Injectable piezoelectric hydrogel for treating osteoarthritis as well as preparation method and application of injectable piezoelectric hydrogel

Abstract

The invention discloses injectable piezoelectric hydrogel for treating osteoarthritis as well as a preparation method and application of the injectable piezoelectric hydrogel. The injectable piezoelectric hydrogel is prepared from an oxidized chondroitin sulfate-3-aminophenylboronic acid polymer hydrogel matrix and piezoelectric nano particles dispersed in the polymer hydrogel matrix, the piezoelectric nano-particle comprises a core layer and a cartilage cell membrane layer, wherein the core layer is composed of biocompatible piezoelectric nano-particles, and the core layer is coated with the cartilage cell membrane layer. The surface of the cartilage cell membrane layer is conjugated with cartilage targeting peptide. The injectable piezoelectric hydrogel material provided by the invention has strong oxidation resistance, and meanwhile, piezoelectric-induced mitochondrial autophagy can reduce the generation of reactive oxygen species from the source, so as to synergistically protect cartilage cells.

Description

Technical Field

[0001] This invention relates to the fields of biomaterials and degenerative joint diseases, and in particular to an injectable piezoelectric hydrogel for treating osteoarthritis, its preparation method, and its application. Background Technology

[0002] Osteoarthritis (OA) is an extremely common degenerative joint disease affecting approximately 500 million people worldwide. It is characterized by the progressive degeneration of articular cartilage, aging of chondrocytes, mitochondrial dysfunction, and chronic inflammation. Currently, there are no drugs (DMOADs) that can alter the course of osteoarthritis. Treatment options primarily aim to relieve symptoms but cannot stop or reverse the progression of the disease.

[0003] A major challenge in the drug treatment of osteoarthritis is effectively delivering medications to chondrocytes, the cells that reside within the avascular cartilage matrix. The dense, negatively charged extracellular matrix (ECM) of cartilage acts as a significant barrier, hindering the entry of drugs administered systemically or intra-articularly. Furthermore, drugs are rapidly drained from the joint cavity via synovial drainage (within 1-4 hours), requiring frequent injections and increasing the risk of infection.

[0004] Mitochondrial dysfunction in chondrocytes is a key pathological feature of osteoarthritis, leading to excessive production of reactive oxygen species (ROS) and promoting cellular senescence. While antioxidants can scavenge existing ROS, they cannot address the root cause—the accumulation of damaged mitochondria. Mitophagy, the selective autophagy of damaged mitochondria, is a crucial cellular process for maintaining mitochondrial quality. Enhancing mitophagy offers a promising strategy for the treatment of osteoarthritis, but effective methods for safely and efficiently inducing it within cartilage are currently lacking.

[0005] Piezoelectric materials generate electrical charges when subjected to mechanical stress, and because they can influence cell behavior through electrical signals, they show potential in biomedical applications. Ultrasound can serve as a non-invasive, deep-penetrating external stimulus to activate these materials. However, their application in the treatment of osteoarthritis is limited by difficulties in targeted delivery to chondrocytes and a lack of understanding of the underlying molecular mechanisms.

[0006] Therefore, there remains an urgent and unmet need in this field for a targeted, sustained-release, and non-invasive therapeutic platform that can effectively penetrate cartilage, remain in the joint for a long period, and target the fundamental pathological processes of osteoarthritis, such as mitochondrial dysfunction and chondrocyte senescence. Summary of the Invention

[0007] This invention solves the problems existing in the prior art and provides an injectable piezoelectric hydrogel for the treatment of osteoarthritis, its preparation method and application. The injectable piezoelectric hydrogel material proposed in this invention has strong antioxidant capacity, and at the same time, piezoelectric-induced mitophagy can reduce the production of reactive oxygen species from the source and synergistically protect chondrocytes.

[0008] The first objective of this invention is to provide an injectable piezoelectric hydrogel for treating osteoarthritis, comprising a chondroitin sulfate-3-aminophenylboronic acid polymer hydrogel matrix and piezoelectric nanoparticles dispersed within the hydrogel matrix, wherein the piezoelectric nanoparticles comprise a core layer composed of biocompatible piezoelectric nanoparticles and a chondrocyte membrane layer covering the core layer; the surface of the chondrocyte membrane layer is conjugated with chondrocyte-targeting peptides.

[0009] The hydrogel matrix provides an injectable scaffold and a biocompatible environment. It is formed by cross-linking periodate-oxidized chondroitin sulfate (OCS) and 3-aminophenylboronic acid (APBA) via dynamic borate ester bonds. These dynamic bonds endow the hydrogel with excellent shear-thinning (thinning upon injection) and self-healing (recovery after injection) properties.

[0010] Piezoelectric nanoparticles are the functional core for generating therapeutic signals and are meticulously designed to achieve targeting and biomimicry.

[0011] Piezoelectric core: composed of biocompatible piezoelectric material, preferably barium titanate nanoparticles (BTO-NPs) in this invention, with the core size of the nanoparticles preferably between 40-60 nm.

[0012] Biomimetic coating (using BTO-NPs as an example of piezoelectric core material): A chondrocyte membrane (CM) is coated around the BTO-NP core, forming CM@BTO. This coating is prepared by co-extruding BTO nanoparticles with isolated chondrocyte membrane vesicles through a polycarbonate membrane (e.g., with a 400 nm pore size). This biomimetic coating helps the nanoparticles evade immune system recognition and improves their biocompatibility.

[0013] Targeted functionalization: Cartilage-targeting peptides are grafted onto the outer surface of CM@BTO via hydrophobic interactions or covalent bonds (e.g., using cholesterol-polyethylene glycol-maleimide (CLS-PEG-MAL) as a linker) to form the final targeted nanoparticles (Cap-CM@BTO). These peptides can specifically bind to cartilage ECM components, greatly enhancing the enrichment and retention of nanoparticles at damaged cartilage.

[0014] Distribution in the hydrogel matrix: The above Cap-CM@BTO nanoparticles were uniformly dispersed in the hydrogel matrix precursor solution and then crosslinked to form the final product.

[0015] The piezoelectric hydrogel proposed in this invention has excellent injectability, making it suitable for minimally invasive applications; active targeting and prolonged retention in damaged cartilage; synergistic piezoelectric and antioxidant effects; and a novel mechanism of action that addresses the root cause of osteoarthritis (OA) at the mitochondrial and epigenetic levels.

[0016] Preferably, the hydrogel matrix further includes collagen.

[0017] To further enhance biocompatibility and biomimetic properties, collagen is incorporated into the matrix, preferably acid-soluble collagen (Col) extracted from tilapia skin. The collagen is integrated into the OCS-APBA network to form an OCS-Col composite hydrogel (Gel for short). This dynamic cross-linked network forms an irregular but interconnected porous structure, which facilitates the transport of nutrients and metabolic waste.

[0018] Preferably, the piezoelectric nanoparticles have a mass fraction of 0.05%-2.00% in the injectable piezoelectric hydrogel.

[0019] Further preferably, the piezoelectric nanoparticles have a mass fraction of 0.1%-1.0% in the injectable piezoelectric hydrogel.

[0020] Preferably, the cartilage-targeting peptide is a CAP peptide with the amino acid sequence DWRVIIPPRPSA.

[0021] A second objective of this invention is to provide a method for preparing the aforementioned injectable piezoelectric hydrogel, comprising the following steps:

[0022] S1. Preparation of piezoelectric nanoparticles: A suspension of biocompatible piezoelectric nanoparticles was mixed with a chondrocyte membrane protein solution. The chondrocyte membrane was then coated onto the biocompatible piezoelectric nanoparticles by extrusion to obtain chondrocyte membrane-coated biocompatible piezoelectric nanoparticles. Then, chondrocyte-targeting peptides were modified onto the surface of the chondrocyte membrane-coated biocompatible piezoelectric nanoparticles to obtain piezoelectric nanoparticles.

[0023] S2. Preparation of hydrogel matrix: Chondroitin sulfate OCS oxidized with periodate was dissolved in water, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EDC and N-hydroxysuccinimide NHS were added. After adjusting the pH, a dimethyl sulfoxide solution of 3-aminophenylboronic acid APBA was added. The reaction was carried out overnight under weakly acidic conditions. The resulting reaction solution was dialyzed and lyophilized to obtain oxidized chondroitin sulfate-3-aminophenylboronic acid APBA-OCS polymer.

[0024] S3. Hydrogel Composite: The APBA-OCS polymer obtained in step S2 is prepared into an APBA-OCS polymer solution. Piezoelectric nanoparticle suspension is added to the APBA-OCS polymer solution and stirred thoroughly to obtain the injectable piezoelectric hydrogel.

[0025] The chondrocyte membrane (CM) extraction steps proposed in this invention are as follows: isolate mouse primary chondrocytes, obtain chondrocyte membrane vesicles using a commercial membrane protein extraction kit (such as the Minute Plasma Membrane Protein Isolation Kit), extract CM protein according to the kit instructions, and finally the concentration of the CM protein solution is 15-25 mg / mL.

[0026] Preferably, the piezoelectric core material is barium titanate nanoparticles (BTO-NPs). The BTO nanoparticle suspension is mixed with the CM protein solution, and the mixture is repeatedly extruded (e.g., 20 times) through a polycarbonate membrane with a specific pore size (e.g., 400 nm) using a liposome extruder, so that the CM is coated on the BTO-NPs core. Excess membrane components are removed by centrifugation to obtain CM@BTO.

[0027] Further optimization involves a BTO nanoparticle suspension concentration of 4-6 mg / mL and a volume ratio of BTO nanoparticle suspension to CM protein solution of 10:1.

[0028] Preferably, the specific steps for modifying the cartilage-targeting peptide onto the surface of the biocompatible piezoelectric nanoparticles coated with the cartilage cell membrane in step S1 are as follows: Cholesterol-polyethylene glycol-maleimide CLS-PEG-MAL and thiolized CAP peptide are mixed in PBS at a molar ratio of 2:1-1:2, and the mixture is reacted with shaking at room temperature for 8-12 hours. After dialysis and lyophilization, powdered CLS-PEG-CAP is obtained. Then, CM@BTO and CLS-PEG-CAP are mixed in PBS buffer at a mass ratio of membrane protein to polymer of 10-20:1 and incubated overnight at 4°C. Surface modification is completed through hydrophobic interactions to obtain piezoelectric nanoparticles Cap-CM@BTO.

[0029] Further optimization involved a 1:1 molar ratio of cholesterol-polyethylene glycol-maleimide CLS-PEG-MAL to thiolated CAP peptide, followed by a 10-hour oscillation reaction at room temperature, and a CM@BTO and CLS-PEG-CAP mixture at a 15:1 mass ratio of membrane protein to polymer.

[0030] The specific preparation steps of chondroitin sulfate OCS oxidized by periodate are as follows: 5 g of chondroitin sulfate (CS) is dissolved in 100 mL of deionized water to obtain a chondroitin sulfate solution; 1.288 g of sodium periodate is dissolved in deionized water and added dropwise to the chondroitin sulfate solution, and stirred in the dark for 12 hours; the resulting solution is dialyzed in deionized water for 72 hours, and then freeze-dried to obtain chondroitin sulfate OCS oxidized by periodate.

[0031] Preferably, step S2 specifically involves: dissolving chondroitin sulfate OCS oxidized with periodate in water to obtain an OCS aqueous solution with a concentration of 3-6 mg / mL; adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EDC and N-hydroxysuccinimide NHS to the OCS aqueous solution, with a mass ratio of OCS:85:50; adjusting the pH to 4-5; and stirring at room temperature in the dark for 0.5-1.5 hours; adding a dimethyl sulfoxide solution of 3-aminophenylboronic acid APBA with a concentration of 10-15 mg / mL, with a mass ratio of APBA to NHS of 1.0-1.5:1; adjusting the pH to 4-5 again; and continuing the reaction overnight at room temperature in the dark; and then dialyzing and lyophilizing the resulting reaction solution to obtain the oxidized chondroitin sulfate-3-aminophenylboronic acid APBA-OCS polymer.

[0032] Further optimization involved dissolving chondroitin sulfate OCS oxidized with periodate in water to obtain an OCS aqueous solution with a concentration of 4 mg / mL. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EDC and N-hydroxysuccinimide NHS were added to 50 mL of the OCS aqueous solution, with a mass ratio of OCS:85:50. After adjusting the pH to 4-5, the mixture was stirred at room temperature in the dark for 1 hour. Then, a 12 mg / mL solution of 3-aminophenylboronic acid APBA in dimethyl sulfoxide was added, with a mass ratio of APBA to NHS of 1.2:1. The pH was adjusted again to 4-5, and the reaction continued overnight at room temperature in the dark. The resulting reaction solution was dialyzed and lyophilized to obtain an orange powder of oxidized chondroitin sulfate-3-aminophenylboronic acid APBA-OCS polymer.

[0033] Preferably, step S3 specifically involves: dissolving collagen in dilute acetic acid with a concentration of 0.01-0.03 M to prepare an acidic collagen solution with a concentration of 4-6 mg / mL; mixing the acidic collagen solution with PBS and adjusting the pH to neutral to form a neutral collagen Col solution; mixing the Col solution with APBA-OCS solution to obtain a mixed solution; adding the piezoelectric nanoparticles obtained in step S1 to the mixed solution; and stirring thoroughly to obtain the injectable piezoelectric hydrogel.

[0034] Further optimization, step S3 specifically involves: dissolving tilapia skin collagen in acetic acid and stirring overnight at 4°C; taking 8 mL of this solution and mixing it with 1 mL of 10×PBS, adjusting the pH to 7.4 with 1 M NaOH to obtain a neutral collagen solution (Col, ~4 mg / mL); dissolving APBA-OCS in deionized water to obtain an APBA-OCS solution with a concentration of 4-6 mg / mL; mixing the Col solution and APBA-OCS solution at a volume ratio of 1:1, and then adding a Cap-CM@BTO nanoparticle suspension with a concentration of 4-6 mg / mL, wherein the concentration of Cap-CM@BTO in the solution is 0.05%-2.00% (w / v); after thorough stirring, transferring the mixture to a 24-well plate and incubating at 37°C for 2-4 minutes to obtain an injectable piezoelectric hydrogel, Cap-M@BTO / Gel.

[0035] Further optimization is achieved by using a concentration of Cap-CM@BTO in the solution of 0.1%-1.0% (w / v).

[0036] A third objective of this invention is to provide the application of the aforementioned injectable piezoelectric hydrogel in the preparation of pharmaceutical formulations for the treatment of osteoarthritis. The piezoelectric hydrogel proposed in this invention possesses shear-thinning properties, allowing for easy injection through fine needles and solidification within the joint cavity due to its self-healing properties, achieving long-term retention.

[0037] A fourth objective of this invention is to provide a pharmaceutical formulation for treating osteoarthritis, using the injectable piezoelectric hydrogel as the active ingredient.

[0038] A fifth object of the present invention is to provide a method of using the aforementioned pharmaceutical preparation for treating osteoarthritis, wherein the pharmaceutical preparation is administered to the joint.

[0039] A sixth object of the present invention is to provide a method for enhancing mitochondrial autophagy in chondrocytes, comprising contacting the chondrocytes with the injectable piezoelectric hydrogel.

[0040] Preferably, an effective amount of injectable piezoelectric hydrogel is injected into the joint, and therapeutic ultrasound is applied to the joint to activate the piezoelectric nanoparticles within the hydrogel, thereby generating local piezoelectric signals to treat osteoarthritis.

[0041] Further optimization yields the following ultrasonic operating parameters: 0.8-1.2 MHz, 2-3 W / cm². 2 Each session lasts 4-6 minutes, three times a day.

[0042] Further optimization yielded the following ultrasonic operating parameters: 1 MHz, 2.5 W / cm². 2 5 minutes each time, three times a day.

[0043] Localized piezoelectric signals generated by ultrasound stimulation induce a therapeutic cascade: they inhibit the mTOR signaling pathway in chondrocytes. This inhibition leads to epigenetic reprogramming, particularly increased histone H3K27 acetylation (H3K27ac) in the GATD3A gene promoter region, relieving transcriptional repression and upregulating GATD3A expression. Upregulation of GATD3A stabilizes mitochondrial transcription factor A (TFAM) and enhances PINK1 / Parkin-mediated mitophagy. This cascade effectively clears damaged mitochondria, reduces oxidative stress and cellular senescence, promotes extracellular matrix (ECM) synthesis, and ultimately improves osteoarthritis.

[0044] The working principle of the injectable piezoelectric hydrogel for treating osteoarthritis proposed in this invention involves a precise cascade of physical energy, chemical signals, and biological responses.

[0045] Its application method includes the following steps:

[0046] a: Inject an effective dose of the Cap-M@BTO / Gel hydrogel composition into the osteoarthritis joint of the patient via intra-articular injection;

[0047] b: Apply therapeutic ultrasound to the injected joint. Ultrasound acts as a non-invasive, deep-penetrating external mechanical stimulus on the hydrogel within the joint.

[0048] The therapeutic cascade mechanism (molecular working principle) involves ultrasound activation of piezoelectric nanoparticles in the hydrogel, triggering a series of molecular events:

[0049] 1. Generation of piezoelectric signals: The mechanical waves of ultrasound cause BTO nanoparticles to deform, generating local piezoelectric signals (micro-electric fields).

[0050] 2. Inhibition of the mTOR pathway: The generated piezoelectric signal effectively inhibits the phosphorylation of key proteins in the mTOR signaling pathway in chondrocytes (i.e., a decrease in p-mTOR levels).

[0051] 3. Epigenetic reprogramming: Repression of mTOR leads to a significant increase in the enrichment of histone H3 lysine acetylation at position 27 (H3K27ac) in the GATD3A gene promoter region. This epigenetic modification alters chromatin conformation, activating the transcriptional repression state of GATD3A.

[0052] 4. Upregulation of GATD3A expression: Epigenetic activation leads to a significant upregulation of GATD3A expression at both the mRNA and protein levels.

[0053] 5. TFAM stabilization and enhanced mitophagy: Upregulated GATD3A protein physically interacts with mitochondrial transcription factor A (TFAM) to form the GATD3A / TFAM complex, thereby stabilizing TFAM.

[0054] The GATD3A / TFAM complex synergistically enhances the PINK1 / Parkin-mediated mitophagy pathway. This is manifested in an increased LC3-II / LC3-I ratio, increased expression of PINK1 and Parkin proteins, and increased colocalization of mitochondria with autophagy markers.

[0055] 6. Final treatment outcome:

[0056] Clearing damaged mitochondria: Enhanced mitophagy effectively clears dysfunctional mitochondria.

[0057] Reduced oxidative stress: The main source of ROS was eliminated, and intracellular ROS levels were significantly reduced.

[0058] Reversing cellular senescence: The relief of oxidative stress and the downregulation of senescence-related genes (p16, p21, p53) jointly reversed the senescent phenotype of chondrocytes.

[0059] Promotes ECM synthesis: chondrocyte function is restored, anabolism is enhanced (e.g., upregulation of COL2A1 and SOX9 expression), and catabolism is suppressed (e.g., downregulation of MMP13 and ADAMTS5 expression), thereby repairing and regenerating the extracellular matrix of chondrocytes.

[0060] This invention also protects a kit for treating osteoarthritis, comprising:

[0061] (1) A syringe pre-loaded with injectable piezoelectric hydrogel;

[0062] (2) Explanation of intra-articular injection and ultrasound application.

[0063] Compared with the prior art, the present invention has the following advantages:

[0064] 1. The biomimetic piezoelectric hydrogel composition proposed in this invention targets cartilage and enhances mitochondrial autophagy by inhibiting mTOR and GATD3A-mediated epigenetic reprogramming, thereby improving osteoarthritis symptoms.

[0065] 2. The biomimetic piezoelectric hydrogel proposed in this invention possesses excellent targeting and retention capabilities. Benefiting from the dual biomimetic design of "cell membrane camouflage" and "CAP peptide targeting," the nanoparticles can actively penetrate and firmly adhere to the cartilage matrix. Experiments show that its cartilage penetration efficiency is 3.2 times that of non-targeted particles, and its in vivo retention time exceeds 5 days. Figure 3 This solved a core problem in drug delivery.

[0066] 3. The biomimetic piezoelectric hydrogel proposed in this invention achieves a minimally invasive and controllable treatment mode. Because the hydrogel is injectable, minimally invasive drug delivery is achieved; the piezoelectric effect is activated on demand by external ultrasound, eliminating the need for implanted electrodes, making it safe and controllable. Figure 2 ).

[0067] 4. The biomimetic piezoelectric hydrogel proposed in this invention provides synergistic antioxidant and cell protection. The material itself possesses strong antioxidant capabilities (ABTS free radical scavenging rate reaches 83.11%). Figure 2 Meanwhile, piezoelectric-induced mitophagy can reduce ROS production at its source, synergistically protecting chondrocytes. Figure 6 ).

[0068] 5. This invention reveals for the first time a novel therapeutic pathway of "piezoelectric stimulation → mTOR inhibition → epigenetic upregulation of GATD3A → enhanced mitophagy" ( Figure 9 This reverses the OA process at the fundamental level of epigenetics and organelle quality control, rather than alleviating symptoms.

[0069] 6. The biomimetic piezoelectric hydrogel proposed in this invention has been proven safe through comprehensive biocompatibility testing (Live / Dead, CCK-8, hemolysis test, etc.). Figure 4 In cell, mouse model (DMM model), and human OA cartilage explant levels, this hydrogel was confirmed to effectively remove senescent cells, promote cartilage ECM regeneration, and inhibit synovitis and osteophyte formation. Figure 5 , 7 8). Attached Figure Description

[0070] Figure 1 This is a schematic diagram illustrating the design strategy of the biomimetic piezoelectric hydrogel and the cartilage regeneration mechanism proposed in this invention.

[0071] Figure 2This document describes the preparation and characterization of biomimetic piezoelectric hydrogels obtained in Examples 1-3 of this invention. Specifically, (A) transmission electron microscopy images of BTO-NPs and CM@BTO obtained in Example 1; (B) average hydrodynamic diameters and surface zeta potentials of BTO-NPs, CM vesicles, CM@BTO, and Cap-CM@BTO in Example 1, measured by dynamic light scattering (DLS) (n = 3 per group); (C) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of BTO-NPs, CM vesicles isolated from fresh mouse chondrocytes, and CM@BTO in Example 1, followed by Coomassie brilliant blue staining; and (D) representative confocal microscopy images of mouse chondrocytes treated with CM@BTO in Example 1, with BTO nuclei labeled with Cy5.5 (red) and CM shells labeled with PKH. 67 (green) marking; (E) Fourier transform infrared spectra of CS, OCS and CS-PBA hydrogels in Example 1; (F) Rheological properties of hydrogels obtained in Examples 1-3, where G′ represents storage modulus and G′′ represents loss modulus; (G) Output voltage of different hydrogels obtained in Examples 1-3; (H) SEM images of the internal structures of different hydrogel scaffolds obtained in Examples 1-3, scale bar, 100 μm; (I) Water contact angle of different hydrogel scaffolds obtained in Examples 1-3; (J) Rapid formation of piezoelectric hydrogels at physiological temperature (3 min); (K) UV-Vis detection of the ability of hydrogel scaffolds obtained in Examples 1-3 to scavenge ATBS free radicals, and analysis of solution color changes and quantitative analysis. Statistical analysis was performed using one-way ANOVA combined with Tukey's post hoc test. *P < 0.05, *P < 0.001, ns, no significant difference.

[0072] Figure 3To demonstrate how CM@BTO obtained in Example 1 prolongs joint retention time through cartilage adhesion and penetration; wherein, (A) representative confocal microscopy images of mouse chondrocytes after 24 hours of incubation with BTO-NPs, CM@BTO, and Cap-CM@BTO (200 μg / mL); (B) flow cytometry analysis and corresponding quantitative mean fluorescence intensity after incubation with BTO-NPs, CM@BTO, and Cap-CM@BTO; (C) representative scanning electron microscopy images of the distribution and adhesion of BTO-NPs, CM@BTO, and Cap-CM@BTO in mouse cartilage tissue; (D) representative scanning electron microscopy images of the distribution and adhesion of BTO-NPs, CM@BTO, and Cap-CM@BTO in human cartilage tissue; (E) representative confocal microscopy images showing cross-sections of mouse cartilage sections after 3 days of incubation, bottom: magnified image of the area within the white box; (red, Cy5.5); cell nuclei (blue, DAPI), scale bar, 50 μm; (F) Schematic diagram of IVIS assessment; (G) Representative IVIS images of C57 mice 5 days after a single intra-articular injection of free BTO-NPs, CM@BTO, and Cap-CM@BTO; (H) Quantitative analysis of normalized fluorescence radiation efficiency over a 5-day period in mouse knee joints (n = 3). Data are expressed as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA combined with Tukey post-hoc test. *P < 0.05, *P < 0.001.

[0073] Figure 4 Biocompatibility evaluation of the hydrogels prepared in Examples 1-3; (A) CLSM images showing live / dead staining of L929 cells on different hydrogels at days 1, 3, and 5, scale bar, 200 μm; (B) CCK-8 assay to study the proliferation of L929 fibroblasts cultured on different hydrogels; (C) Cytoskeleton staining of L929 cells on various hydrogels shown in CLSM images, scale bar, 200 μm; (D) Flow cytometry detection of apoptosis of L929 cells cultured on different hydrogels; (E) EdU staining of L929 cells on various hydrogels shown in CLSM images, scale bar, 200 μm; (F) Quantitative analysis of hemolysis rate of samples. Data are expressed as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA, with Tukey post-hoc test. *P < 0.05, *P < 0.001, ns, no statistically significant difference.

[0074] Figure 5Example 1 describes the evaluation of the piezoelectric hydrogel's ability to alleviate IL-1β-induced extracellular matrix degradation and aging in chondrocytes. The results include: (A) a representative image of β-galactosidase staining in chondrocytes on the hydrogel, scale bar, 200 μm; (B) a representative immunofluorescence image showing the chondrocyte aging marker P16, scale bar, 50 μm; (C) the effect of the piezoelectric hydrogel on the expression of P16, P21, and P53 in mouse chondrocytes under IL-1β stimulation or without stimulation, detected by qRT-PCR; (D, E) the effect of the piezoelectric hydrogel on the expression of MMP 13 (red) and COL 2A 1 (green) in mouse chondrocytes under IL-1β stimulation or without stimulation, scale bar, 50 μm; (F) the detection of the effects of the piezoelectric hydrogel on COL 2A 1 and SOX 9 in mouse chondrocytes, and the detection of Adamt 5 and MMP 1 in mouse chondrocytes before and after IL-1β stimulation using qRT-PCR. The data are expressed as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA combined with Tukey's posthoc test. *P < 0.001 ns, indicating no significant difference.

[0075] Figure 6 To demonstrate how the piezoelectric hydrogel obtained in Example 1 triggers the piezoelectric effect, thereby enhancing mitophagy and improving chondrocyte function; (A) Representative image of TMRE (red) immunofluorescence staining in chondrocytes, scale bar, 50 μm; (B) Representative image of LC3 (red) immunofluorescence staining in chondrocytes, scale bar, 50 μm; (C) Representative image of reactive oxygen species (ROS) immunofluorescence staining in different groups, scale bar, 200 μm; (D) Flow cytometry analysis of ROS levels in different groups; (E) Representative transmission electron microscopy images of mitochondria in chondrocytes from different groups, scale bar, 200 nm; (F) Representative images of live / dead chondrocyte immunofluorescence staining in different groups, scale bar, 200 μm; (G) Schematic diagram (left) of the LC3 dual-labeling system used to assess autophagy flux, cultured with mCherry-GFP-LC under IL-1β conditions. 3. Transfected chondrocytes, treated with or without piezoelectric hydrogel, were then observed under a confocal microscope. Red dots represent autophagosomes, and yellow dots represent autophagic vesicles (right). Scale bar: 100 μm. Data are expressed as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA combined with Tukey's post-hoc test. *P < 0.05, *P < 0.001.ns, no significant difference was found.

[0076] Figure 7The piezoelectric hydrogel obtained in Example 1 was shown to alleviate the progression of osteoarthritis in mice. The study included: (A) the design of the piezoelectric hydrogel treatment for osteoarthritis in mice; (B) representative 3D microCT images (n=6) showing the joint surface morphology, with 2D microCT images at the bottom, showing the microstructure of osteophytes (red arrows) and subchondral bone, scale bar 1 mm; (C) SO&FG staining of the knee joints in each group after 4 and 8 weeks of treatment, scale bar 200 μm; (D) HE staining of the synovium in each group after 4 and 8 weeks of treatment, scale bar 200 μm; (E) COL2a immunofluorescence staining of the knee cartilage in mice after 4 and 8 weeks of treatment, scale bar 50 μm; and (F) MMP 13 immunohistochemical staining of the knee cartilage in mice after 4 and 8 weeks of treatment, scale bar 200 μm. Statistical analysis was performed using one-way ANOVA combined with Tukey's post-hoc test. *P < 0.05, *P < 0.001. ns, no significant difference.

[0077] Figure 8 This study illustrates the therapeutic effect of the piezoelectric hydrogel obtained in Example 1 on human osteoarthritis cartilage samples. (A) Schematic diagram of the experimental design: human osteoarthritis cartilage samples were cultured in vitro for one week, with the hydrogel replaced weekly. Cartilage samples were collected after three weeks. (B) SO&FG staining of different groups of cartilage samples, scale bar, 200 μm. (C, D, E) represent the immunohistochemical staining results of P16, COL2A1, and MMP 13, respectively, scale bar, 200 μm. Statistical analysis was performed using one-way ANOVA combined with Tukey's post-hoc test. *P < 0.05, *P < 0.001. No significant difference was observed within ns.

[0078] Figure 9The molecular mechanism by which the piezoelectric hydrogel obtained in Example 1 promotes cartilage repair in mice is as follows: (A) Quantitative analysis of DEG in chondrocytes of the IL-1β group and the piezoelectric hydrogel group; (B) Analysis of the KEGG pathway in chondrocytes of the IL-1β group and the piezoelectric hydrogel group; (C) Gene set enrichment analysis (GSEA) of the mTOR signaling pathway in chondrocytes of the IL-1β group and the piezoelectric hydrogel group; (D) Immunofluorescence image of P-mTOR (green), scale bar, 100 μm; (E) qRT-PCR analysis of P-mTOR levels in chondrocytes; (F) Volcano plot of RNASeq data; (G) qRT-PCR analysis of GATD 3A expression in IL-1β-stimulated mouse chondrocyte samples (24 hours); (H) Immunohistochemical staining of GATD 3A on the knee joints of mice in the control group and 4 and 8 weeks after DMM, scale bar, 100 μm; (I) Immunohistochemical staining of GATD 3A in human osteoarthritis chondrocytes, scale bar, 100 μm. (J) Immunofluorescence staining of GATD 3A expression in IL-1β-stimulated mouse chondrocyte samples, scale bar, 100 μm; (K) Correlation analysis between GATD 3A and LC 3 using the GEPIA database; (L) Immunohistochemical staining of GATD 3A and GATD 3A levels in chondrocytes using ultrasound combined with piezoelectric hydrogel and MHY 1485 (mTOR activator) (with or without IL-1β stimulation), scale bar, 100 μm; qRT-PCR analysis of GATD 3A levels in chondrocytes using ultrasound combined with piezoelectric hydrogel and MHY 1485 (mTOR activator) (with or without IL-1β stimulation); qPCR analysis showing the enrichment level of H3K27 ac in the GATD 3A promoter region of chondrocytes under different treatment conditions; (M) Immunohistochemical staining of GATD 3A in chondrocytes using ultrasound combined with piezoelectric hydrogel and MHY1485 (mTOR activator) (with or without IL-1β stimulation). (N) ChIP-qPCR analysis of H3K27ac enrichment levels of the GATD3A promoter region in chondrocytes under different treatment conditions; (O) Co-IP analysis of GATD3A (red) and TFAM (green) in chondrocyte lysates using anti-GATD3A antibody or normal IgG (negative control). Immunoblot analysis using TFAM and GAPDH antibodies. (P) Representative immunofluorescence images showing subcellular localization of GATD3A (red) and TFAM (green) in chondrocytes. Scale bar, 50 μm. (Q) LC 3-I / II, PINK 1 / Parkin, TFAM and GATD3A protein levels in chondrocytes under different treatment conditions. Statistical analysis was performed using one-way ANOVA combined with Tukey's post-hoc test.*P < 0.05, *P < 0.001. ns, no significant difference.

[0079] Figure 10 The molecular mechanism by which the piezoelectric hydrogel obtained in Example 1 promotes cartilage repair in mice is as follows: (A) Quantitative analysis of DEG in chondrocytes of the IL-1β group and the piezoelectric hydrogel group; (B) Analysis of the KEGG pathway in chondrocytes of the IL-1β group and the piezoelectric hydrogel group; (C) Gene set enrichment analysis (GSEA) of the mTOR signaling pathway in chondrocytes of the IL-1β group and the piezoelectric hydrogel group. Detailed Implementation

[0080] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0081] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental materials and reagents used herein are commercially available products conventional in this field. Thioylated CAP peptides were purchased from Nanjing Yuanpeptide Biotechnology Co., Ltd.

[0082] In the following examples or comparative examples, the core layer material is barium titanate nanoparticles (BTO-NPs).

[0083] like Figure 1 As shown, the preparation method of injectable piezoelectric hydrogel includes the following steps:

[0084] S1. Preparation of piezoelectric nanoparticles (Cap-CM@BTO)

[0085] S11. Chondrocyte membrane (CM) extraction: Mouse primary chondrocytes were isolated, and chondrocyte membrane vesicles were obtained using a commercial membrane protein extraction kit (such as the Minute Plasma Membrane Protein Isolation Kit). CM protein was extracted according to the kit instructions. The final concentration of the CM protein solution was 15-25 mg / mL, preferably 20 mg / mL.

[0086] S12. Membrane Coating (CM@BTO Preparation): A BTO nanoparticle suspension is mixed with a CM protein solution. Using a liposome extruder, the mixture is repeatedly extruded (e.g., 20 times) through a polycarbonate membrane with a specific pore size (e.g., 400 nm) to coat the BTO core with CM. Excess membrane components are removed by centrifugation to obtain CM@BTO.

[0087] S13, Targeted peptide functionalization (preparation with Cap-CM@BTO):

[0088] S131. Synthesis of targeted polymer: CLS-PEG-MAL and CAP-SH (thiolized CAP peptide) were mixed in PBS at a molar ratio of 2:1-1:2 and reacted with shaking at room temperature for 8-12 hours. After dialysis and lyophilization, powdered CLS-PEG-CAP was obtained.

[0089] S132. Surface modification: CM@BTO and CLS-PEG-CAP were mixed in PBS at a mass ratio of membrane protein to polymer of 10-20:1 and incubated overnight at 4°C. Surface modification was completed through hydrophobic interactions to obtain Cap-CM@BTO.

[0090] S2. Preparation of hydrogel matrix

[0091] S21, Synthesis of APBA-OCS: OCS was dissolved in water, EDC and NHS were added to activate the carboxyl groups, the pH was adjusted, and APBA dissolved in DMSO was added. The reaction was carried out overnight under weakly acidic conditions. The reaction solution was dialyzed and lyophilized to obtain APBA-OCS polymer.

[0092] S22. Collagen Extraction: Following the method described in an, P., et al., Ultrasound-activated piezoelectric hydrogel scaffold for synergistic immunomodulation and angiogenesis in accelerated wound healing. Acta Biomaterialia, 2025. 204: p.216-233, acid-soluble collagen was extracted and purified from tilapia skin through steps including alkali treatment, defatting, acid dissolution, salting out, dialysis, and freeze-drying.

[0093] S23, Hydrogel Composite:

[0094] S231. Dissolve collagen in dilute acetic acid to prepare an acidic collagen solution;

[0095] S232. Mix the acidic collagen solution with PBS buffer solution, and adjust the pH to neutral with NaOH solution to form a neutral collagen solution (Col).

[0096] S233. Mix the Col solution with the APBA-OCS solution;

[0097] S234. Add a Cap-CM@BTO nanoparticle suspension with a concentration of 4-6 mg / mL to the above mixture, stir thoroughly to obtain the final solution, and the concentration of Cap-CM@BTO in the final solution is 0.1%-1% (w / v).

[0098] S235. The final solution is injected into the mold, and a stable hydrogel, Cap-M@BTO / Gel, can be formed in about 2-4 minutes at physiological temperature (37°C).

[0099] Furthermore, in step S12, the concentration of the BTO nanoparticle suspension is 4-6 mg / mL, and the volume ratio of the BTO nanoparticle suspension to the CM protein solution is 10:1.

[0100] Further, in step S131, the specific steps for synthesizing the targeted polymer are as follows: CLS-PEG-MAL and CAP-SH (thiolized CAP peptide) are mixed in PBS at a molar ratio of 1:1, and the mixture is shaken at room temperature for 10 hours. After dialysis and lyophilization, powdered CLS-PEG-CAP is obtained.

[0101] Further, in step S132, CM@BTO and CLS-PEG-CAP are mixed at a mass ratio of membrane protein to polymer of 15:1.

[0102] Further, in step 231, tilapia skin collagen is dissolved in acetic acid and stirred overnight at 4°C to obtain an acidic collagen solution. The mass-to-volume ratio of tilapia skin collagen to acetic acid is 4-6 mg / mL, and the molar concentration of acetic acid is 0.01-0.03 M.

[0103] Furthermore, the mass-to-volume ratio of tilapia skin collagen to acetic acid is 5 mg / mL, and the molar concentration of acetic acid is 0.02 M.

[0104] Further, in step S232, the volume ratio of acidic collagen solution to 10×PBS buffer solution is 7-9:1.

[0105] Further, in step S233, chondroitin sulfate OCS oxidized with periodate is dissolved in water to obtain an OCS aqueous solution with a concentration of 3-6 mg / mL. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide EDC and N-hydroxysuccinimide NHS are added to the OCS aqueous solution, with a mass ratio of OCS, EDC, and NHS of 150-300:85:50. After adjusting the pH to 4-5, the mixture is stirred at room temperature in the dark for 0.5-1.5 hours. A dimethyl sulfoxide solution of 3-aminophenylboronic acid APBA with a concentration of 10-15 mg / mL is added, with a mass ratio of APBA to NHS of 1.0-1.5:1. The pH is adjusted to 4-5 again, and the reaction continues overnight at room temperature in the dark. The resulting reaction solution is dialyzed and lyophilized to obtain the oxidized chondroitin sulfate-3-aminophenylboronic acid APBA-OCS polymer.

[0106] Further optimization involved dissolving chondroitin sulfate OCS oxidized with periodate in water to obtain an OCS aqueous solution with a concentration of 4 mg / mL. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EDC and N-hydroxysuccinimide NHS were added to 50 mL of the OCS aqueous solution, with a mass ratio of OCS:85:50. After adjusting the pH to 4-5, the mixture was stirred at room temperature in the dark for 1 hour. Then, a 12 mg / mL solution of 3-aminophenylboronic acid APBA in dimethyl sulfoxide was added, with a mass ratio of APBA to NHS of 1.2:1. The pH was adjusted again to 4-5, and the reaction continued overnight at room temperature in the dark. The resulting reaction solution was dialyzed and lyophilized to obtain an orange powder of oxidized chondroitin sulfate-3-aminophenylboronic acid APBA-OCS polymer.

[0107] Furthermore, the concentration of the APBA-OCS solution is 4-6 mg / mL.

[0108] Furthermore, the concentration of the APBA-OCS solution is 5 mg / mL.

[0109] Example 1

[0110] like Figure 1 As shown, the preparation method of injectable piezoelectric hydrogel includes the following steps:

[0111] Preparation of S1 and Cap-CM@BTO nanoparticles

[0112] S11, CM extraction: Take 10^7 mouse primary chondrocytes and extract CM protein according to the kit instructions. The final concentration was adjusted to 20 mg / mL.

[0113] S12. Membrane Coating: Mix 200 μL of 5 mg / mL BTO suspension with 20 μL of 20 mg / mL CM protein solution. Using a 400 nm pore size polycarbonate membrane, extrude the mixture 20 times in a liposome extruder. Centrifuge (11,000 g, 15 min), discard the supernatant, and resuspend the precipitate in PBS to obtain CM@BTO.

[0114] S13, Targeted peptide functionalization (Preparation of Cap-CM@BTO): Weigh out CLS-PEG-MAL (M W34 mg (10 μmol) of CM@BTO (MW: 3400 Da) and 6.93 mg (5 μmol) of CAP-SH (MW: 1386.6 Da) were dissolved together in 5 mL of PBS (pH 7.4). The molar ratio of reactants was 1:0.5; a small amount of TCEP (5 mM) was added to prevent thiol oxidation. The reaction was carried out at room temperature with shaking for 10 hours to synthesize the CLS-PEG-CAP targeting polymer. The reaction solution was transferred to a dialysis bag (MWCO: 3000 Da) and dialyzed against PBS for 24 hours to remove unreacted small molecules. After lyophilization, powdered CLS-PEG-CAP was obtained. This ratio is suitable for ensuring sufficient reaction of CLS-PEG-MAL. CM@BTO and CLS-PEG-CAP were incubated at a membrane protein to polymer mass ratio of 15:1. Surface modification was completed through hydrophobic interactions to obtain Cap-CM@BTO.

[0115] Preparation of S2, 1% Cap-M@BTO / Gel hydrogel

[0116] S21. Preparation of collagen solution: Dissolve 50 mg of tilapia skin collagen in 10 mL of 0.02 M acetic acid and stir overnight at 4°C. Take 8 mL of this solution and mix it with 1 mL of 10×PBS. Adjust the pH to 7.4 with 1 N NaOH to obtain a neutral collagen solution (Col, ~4 mg / mL).

[0117] S22. Preparation of APBA-OCS solution: Add EDC (0.085 g) and NHS (0.05 g) to an OCS aqueous solution (concentration 4 mg / mL, 50 mL). Adjust the pH of the mixture to 4-5 and stir at room temperature in the dark for 1 hour. Then, add APBA solution (concentration 0.012 g / mL, volume 5 mL in DMSO) to the above mixture. Adjust the pH to 4-5 again and continue the reaction overnight at room temperature in the dark. Finally, dialyze the reaction solution (molecular weight cutoff 3500) and freeze-dry to obtain an orange powder of APBA-OCS. Dissolve 50 mg of APBA-OCS in 10 mL of deionized water to obtain an APBA-OCS solution.

[0118] S23. Composite: Mix Col solution and APBA-OCS solution in a volume ratio of 1:1, then add Cap-CM@BTO nanoparticle suspension, and stir thoroughly to obtain a mixed solution. The concentration of nanoparticles in the mixed solution is 1% (w / v).

[0119] S24, Gelation: Transfer the mixture to a 24-well plate and incubate at 37°C. After about 3 minutes, the final injectable piezoelectric hydrogel (1% Cap-M@BTO / Gel) will be formed.

[0120] Example 2

[0121] Similar to Example 1, except that in step S23, the Col solution and APBA-OCS solution are mixed, and then the Cap-CM@BTO nanoparticle suspension is added to make the mass fraction of nanoparticles in the final piezoelectric hydrogel 0.05%. After thorough stirring, a mixture is obtained. In step S24, the final injectable piezoelectric hydrogel (0.1% Cap-M@BTO / Gel) is formed.

[0122] Example 3

[0123] Similar to Example 1, except that in step S23, the Col solution and APBA-OCS solution are mixed, and then the Cap-CM@BTO nanoparticle suspension is added so that the mass fraction of nanoparticles in the final piezoelectric hydrogel is 0.1%. After thorough stirring, a mixture is obtained. In step S24, the final injectable piezoelectric hydrogel (0.5% Cap-M@BTO / Gel) is formed.

[0124] Example 4

[0125] Similar to Example 1, except that in step S23, the Col solution and APBA-OCS solution are mixed, and then the Cap-CM@BTO nanoparticle suspension is added so that the mass fraction of nanoparticles in the final piezoelectric hydrogel is 0.1%. After thorough stirring, a mixture is obtained. In step S24, the final injectable piezoelectric hydrogel (2% Cap-M@BTO / Gel) is formed.

[0126] Experimental Example 1

[0127] The hydrogels obtained in Examples 1-3 were characterized and tested as follows:

[0128] 1. Transmission Electron Microscopy (TEM): The morphological characterization of CM@BTO was performed using transmission electron microscopy (TEM, Themis Z(3.2)). For TEM samples, CM@BTO nanopowder was dissolved in anhydrous ethanol (5 mg / mL) for 10 minutes, and then added dropwise to a copper grid (accelerating voltage: 300 kV).

[0129] 2. Scanning Electron Microscopy (SEM): Emission scanning electron microscopy (FE-SEM; Zeiss, Sigma, Germany) was used to characterize the cross-sectional morphology of the piezoelectric hydrogel. Energy dispersive spectrometer (UltimMax40, Oxford, UK) was used to scan the distribution of C, O, Ba, and Ti elements in the scaffold.

[0130] 3. Piezoelectric properties of the hydrogel scaffold: The output voltage of the hydrogel was measured using a high-precision multimeter (DMM7510, Keithley, USA). The sample was cylindrical (3 mm thick, 10 mm in diameter), with the hydrogel layered at the top and bottom, and copper foil as the conductive material. The piezoelectric coefficient (d33) was determined using a piezoelectric testing instrument (ZJ-3AN, JKZC, Beijing, China).

[0131] 4. Biocompatibility Testing: The biocompatibility of the hydrogels was evaluated using the CCK-8 assay and the live / dead staining method. Three × 10⁻⁶ samples were used per sample. 3 L929 cells were seeded at a density of [number] cells onto different hydrogel surfaces in a 96-well plate. The cells were then sonicated (1 MHz, 2.5 W / cm²). 2 Cells on the hydrogel were stimulated by sonication for 5 minutes every 4 hours, three times a day. After culturing for 1, 3, and 5 days, 10 µL of CCK-8 solution was added to each well, followed by incubation for another hour. Absorbance was measured at a test wavelength of 450 nm using a plate reader, in triplicate. In the live / dead staining assay, 1 × 10⁻⁶ cells were used per sample. 4 L929 cells were seeded at specific cell densities onto different piezoelectric hydrogel surfaces in 24-well plates and cultured for 1, 3, and 5 days. Cells on the hydrogels were stimulated with sonication for 5 min every 4 h, three times daily. After removing the culture medium and washing with PBS, all cell groups were stained with live / dead dyes, with Calcein-AM staining live cells green and propidium iodide (PI) staining dead cells red. The samples were then observed using a high-content imaging system (Operetta CLS), with at least three fields selected for each sample.

[0132] 5. Cell proliferation assay: The proliferation of L929 cells on different hydrogels was assessed using the Edu Cell Proliferation Imaging Analysis Kit (KTA2030). Cells on the hydrogels were stimulated with ultrasound for 5 min every 4 h, three times a day. Cells were then photographed using a laser confocal high-content screening system. At least three fields of view were selected for each sample, and the average percentage of EdU-positive cells was calculated using ImageJ.

[0133] 6. Cell Targeting and Internalization: To observe the targeting and internalization abilities of different types of nanoparticles on chondrocytes, primary mouse chondrocytes were co-incubated with Cy5.5-labeled nanoparticles at 37°C. Confocal fluorescence microscopy analysis was performed. The treated cells were washed three times with PBS, stained with DAPI, and then imaged under a confocal microscope. In flow cytometry analysis, the same treated cells were centrifuged at 1000 rpm for 3 minutes, resuspended in fluorescence-activated cell sorting buffer (DMEM medium supplemented with 2% FBS), and then analyzed on a flow cytometer (BD Biosciences).

[0134] 7. Knee Joint Retention Test: To conduct long-term in vivo retention tests and biodistribution experiments, 5 mg / mL of Cy5.5-labeled nanoparticles were injected into the knee joints of mice. Over a 5-day period, the radiation efficiency within each joint was continuously monitored and quantified using Maestro IVIS (CRI, Warburn, USA) and Living Image software.

[0135] 8. RNA-mediated interference: RNA interference experiments are performed using the following method:

[0136] (1) Seeding cells: Seed cells in culture plates 8-16 hours in advance so that the cells are in the logarithmic growth phase and the cell density reaches about 50%.

[0137] (2) Preparation of siRNA / miRNA premix for transfected cells: Take 8.5 μL of buffer and place it in a sterile enzyme-free EP tube. Add 15 pmol siRNA / miRNA and mix well by pipetting to obtain the siRNA / miRNA premix.

[0138] To prepare the RNA / plus complex, add 1.5 μL of plus transfection reagent to each siRNA / miRNA premixed solution in the EP tubes mentioned above, and immediately mix by pipetting dozens of times to obtain the RNA / plus complex.

[0139] The prepared RNA / plus complex was added to the cells, and the 24-well plate was gently shaken to disperse it evenly.

[0140] (3) Effect detection: Changes in the target gene can be quantitatively detected 24-48 hours after siRNA transfection.

[0141] When chondrocyte fusion reached 50%, siRNA oligonucleotides were transfected into the cells using Lipofectamine RNAiMAX (Thermo Fisher Scientific, product number 13778075). The siRNA sequence was provided by GenePharma. A Stealth siRNA negative control (siNC) duplex with similar GC content was used as a control.

[0142] 9. Age-associated β-galactosidase (SA-βGal) staining: SA-βGal activity was determined using a staining kit (Solepro Biosciences). Chondrocytes were seeded in the wells of 6-well plates, fixed at room temperature for 10 minutes, washed, and then incubated overnight at 37°C with the staining solution.

[0143] 10. Human Samples: After obtaining approval from the Clinical Research Ethics Committee of the Third Affiliated Hospital of Southern Medical University (2025-ER-020), tibial plateau samples were collected from five male patients (aged 50 to 80 years) who underwent total knee arthroplasty (TKA). Histological analysis was performed on these samples after treatment with piezoelectric hydrogel.

[0144] 11. Transcriptome Analysis: RNA sequencing was performed to investigate gene expression patterns in chondrocytes cultured on different hydrogel scaffolds. Briefly, 5 × 10^6 chondrocytes per mL were co-cultured with different hydrogel scaffold samples in 6-well plates. Total RNA was then collected from each group of chondrocytes using TRIzol reagent, following the manufacturer's instructions. These RNA samples were stored at -80°C prior to sequencing. Total RNA extraction, purification, library construction, and sequencing were all performed by Beijing Qingke Biotechnology Co., Ltd.

[0145] result:

[0146] 1. Transmission electron microscopy (TEM) analysis

[0147] The morphology of CM@BTO nanoparticles was characterized using transmission electron microscopy (TEM, Themis Z). Figure 2As shown in Figure A, the average particle size is 50.14 ± 4.67 nm. After being coated with chondrocyte membranes, a clear membrane structure is visible around the CM@BTO particles, indicating successful membrane coating. Dynamic light scattering (DLS) further revealed that the hydrated particle size of CM@BTO is 250.60 ± 0.25 nm, which is larger than that of BTO-NPs (200.12 ± 3.28 nm), and the surface potential decreases from -12.21 mV to -24.40 mV, close to the cell membrane potential, confirming complete membrane encapsulation. Figure 2 B).

[0148] 2. Scanning Electron Microscopy (SEM) and Elemental Distribution

[0149] The microstructure of the hydrogel cross-section was observed using field emission scanning electron microscopy (FE-SEM). Figure 2 As shown in H, all hydrogels exhibit an irregular but interconnected porous network, and the pore size distribution facilitates the transport of nutrients and metabolites.

[0150] 3. Piezoelectric performance testing

[0151] The piezoelectric output of the hydrogel was measured using a high-precision multimeter and a piezoelectric meter. As the Cap-CM@BTO content increased from 0.1% to 1% (w / v), the output voltage of the hydrogel increased from 10.12 mV to 30.25 mV. Figure 2 G). This demonstrates that the synergistic polarization effect of nanoparticles can effectively improve the force-to-electricity conversion efficiency.

[0152] 4. Cell activity and proliferation

[0153] The biocompatibility of the hydrogels was evaluated using the CCK-8 assay and live / dead staining. L929 cells showed high viability (>95%) on all hydrogel surfaces, with live cells exhibiting green fluorescence and very few dead cells. Figure 4 A). CCK-8 results showed that the number of cells increased significantly over time, especially the 1% Cap-CM@BTO / Gel group, which had the highest proliferation rate. Figure 4 B). EdU staining further confirmed that ultrasound-activated piezoelectric hydrogels significantly increased the proportion of EdU-positive cells, indicating that piezoelectric stimulation promotes cell cycle progression (B). Figure 4 E).

[0154] 5. Cell targeting and internalization

[0155] Cy5.5-labeled nanoparticles were co-incubated with primary mouse chondrocytes. Confocal microscopy revealed that the Cap-CM@BTO group exhibited a strong red fluorescence signal, while the unmodified BTO group showed weak fluorescence. Figure 3A). Flow cytometry quantitative analysis showed that the mean fluorescence intensity of cells treated with Cap-CM@BTO was significantly higher than that of the BTO and CM@BTO groups ( Figure 3 (B) demonstrates that targeted peptide modification significantly enhances the chondrocyte endocytosis efficiency of nanoparticles.

[0156] 6. Joint retention test

[0157] Cy5.5-labeled nanoparticles were injected into the knee joints of mice, and the results were monitored continuously for 5 days using an in vivo imaging system (IVIS). The Cap-CM@BTO group showed the strongest fluorescence signal at all time points after injection, and maintained significant enrichment until day 5. Figure 3 (F–H) indicates that it has excellent joint retention ability, effectively overcoming the problem of rapid clearance of traditional formulations.

[0158] 7. RNA interference and gene expression regulation

[0159] GATD3A-specific siRNA was transfected into chondrocytes using Lipofectamine RNAiMAX. After GATD3A knockdown, the piezoelectric hydrogel-induced upregulation of LC3-II and the enhanced mitophagy were significantly inhibited, demonstrating that GATD3A plays a crucial role in the piezoelectric-autophagy signaling axis. Figure 9 Q).

[0160] 8. Staining of aging-associated β-galactosidase (SA-β-Gal)

[0161] IL-1β-induced SA-β-Gal activity in chondrocytes was significantly increased, while the blue staining area was significantly reduced after 3 days of piezoelectric hydrogel treatment. Figure 5 A) indicates that hydrogels can effectively alleviate the cellular senescence phenotype.

[0162] 9. Evaluation of clinical OA cartilage samples

[0163] Cartilage tissue was obtained from patients who underwent total knee arthroplasty. After 3 weeks of in vitro culture in piezoelectric hydrogels, hematoxylin-eosin (H&E) and safranin O-fast green staining showed that the cartilage surface in the hydrogel group was smoother and the proteoglycan staining was deeper. Figure 8 B). Immunohistochemistry further confirmed the upregulation of COL2A1 and SOX9 expression, while the expression of MMP13 and P16 decreased. Figure 8 (C–E), suggesting that the hydrogel has clinical translational potential.

[0164] 10. Transcriptome sequencing and pathway enrichment

[0165] RNA-Seq analysis of IL-1β-induced chondrocytes revealed 3664 upregulated genes and 3469 downregulated genes after piezoelectric hydrogel intervention. Figure 9 A). KEGG enrichment analysis showed significant changes in the mTOR and AMPK signaling pathways (A). Figure 9 (BC) suggests that piezoelectric stimulation triggers downstream epigenetic and autophagy remodeling by inhibiting the mTOR pathway.

[0166] Experiment 2: Treatment of Osteoarthritis in Mice

[0167] Modeling: Right knee medial meniscus instability (DMM) surgery was performed on 12-week-old male C57BL / 6 mice to induce OA.

[0168] Treatment: Starting from the 4th week after surgery, 50 μL of 1% Cap-M@BTO / Gel hydrogel prepared in Example 1 was injected into the joint cavity once a week.

[0169] Assessment: Samples were taken 8 weeks after treatment. Micro-CT scans showed reduced osteophyte formation and improved bone microstructure in the treatment group (see attached image). Figure 7 B); Histological staining (Safranin O) and OARSI score showed a significant reduction in cartilage degeneration (see appendix). Figure 7 C); Immunohistochemistry showed increased COL2A1 expression and decreased MMP13 expression (see appendix). Figure 7 E, F).

[0170] Example 5

[0171] Same as Example 1, except that:

[0172] In step S11, the concentration of the CM protein solution is 15 mg / mL.

[0173] In step S12, the concentration of the BTO nanoparticle suspension is 4 mg / mL.

[0174] In step S131: the molar ratio of CLS-PEG-MAL to CAP-SH (thiolized CAP peptide) is 2:1, and the reaction is carried out at room temperature with shaking for 8 hours.

[0175] In step S132, CM@BTO and CLS-PEG-CAP are mixed at a mass ratio of membrane protein to polymer of 10:1.

[0176] In step S22, chondroitin sulfate OCS oxidized with periodate was dissolved in water to obtain an OCS aqueous solution with a concentration of 6 mg / mL. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide EDC and N-hydroxysuccinimide NHS were added to the OCS aqueous solution, with a mass ratio of OCS:85:50. After adjusting the pH to 4-5, the mixture was stirred at room temperature in the dark for 1.5 hours. A dimethyl sulfoxide solution of 3-aminophenylboronic acid APBA with a concentration of 15 mg / mL was added, with a mass ratio of APBA to NHS of 1.5:1. The pH was adjusted to 4-5 again, and the reaction was continued overnight at room temperature in the dark. The resulting reaction solution was dialyzed and lyophilized to obtain the oxidized chondroitin sulfate-3-aminophenylboronic acid APBA-OCS polymer.

[0177] In step S231, the mass-to-volume ratio of tilapia skin collagen to acetic acid is 4 mg / mL, and the molar concentration of acetic acid is 0.01 M.

[0178] In step S232, the volume ratio of acidic collagen solution to 10×PBS buffer solution is 7:1.

[0179] In step S233, the concentration of the APBA-OCS solution is 4 mg / mL.

[0180] Example 6

[0181] Same as Example 1, except that:

[0182] In step S11, the concentration of the CM protein solution is 25 mg / mL.

[0183] In step S12, the concentration of the BTO nanoparticle suspension is 6 mg / mL.

[0184] In step S131: the molar ratio of CLS-PEG-MAL to CAP-SH (thiolized CAP peptide) is 1:2, and the reaction is carried out at room temperature with shaking for 12 hours.

[0185] In step S132, CM@BTO and CLS-PEG-CAP are mixed at a mass ratio of membrane protein to polymer of 20:1.

[0186] In step S22, chondroitin sulfate OCS oxidized with periodate was dissolved in water to obtain an OCS aqueous solution with a concentration of 3 mg / mL. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide EDC and N-hydroxysuccinimide NHS were added to the OCS aqueous solution, with a mass ratio of OCS:85:50. After adjusting the pH to 4-5, the mixture was stirred at room temperature in the dark for 0.5 hours. A dimethyl sulfoxide solution of 3-aminophenylboronic acid APBA with a concentration of 10 mg / mL was added, with a mass ratio of APBA to NHS of 1.0:1. The pH was adjusted to 4-5 again, and the reaction was continued overnight at room temperature in the dark. The resulting reaction solution was dialyzed and lyophilized to obtain the oxidized chondroitin sulfate-3-aminophenylboronic acid APBA-OCS polymer.

[0187] In step S231, the mass-to-volume ratio of tilapia skin collagen to acetic acid is 6 mg / mL, and the molar concentration of acetic acid is 0.03 M.

[0188] In step S232, the volume ratio of acidic collagen solution to 10×PBS buffer solution is 9:1.

[0189] In step S233, the concentration of the APBA-OCS solution is 6 mg / mL.

[0190] The above description of the embodiments is only for the purpose of helping to understand the technical solution and core idea of ​​the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. An injectable piezoelectric hydrogel for treating osteoarthritis, characterized in that, The invention comprises a chondroitin sulfate-3-aminophenylboronic acid polymer hydrogel matrix and piezoelectric nanoparticles dispersed within the polymer hydrogel matrix. The piezoelectric nanoparticles include a core layer composed of biocompatible piezoelectric nanoparticles and a chondrocyte membrane layer covering the core layer. The surface of the chondrocyte membrane layer is conjugated with chondrocyte-targeting peptides.

2. The injectable piezoelectric hydrogel according to claim 1, characterized in that, The hydrogel matrix further includes collagen; the piezoelectric nanoparticles have a mass fraction of 0.05%-2.00% in the injectable piezoelectric hydrogel.

3. The method for preparing the injectable piezoelectric hydrogel according to claim 1 or 2, characterized in that, Includes the following steps: S1. Preparation of piezoelectric nanoparticles: A suspension of biocompatible piezoelectric nanoparticles was mixed with a chondrocyte membrane protein solution. The chondrocyte membrane was then coated onto the biocompatible piezoelectric nanoparticles by extrusion to obtain chondrocyte membrane-coated biocompatible piezoelectric nanoparticles. Then, chondrocyte-targeting peptides were modified onto the surface of the chondrocyte membrane-coated biocompatible piezoelectric nanoparticles to obtain piezoelectric nanoparticles. S2. Preparation of hydrogel matrix: Chondroitin sulfate OCS oxidized with periodate was dissolved in water, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EDC and N-hydroxysuccinimide NHS were added. After adjusting the pH, a dimethyl sulfoxide solution of 3-aminophenylboronic acid APBA was added. The reaction was carried out overnight under weakly acidic conditions. The resulting reaction solution was dialyzed and lyophilized to obtain oxidized chondroitin sulfate-3-aminophenylboronic acid APBA-OCS polymer. S3. Hydrogel Composite: The APBA-OCS polymer obtained in step S2 is prepared into an APBA-OCS polymer solution. Piezoelectric nanoparticle suspension is added to the APBA-OCS polymer solution and stirred thoroughly to obtain the injectable piezoelectric hydrogel.

4. The preparation method according to claim 3, characterized in that, The specific steps for modifying the cartilage-targeting peptide onto the surface of the cartilage cell membrane-coated biocompatible piezoelectric nanoparticles in step S1 are as follows: Cholesterol-polyethylene glycol-maleimide CLS-PEG-MAL and thiolized CAP peptide are mixed in PBS at a molar ratio of 2:1-1:2, and the mixture is reacted with shaking at room temperature for 8-12 hours. After dialysis and lyophilization, powdered CLS-PEG-CAP is obtained. Then, the cartilage cell membrane-coated biocompatible piezoelectric nanoparticles are mixed with CLS-PEG-CAP at a mass ratio of membrane protein to polymer of 10-20:1, and incubated overnight at 4°C. Surface modification is completed through hydrophobic interactions to obtain piezoelectric nanoparticles Cap-CM@BTO.

5. The preparation method according to claim 3 or 4, characterized in that, Step S3 is as follows: Collagen is dissolved in dilute acetic acid to prepare an acidic collagen solution. The acidic collagen solution is mixed with PBS and the pH is adjusted to neutral to form a neutral collagen Col solution. The Col solution is mixed with APBA-OCS solution to obtain a mixed solution. The piezoelectric nanoparticles obtained in step S1 are added to the mixed solution and stirred thoroughly to obtain the injectable piezoelectric hydrogel.

6. The use of the injectable piezoelectric hydrogel according to claim 1 or 2 or the injectable piezoelectric hydrogel obtained by the preparation method according to any one of claims 3-5 in the preparation of pharmaceutical formulations for the treatment of osteoarthritis.

7. A pharmaceutical preparation for treating osteoarthritis, characterized in that, The active ingredient is the injectable piezoelectric hydrogel as described in claim 1 or 2, or the injectable piezoelectric hydrogel obtained by the preparation method described in any one of claims 3-5.

8. The method of using the pharmaceutical preparation for treating osteoarthritis according to claim 7, characterized in that, The pharmaceutical preparation is administered to the bone and joint.

9. A method for enhancing mitochondrial autophagy in chondrocytes, comprising contacting the chondrocytes with the injectable piezoelectric hydrogel of claim 1 or 2 or the injectable piezoelectric hydrogel prepared by any one of claims 3-5.

10. A kit for treating osteoarthritis, characterized in that, include: (1) A syringe pre-loaded with the injectable piezoelectric hydrogel as described in claim 1 or 2 or the injectable piezoelectric hydrogel obtained by the preparation method according to any one of claims 3-5; (2) Instructions on intra-articular injection.

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