generates sufficient positively charged nanoparticles in diseased tissues to deliver adequate drug. In healthy tissues, due to insufficient stress triggering, charge reversal does not occur, resulting in very few positively charged nanoparticles. This greatly reduces drug release into healthy tissues and significantly minimizes side effects.

Claims

1. A method for preparing stress-triggered selectively targeted hydrogel microspheres, characterized in that, Includes the following steps: (1) Nanoliposomes were prepared by thin film hydration using lecithin, cholesterol, LSKL peptide, N-(benzoylthio)benzamide and DSPE-PEG-Nap-N3 as raw materials; (2) Methacrylated hyaluronic acid and thiolized hyaluronic acid were constructed as the basic framework of the dual-network hydrogel system by esterification reaction. Combined with microfluidic technology, double-bond free radical polymerization of the methacrylated hyaluronic acid polymer network and thiolized hyaluronic acid polymer network were initiated by ultraviolet light to construct dual-network micro-nano hydrogel microspheres loaded with "charge reversal" secondary nanostructures.

2. The preparation method according to claim 1, characterized in that, The weight ratio of lecithin, cholesterol and DSPE-PEG-Nap-N3 in step (1) is 4:1:0.1-10, the concentration of LSKL peptide is 0.1-100 mg / mL, and the concentration of N-(benzoylthio)benzamide is 0.1-100 mg / mL.

3. The preparation method according to claim 1, characterized in that, The reaction conditions for the thin film hydration method in step (1) are hydration at 37°C for 0.5-2.0 h, followed by ultrasonic power of 5-80% and ultrasonic time of 5-30 min.

4. The preparation method according to claim 1, characterized in that, The preparation process of thiolated hyaluronic acid in step (2) is as follows: add HOBt and mercaptoethylamine to sodium hyaluronate solution, adjust the pH to 3.0-6.5, then add EDC, react at room temperature for 24 h, after the reaction is completed, adjust the pH to 7.5-11.0, add dithiothreitol, and continue the reaction for 24 h.

5. The preparation method according to claim 1, characterized in that, The weight ratio of methacrylated hyaluronic acid and thiolized hyaluronic acid in step (2) is 1:0.1-10.

6. The preparation method according to claim 1, characterized in that, The conditions for the microfluidic reaction in step (2) are: the flow rate ratio is controlled to be water phase: oil phase = 1:1-10.

7. The preparation method according to claim 1, characterized in that, In step (2), the conditions for UV initiation are curing by irradiation with 365nm UV light for 5-30 minutes.

8. The preparation method according to claim 1, characterized in that, The concentration of the dual-network hydrogel system in step (2) is 3.0-5.0 wt%.

9. Stress-triggered selectively targeted hydrogel microspheres prepared by the method according to any one of claims 1-8.

10. The use of the stress-triggered selective targeting hydrogel microspheres according to claim 9 in the preparation of a drug for treating osteoarthritis.

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