High permeability micro-nano hydrogel microspheres and preparation method thereof

Abstract

The application provides a kind of high permeability micro-nano hydrogel microspheres and its preparation method and application, belong to the field of biological medicine technology.The preparation method of the high permeability micro-nano hydrogel microspheres of the application is as follows: (1) triphenylphosphine is reacted with selenium-doped carbon quantum dots to obtain triphenylphosphine functionalized selenium-doped carbon quantum dots; (2) aldehyde hyaluronic acid is mixed with methacrylic anhydride to obtain double bond modified aldehyde hyaluronic acid hydrogel; (3) hydrogel microspheres are prepared by microfluidic method, and the microspheres are crosslinked under ultraviolet light to obtain high permeability micro-nano hydrogel microspheres.The high permeability micro-nano hydrogel microspheres of the application can fully penetrate into subchondral bone, inhibit osteoclast differentiation and function, inhibit early chondrocyte apoptosis, and the microsphere delivery system can also inhibit osteoclastogenesis and H-vascular invasion, thereby regulating the occurrence and process of abnormal bone remodeling and inhibiting osteoarthritic cartilage degeneration.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a highly permeable micro / nano hydrogel microsphere, its preparation method, and its application. Background Technology

[0002] Abnormal subchondral bone (SB) remodeling can significantly induce degenerative changes in articular cartilage, characterized by early bone loss due to bone resorption, late-stage subchondral sclerosis, and osteophytes formed during bone formation. Traditional intra-articular injections used to treat osteoarthritis cannot directly penetrate the cartilage barrier to reach the SB; compared to cartilage-based treatments, clinical interventions have greater potential for treating SB. However, cartilage-based treatments cannot fundamentally reverse the pathological process of SB during osteoarthritis (OA). Furthermore, abnormal SB remodeling exacerbates the breakdown of the cartilage matrix. The high anisotropy, density, and avascularity of cartilage pose challenges to overcoming steric hindrance and penetrating the SB to exert therapeutic effects. Therefore, effectively penetrating the SB and remodeling its bone metabolism by overcoming the spatial hindrance of cartilage is a key factor in OA treatment.

[0003] Under physiological conditions, the bone matrix (SB) maintains a low osteogenic rate and has few osteoclasts. Osteoclast-induced abnormal bone remodeling in the SB is a crucial pathological event in osteoarthritis (OA). Osteoclast precursor cells migrate to the cartilage layer, directly interacting with hypertrophic chondrocytes, damaging the articular cartilage matrix and osteochondral junctions, further promoting the release of large amounts of growth factors that interfere with chondrocyte metabolism, such as transforming growth factor β1 (TGF-β1), insulin-like growth factor 1 (IGF-1), and platelet-derived growth factor-bb (PDGF-BB). Conversely, osteoclast-induced H-type angiogenesis may promote the transformation from bone resorption to osteosclerosis in the SB microenvironment of OA. Accompanying H-type angiogenesis is the invasion of cartilage, along with the invasion of numerous cytokines and inflammatory cells into chondrocytes, collectively disrupting their differentiation phenotype and metabolic homeostasis. Although the phenotype of OA remains unclear, numerous animal and clinical trials have shown that early activation of osteoclasts can block abnormal SB remodeling, thereby preventing articular cartilage degeneration.

[0004] Mitochondrial reactive oxygen species (ROS) act as secondary messengers, initiating the differentiation of hematopoietic stem cells or monocyte / macrophage progenitors into osteoclasts. Currently, targeting mitochondrial-generated ROS can effectively inhibit osteoclast activation. Abnormal ROS signaling exhibits a spatiotemporal distribution pattern from the joint surface to the bone sclera (SB). Therefore, clearing overexpressed ROS throughout the joint while inhibiting abnormal cartilage and bone metabolism is crucial for the treatment of osteoarthritis (OA).

[0005] Initially, antioxidant molecules were used to neutralize reactive oxygen species (ROS), reduce oxidative stress, inhibit cartilage degradation, and slow the progression of osteoarthritis (OA). However, due to steric hindrance, most antioxidants cannot effectively penetrate cartilage to enter the synovial artery (SB), limiting their efficacy. Carbon quantum dots (CQDs) are zero-dimensional, ultra-small nanomaterials that hold promise for penetrating the dense structure of cartilage. Selenium-doped carbon quantum dots (Se-CQDs) possess redox-modulating functions and can effectively scavenge ROS under oxidative stress. However, ultra-small CQDs are easily cleared and metabolized by capillaries and lymphatic vessels in the synovium, severely affecting their bioavailability and reducing their protective effect on cartilage. Therefore, selenium-doped carbon quantum dots cannot solve the problem of short residence time in the joint cavity and low drug bioavailability. Developing effective strategies to prolong the residence time of biologics in the joint cavity is crucial for the treatment of osteoarthritis.

[0006] Another approach to treating osteoarthritis involves precisely delivering drugs to cartilage using mitochondrial-targeting drug delivery systems, thereby scavenging reactive oxygen species (ROS). Using triphenylphosphine (TPP) as a mitochondrial targeting device, modified onto the surface of drug molecules or nanoparticles, is the most frequently reported strategy. However, this method suffers from drawbacks such as low targeting efficiency, disruption of the mitochondrial electron transport chain, and cytotoxicity.

[0007] To improve the safety of mitochondrial-targeting nanoparticles, researchers have discovered a novel mitochondrial-targeting peptide that can replace TPP (tracerebrospinal polymer) to perform the corresponding function. For example, patent document CN 114042147 B discloses a micro / nano hydrogel microsphere targeting and regulating the mitochondrial respiratory chain and its preparation method. It uses a mitochondrial-targeting peptide to prepare nanoliposomes, which are then loaded onto hyaluronic acid hydrogel microspheres. SS-31 peptide is a tetrapeptide that can be taken up by cells and can specifically bind to the inner mitochondrial membrane, improving the cristae curvature and restoring the vitality of the mitochondrial electron transport chain, thus reducing ROS production. After the specific type II collagen-targeting peptide (Wyrgrl peptide) binds to SS-31 peptide, the liposomes can target the mitochondria of articular chondrocytes. Furthermore, SS-31 peptide can stabilize the cardiolipin (CL) structure of the inner mitochondrial membrane, thereby regulating the electron transport efficiency of the mitochondrial respiratory chain, and holds promise for the effective treatment of degenerative diseases such as osteoarthritis, which are associated with mitochondrial respiratory chain dysfunction. However, this hydrogel microsphere delivery system has low penetration into the subchondral bone and cannot penetrate into the subchondral bone in large quantities, thus affecting its efficacy against OA at the SB level.

[0008] Therefore, how to provide a hydrogel delivery system that has good cartilage targeting and good permeability to subchondral bone, can penetrate the cartilage barrier, and efficiently penetrate into the subchondral bone, in order to improve the poor permeability of existing hydrogel microspheres in the treatment of diseases such as osteoarthritis, has become an urgent technical problem to be solved. Summary of the Invention

[0009] The present invention aims to solve the aforementioned technical problems by providing highly permeable micro / nano hydrogel microspheres, their preparation method, and applications. The technical objective of this invention is to address the shortcomings of existing hydrogel microsphere delivery systems in treating osteoarthritis, which suffer from poor permeability, inability to efficiently penetrate subchondral bone, and consequently, failure to regulate the function of subchondral bone cells, thus affecting the therapeutic effect on osteoarthritis.

[0010] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows:

[0011] This invention first provides a method for preparing highly permeable micro / nano hydrogel microspheres, comprising the following steps:

[0012] (1) Triphenylphosphine was activated by EDC / NHS and then reacted with selenium-doped carbon quantum dots to prepare triphenylphosphine-functionalized selenium-doped carbon quantum dots.

[0013] (2) Acetaldehyde was used to modify hyaluronic acid to obtain aldehyde-based hyaluronic acid. Then, the aldehyde-based hyaluronic acid was mixed with methacrylic anhydride to prepare a double-bond modified aldehyde-based hyaluronic acid hydrogel.

[0014] (3) Using the triphenylphosphine-functionalized selenium-doped carbon quantum dots prepared in step (1), the double bond-modified aldehyde hyaluronic acid hydrogel prepared in step (2), and the photoinitiator as the aqueous phase, and a mixture of Span 80 and paraffin oil as the oil phase, hydrogel microspheres were prepared by microfluidic method. The microspheres were cross-linked under ultraviolet light to obtain highly permeable micro-nano hydrogel microspheres.

[0015] This invention provides a method for preparing highly permeable micro / nano hydrogel microspheres. Initially, the inventors believed that using substances with mitochondrial targeting to prepare hydrogel microspheres would significantly enhance their ability to penetrate cartilage. However, as shown in the comparative examples of this invention, using two targeting peptides to prepare hydrogel microspheres did not significantly increase their ability to penetrate subchondral bone. Through extensive experimentation, the inventors finally discovered that the micro / nano hydrogel microspheres prepared according to the method described above can efficiently penetrate subchondral bone, achieving regulatory function on subchondral osteocytes. Their high permeability ensures that the microspheres overcome the cartilage barrier to reach the subchondral bone, inhibiting osteoclast differentiation and function, and suppressing early chondrocyte apoptosis. Furthermore, this microsphere delivery system can also inhibit osteoclast formation and H-type vascular invasion, thereby regulating the occurrence and process of abnormal bone remodeling, inhibiting cartilage degeneration in osteoarthritis, and exerting a highly effective therapeutic effect.

[0016] Existing hydrogel microsphere drug delivery systems generally achieve cartilage targeting through mitochondrial targeting. However, this type of system suffers from poor permeability. As shown in the comparative example of this invention, when two mitochondrial-targeting peptides are selected to prepare microspheres, selenium-carbon quantum dots cannot fully utilize their cartilage permeability. The highly permeable micro / nano hydrogel microspheres provided by this invention, in addition to possessing mitochondrial targeting, also exhibit highly efficient penetration into subchondral bone, making them well-suited for regulating subchondral bone metabolism to treat osteoarthritis.

[0017] In vitro experiments confirmed that the highly permeable micro / nano hydrogel microspheres of this invention can reach deep cartilage layers and release selenium atoms to scavenge reactive oxygen species (ROS) in the mitochondria of mononuclear macrophages, inhibit osteoclast differentiation and function, suppress early chondrocyte apoptosis, and maintain the balance between cartilage matrix synthesis and catabolism. In vivo experiments demonstrated that this delivery system can inhibit osteoclast formation and H-type vascular invasion, thereby regulating the occurrence and process of abnormal bone remodeling and inhibiting cartilage degeneration in osteoarthritis. In summary, these micro / nano hydrogel microspheres promote effective penetration of sclerosing hormone (SB) into joints and can be used to regulate SB metabolism to treat osteoarthritis.

[0018] After extensive experimentation, the inventors ultimately selected acetaldehyde and methacrylic anhydride-modified hyaluronic acid and triphenylphosphine-grafted selenium-doped carbon quantum dots to design and synthesize a highly permeable micro / nano-hydrogel microsphere to regulate osteoclast activation within the subchondral bone. In the weakly acidic environment of osteoarthritis (OA), the Schiff base bonds of these highly permeable micro / nano-hydrogel microspheres break, allowing for the continuous release of supercritical chondrocytes (SCTs), overcoming cartilage steric hindrance to reach the subchondral bone matrix (SB), and precisely regulating the synthesis and metabolism of the cartilage matrix. Furthermore, these highly permeable micro / nano-hydrogel microspheres can adhere to the site of articular cartilage injury, thereby enhancing therapeutic efficacy. After intra-articular injection, the microspheres of this invention can overcome cartilage steric hindrance, penetrate the cartilage, act on the SB, and reshape the homeostatic balance of the OA microenvironment. This allows them to serve as a highly efficient, rapid, and long-lasting superpermeable drug delivery system for OA treatment.

[0019] Furthermore, the weight ratio of triphenylphosphine to selenium-doped carbon quantum dots in step (1) is 1:17.5.

[0020] Furthermore, the reaction time described in step (1) is 12 hours.

[0021] Furthermore, the preparation steps of the selenium-doped carbon quantum dots in step (1) are as follows: L-selenocysteine ​​is mixed with water to form an aqueous dispersion, the pH is adjusted to 10 with sodium hydroxide, and then the reaction is carried out at 60°C for 20 hours under nitrogen protection. The resulting product is centrifuged and dialyzed to obtain selenium-doped carbon quantum dots.

[0022] Furthermore, the preparation steps of the aldehyde-based hyaluronic acid in step (2) are as follows: hyaluronic acid is dissolved in water, sodium periodate solution is added to it, the mixture is stirred and reacted at room temperature for 2 hours, and the resulting product is dialyzed and then freeze-dried to obtain aldehyde-based hyaluronic acid.

[0023] Furthermore, the preparation steps of the double bond modified aldehyde hyaluronic acid hydrogel in step (2) are as follows: dissolve hyaluronic acid, add methacrylic anhydride, and then keep the mixture in an ice bath at pH=8-8.5 for 12 hours. After the reaction is completed, dialyze and then freeze dry to obtain the double bond modified aldehyde hyaluronic acid hydrogel.

[0024] Furthermore, the weight ratio of the triphenylphosphine-functionalized selenium-doped carbon quantum dots, the double-bond modified aldehyde-based hyaluronic acid hydrogel, and the photoinitiator in step (3) is 1:5:1.

[0025] Furthermore, the flow rate ratio of the water phase to the oil phase in step (3) is 1:30.

[0026] The second objective of this invention is to provide a highly permeable micro / nano hydrogel microsphere prepared by any of the methods described above.

[0027] A third objective of this invention is to provide the application of the highly permeable micro / nano hydrogel microspheres described above in the preparation of a medicament for treating osteoarthritis.

[0028] The beneficial effects of this invention are as follows:

[0029] (1) The present invention provides a highly permeable micro-nano hydrogel microsphere drug delivery system. In addition to mitochondrial targeting, the microsphere drug delivery system also has highly efficient permeability to subchondral bone. The microspheres can break through the cartilage barrier to reach the subchondral bone and exert a highly effective therapeutic effect. It can be well used for the treatment of bone and joint injuries and can exert a good therapeutic effect on bone and joint injuries.

[0030] (2) This invention provides a method for preparing highly permeable micro / nano hydrogel microspheres, which has the advantages of simple preparation process, controllable operation, small particle size of microsphere products, and the ability to overcome the steric hindrance of cartilage after intra-articular injection, penetrate cartilage, act on SB, and reshape the homeostatic balance of OA microenvironment. It can be used as a highly efficient, rapid and long-acting super-permeable drug delivery system for OA treatment. Attached Figure Description

[0031] Figure 1 The diagram illustrates the synthesis and application of micro / nano hydrogel microspheres; A) Synthesis of SCT; B) Preparation of SCT-HA using microfluidic technology, including the synthesis process of AHAMA; C) SCT-HA continuously releases SCT in the weakly acidic environment within the joint cavity, enabling it to penetrate the cartilage matrix and reach the SB for OA treatment.

[0032] Figure 2 Characterization of highly permeable micro / nano hydrogel microspheres; A) TEM image of SCT; B) UV absorption and fluorescence spectra of SCT; C) ζ-potential measurement of aqueous solutions of SC and SCT; D) Photographs of SC and SCT in solution; E) XPS spectra of Se, P, and N in SCT; F) Light microscopic observation of SCT-HA hydrogel microspheres; G) Light micrograph of a single SCT-HA hydrogel microsphere; H) Fluorescence micrograph of a single SCT-HA hydrogel microsphere; I) Photograph of a single SCT-HA hydrogel microsphere under a laser confocal microscope; J) 3D rendering of a single SCT-HA hydrogel microsphere using Imris software; K) SEM image and elemental mapping of SCT-HA; L) Release curve of SCT-HA microspheres; M) Microsphere particle size distribution; N) AHA, AHA, and HA... 1 HNMR spectrum.

[0033] Figure 3For the diameter comparison between SC and SCT (n=3), the one-student t-test was used to calculate the differences between groups. The results are expressed as mean ± standard deviation. The result value is expressed as mean ± standard deviation. p < 0.05 is considered statistically significant. NS indicates no significant difference.

[0034] Figure 4 The following images illustrate the penetration of SCT-HA into articular cartilage grafts in vitro and in vivo: A) Schematic diagram of SCT-HA penetration experiment in cartilage grafts; B) Laser confocal images of CQ-HA, SC-AHAMA (SC-AH), and SCT-HA penetrating articular cartilage grafts; C) Fluorescence intensity analysis of the three materials penetrating the superficial, transitional, deep, and calcified cartilage regions of the articular cartilage grafts; D) Laser confocal observation and three-dimensional surface reconstruction of mouse knee joint sections after CQ-HA and SCT-HA were injected into the joint cavity, observing the entire joint, meniscus, cartilage, SB, and medullary cavity; E) Fluorescence intensity analysis of CQ-HA and SCT-HA penetrating the meniscus, cartilage, SB, and medullary cavity of the mouse knee joint; NS: Not significant, p values ​​< 0.05, 0.01, 0.001, and 0.0001 are represented by *, **, ***, and ****, respectively.

[0035] Figure 5 The results for day 1 and day 3 are: A) chondrocyte and B) RAW246.7 dead (red) and live (green) staining fluorescence results; C) live cell counts of chondrocytes and RAW246.7 obtained by live / dead staining method (n=3) (NS: no statistical significance); D) CCK-8 results of chondrocytes and RAW246.7 on day 1 and day 3 (n=3) (NS: no statistical significance).

[0036] Figure 6To protect chondrocytes from H2O2-induced ROS damage using highly permeable micro / nano-hydrogel microspheres; A) SCT-HA targeting mouse chondrocyte mitochondria: laser confocal images showing Mito-tracker mitochondrial staining (red), SCT autofluorescence (green), and merging (yellow); mitochondria, SCT, and cell outlines reconstructed using Imris (red, green, blue); B) Inhibitory effect of micro / nano-hydrogel microspheres on H2O2-induced ROS production; DCFH-DA staining of chondrocytes (green) indicates H2O2-induced ROS production; scale bar = 100 μm; C) JC-1 staining image of depolarized mitochondrial membranes after hydrogen peroxide intervention in chondrocytes; scale bar = 100 μm; D) 50 μm Representative images of Col2α1 chondrocytes after H2O2 treatment and co-culturing with AHAMA, CQ-HA, and SCT-HA for 12 h; scale bar = 100 μm; E) SCT-HA targeting chondrocyte mitochondria inhibits ROS generation, promotes chondrocyte anabolic metabolism, and inhibits chondrocyte catabolism and inflammation; F) DCFH-DA fluorescence intensity analysis after H2O2 intervention in chondrocytes; G) Statistical analysis of fluorescence intensity of JC-1 aggregates (red) and monomers (green) after H2O2 intervention in chondrocytes, 50 μm. Changes in the expression of HL, IL-6 (H), MMP13 (I), ADAMTS5 (J), Col2α1 (K), and Aggrecan (L) in chondrocytes after 12 h of H2O2 treatment; Statistical analysis of the immunofluorescence intensity of Col2α1 protein in chondrocytes after H2O2 intervention. NS: No significance. p < 0.05, 0.01, 0.001, and 0.0001 are represented by *, **, ***, and ****, respectively.

[0037] Figure 7To inhibit osteoclast differentiation and function using highly permeable micro / nano hydrogel microspheres; A) TRAP staining showed that SCT-HA inhibited RANKL-induced osteoclast differentiation; B) Green DCFH-DA staining demonstrated that SCT-HA inhibited RANKL-induced ROS production in RAW246.7 cells; C) Using confocal laser images with Mito-tracker dye (red), SCT autofluorescence (green), and combined (yellow), SCT-HA specifically targets the mitochondria of RAW246.7 cells, and Imris reconstruction images show mitochondrial staining, SCT, and cell outlines (red, green, blue); D) SCT-HA targets the mitochondria of monocytes and macrophages, leading to inhibition of ROS production and inhibition of osteoclast differentiation-related signaling pathways; E) Statistical analysis of TRAP staining in different treatment groups involved in osteoclast differentiation; F) Estimation of osteoclast-related genes NFATc1, PDGF-BB, and Cathepsin in different subgroups after 3 days of treatment. Expression levels of K and TRAP; NS: not significant, p-values ​​< 0.05, 0.01, 0.001, and 0.0001 are represented by *, **, ***, and ****, respectively.

[0038] Figure 8 Analysis of DCFH-DA fluorescence intensity in RAW246.7 cells (n=3) (NS: no statistical significance, * indicates p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0039] Figure 9 Imaging evaluation of SCT-HA treatment for OA: A) Representative anterior and posterior 3D reconstructed Micro-CT images of the knee joint, with red arrows indicating the presence of osteophytes; B) Representative 2D Micro-CT coronal and sagittal images of the knee joint, with red arrows indicating the presence of osteophytes and red triangles indicating articular surface collapse; C) Statistical analysis of osteophyte volume; D) Statistical analysis of bone mineralization density (BMD) of the SB plate in each treatment group; E) Statistical analysis of bone volume fraction (BV / TV), F) Trabecular bone separation (Tb.pf), and G) SB plate thickness (SBP.Th); NS: No significance; p-values ​​< 0.05, 0.01, 0.001, and 0.0001 are represented by *, **, ***, and ****, respectively.

[0040] Figure 10To inhibit abnormal bone remodeling in the tibia and prevent cartilage degradation using SCT-HA; A) Representative images stained with toluidine blue and H&E; B) Immunohistochemical staining levels of Col2α1 and MMP13; C) Double labeling with calcein green and alizarin red under a fluorescence microscope; D) Representative trap-stained SB sections of the tibia; E) Grading; F) OARSI score; G) Summary of relative Col2α1 expression results; H) MMP13 + Cell count summary; I) MAR quantification; J) TRAP + Quantitative analysis of multinucleated cells; NS: not significant; p-values ​​< 0.05, 0.01, 0.001, and 0.0001 are represented by *, **, ***, and ****, respectively.

[0041] Figure 11 To illustrate SCT-HA's role in inhibiting SB angiogenesis: A) Immunofluorescence images of SB H-vessels (CD31hiEmcnhi) in each treatment group; B) SB micromembrane MV-122 angiography after Micro-CT 3D reconstruction in each treatment group; C) Schematic diagram of SCT-HA's inhibition of abnormal SB remodeling. SCT-HA inhibits the abnormal invasion of SB vessels in OA by inhibiting the differentiation of monocytes and macrophages into osteoclasts and the production of PDGF-BB. SCT-HA also prevents abnormal SB bone remodeling by inhibiting vessels, which act as the "executors" of abnormal bone remodeling; D) Quantitative statistical analysis of H-vessels in SB in each group; E) Vessel volume relative to SB tissue volume (VV / TV); F) Number of vessels (VN); NS: No significance; p-values ​​<0.05, 0.01, 0.001, and 0.0001 are represented by *, **, ***, and ****, respectively.

[0042] Figure 12 Transcriptional profiling analysis of SCT-HA regulating SB function in a mouse OA model; A) Volcano plot comparing the PBS group and the Sham group, and the SCT-HA group and the PBS group, with upregulated genes marked in red and downregulated genes marked in blue; B) Heatmap of gene expression differences between the PBS group and the Sham group, and between the SCT-HA group and the PBS group; C) Functional enrichment analysis of the KEGG pathway between the PBS group and the Sham group using differentially expressed genes (DEGs); D) SCT-HA and PBS groups; E) Changes in osteoclast activation-related genes between the SCT-HA group and the PBS group, with blue indicating downregulation of osteoclast-related genes and purple indicating upregulation of osteoclast-related genes; F) GSEA of the KEGG pathway between the SCT-HA group and the PBS group, with normalized enrichment scores (NES) representing the combined dataset of KEGG pathway gene sets; (G,H,I) GSEA after threshold screening. Detailed Implementation

[0043] 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.

[0044] Example 1

[0045] I. Experimental Methods (I) Synthesis of High-Permeability Micro / Nano Hydrogel Microspheres

[0046] 1. Synthesis of SCT

[0047] Selenium-doped carbon quantum dots (Se-CQDs) were synthesized via a hydrothermal method, with the following steps: 0.3 g of L-selenocysteine ​​was mixed with 35 mL of water to form an aqueous dispersion, and the pH was adjusted to 10 using 0.1 M sodium hydroxide. Then, under nitrogen protection, the reaction mixture was heated at 60 °C for 20 hours. After the reaction was complete, the brown solution was recovered by centrifugation at 12000 rpm for 10 min, and the supernatant was dialyzed to obtain Se-CQDs (abbreviated as SC).

[0048] Triphenylphosphine (TPP, 2.0 mL, 3.5 mM) was activated for 3 hours using 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide (EDC / NHS) as an activator. The activated TPP was then reacted with Se-CQDs (weight ratio 1:17.5) for 12 hours to generate TPP-functionalized Se-CQDs (SCT). Excess TPP was removed by dialysis.

[0049] 2. Preparation of AHAMA hydrogel

[0050] The synthesis method of aldehyde hyaluronic acid (AHA) is as follows: 1 g of hyaluronic acid (HA) was completely dissolved in 100 ml of water, and 5 ml of sodium periodate (0.5 M) was added dropwise. The mixture was then stirred for 2 hours under light protection at room temperature. 1 ml of ethylene glycol was added, and the reaction was continued for 1 hour to deactivate unreacted periodate. The reaction product was dialyzed for 3 days and then freeze-dried to obtain AHA.

[0051] The synthesis method of double-bond modified AHA is as follows: 1 g of hyaluronic acid (HA) was completely dissolved in 100 ml of water, and then 1 ml of methacrylic anhydride was added. The mixture was then maintained at pH 8-8.5 for 12 hours under ice-cold conditions. After the reaction was completed, the solution was dialyzed for two days and then lyophilized to obtain AHAMA. The sample was dissolved in D2O and quantitatively analyzed. 1The percentage of double bonds was determined by ¹H NMR, and the content of AHAMA aldehyde was determined by tert-butylammonium diazoate and trinitrobenzenesulfonic acid (TNBS) solution.

[0052] 3. Preparation of highly permeable micro / nano hydrogel microspheres (SCT-HA)

[0053] SCT-HA was prepared using a microfluidic device, specifically as follows: SCT was mixed with a hydrogel solution containing AHAMA, SCT, and a photoinitiator (SCT:AHAMA:photoinitiator weight ratio of 1:5:1) as the aqueous phase. Span 80 was mixed with paraffin oil as the oil phase. The mixture was injected into the inlet of the microfluidic device using a syringe, controlling the flow rate ratio between the aqueous and oil phases at 1:30. The droplets flowing out of the microfluidic device were frozen at -30°C and then crosslinked using ultraviolet light to form highly permeable micro / nano hydrogel microspheres, denoted as SCT-HA. Liquid paraffin was removed with ether, and the microspheres were suspended in water and freeze-dried for storage.

[0054] (II) Characterization of highly permeable micro / nano hydrogel microspheres

[0055] 1. Characterization of SCT and SCT-HA microspheres

[0056] The characterization methods for SCT and SCT-HA microspheres are as follows:

[0057] (i) The morphology of SCT was characterized using transmission electron microscopy (TEM, FEI TF20, USA) at an accelerating voltage of 200 kV (2100 F). (ii) The zeta potential and size distribution of SCT particles were characterized using a nanoparticle size potentiometer (NanoBrook 90plus PALS, Brookhaven, USA). (iii) UV-Vis absorption experiments were performed using a UV spectrophotometer (UH5300) in a 1 cm quartz cell, and the fluorescence spectrum of SCT was measured using an F-4600 fluorescence spectrophotometer. (iv) The elemental valence state and content of SCT were detected using X-ray photoelectron spectroscopy (XPS). (v) The wet morphology and autofluorescence of SCT-HA microspheres were observed using a microscope (LSM800, ZEISS, Germany), and the particle size distribution of 100 SCT-HA microspheres was measured using ImageJ. (vi) The dried morphology of SCT-HA microspheres was observed using scanning electron microscopy combined with energy dispersive spectroscopy to verify the elemental distribution (O, N, Se, P). (vii) The autofluorescence of SCT in the microspheres was observed using laser scanning confocal microscopy (LSCM) (Zeiss). (viii) To quantify the release of SCT from the SCT-HA microspheres, the SCT-HA microspheres were first immersed in weakly acidic phosphate buffer (pH 6.8) and stirred at 80 rpm using a vibrating sieve. The supernatant was then replaced with PBS every two days. The collected supernatant was analyzed for selenium content using inductively coupled plasma atomic emission spectrometry (iCAP7600, Thermo, USA).

[0058] 2. Isolation of primary chondrocytes

[0059] To isolate mouse primary chondrocytes (MCCs), the tibial plateau, femoral condyle, and femoral head of mice were harvested, and MCCs were isolated using cartilage fragments. The fragments were cut into small pieces, digested with 0.25% trypsin at 37°C for 30 minutes, washed with PBS, and then digested with 0.25% collagenase II at 37°C for 8 hours. The cells were then filtered through a 70 μm cell filter, centrifuged at 1000 rpm for 5 minutes, and the MCCs were collected. The isolated cells were cultured in DMEM medium containing a double antibody (1%) and fetal bovine serum (10%). Finally, third-generation chondrocytes were used for subsequent experiments.

[0060] 3. Mitochondrial targeting of SCT-HA microspheres

[0061] Cells were incubated in DMEM medium containing 10% FBS for 4 hours and washed, after which the medium was replaced. Then, cells were incubated with SCT-HA microspheres for 4 hours and stained with 0.5 μM Mito-Tracker for 4 hours to analyze mitochondrial colocalization. Staining was performed using a DeepRed FM microscope (Biyuntian, China). Finally, LSCM was performed using a Zeiss Axio Imager M1 microscope (Germany). The same method was used to observe mitochondrial targeting in Raw264.7 cells.

[0062] 4. Cartilage permeability

[0063] In vitro experiments were conducted using porcine articular cartilage explants to test cartilage penetration. Articular cartilage was obtained from the patellar groove of the femur using a 4 mm diameter trephine drill. The extracted articular cartilage was washed with sterile PBS. The penetration test was performed in a self-designed one-way diffusion chamber. Figure 2 (Part A). The articular cartilage explant was sealed within the cavity and consisted of two layers. The lower layer and the area surrounding the cartilage were filled with Ab adhesive, while the upper layer, containing SCT-HA microspheres soaked in PBS, was placed on the cartilage surface. After 48 hours, a 1 mm thick section was excised from the center of the cartilage and immediately observed using a laser confocal microscope (LSCM) (Zeiss, Axio Imager M1, Germany).

[0064] Further in vivo cartilage penetration experiments were conducted on 10-week-old male wild-type C57BL / 6 mice. After surgery in the OA model (see the OA animal model section for details), SCT-HA microspheres were injected into the joint cavity. Seven days later, sections of the mouse knee joint were taken and observed under a Zeiss laser confocal microscope.

[0065] 5. Cell compatibility

[0066] To evaluate the effects of AHAMA, CQDs@AHAMA (CQ-HA), and SCT-HA on primary chondrocyte proliferation, live / dead staining was performed using the following methods:

[0067] In a 24-well perforated plate, 1.0 × 10⁻⁶ ppm is added to the low cavity. 4 / mL of primary mouse chondrocytes (MCCs) were added to the supernatant with AHAMA, CQ-HA, and SCT-HA. Cells were then incubated for 30 minutes with Calcein AM / PI assay working solution (250 μL) (Biyuntian). After 1 or 3 days of culture, cells were observed under a microscope. Cell proliferation was measured after incubation with AHAMA, CQ-HA, and SCT-HA. After incubation in 96-well perforated plates for 1 or 3 days, CCK-8 solution (10 μL) was added to each well, and the plates were incubated at 37°C for 1 hour. The absorbance at 450 nm was then detected and recorded using a FlexStaston3 enzyme labeler (Molecular Devices, Japan).

[0068] The effects of AHAMA, CQ-HA, and SCT-HA on the proliferation of Raw264.7 cells were evaluated using a similar approach.

[0069] 6. Measurement of intracellular reactive oxygen species and mitochondrial membrane potential

[0070] First, MCCs were seeded in 6-well plates and cultured with H2O2 to induce oxidative stress. Cells were then co-cultured with different groups (PBS, AHAMA, CQ-HA, and SCT-HA) for 12 hours. Afterward, all cells were treated with the fluorescent dye DCFH-DA (10 μM) (DCFH-DA, Beyotime, China). Cells were washed three times and observed under a microscope (LSM800, Zeiss, Germany). After the same 12-hour treatment, cells were stained with JC-1 (Beyotime) for 30 minutes and observed under an LSM800 microscope. The effects of AHAMA, CQ-HA, and SCT-HA on ROS in Raw264.7 cells were assessed using a similar method.

[0071] 7. Detection of genes and proteins related to the OA model

[0072] To evaluate the role of SCT-HA micro / nanohydrogel microspheres in OA, MCCs were first treated with H2O2 (50 μM) and then exposed to different treatments (PBS, AHAMA, CQ-HA, and SCT-HA) for 12 hours. Total RNA was extracted from MCCs and reverse transcribed, followed by RT-qPCR. Primer sequences for Aggrecan, Col2α1, MMP-13, ADATMS-5, and IL-6 are shown in Table 1. The experiment was repeated three times. After 12 hours of treatment, the Col2α1 level in chondrocytes was detected by immunofluorescence staining. After washing twice with PBS, fixation with 4% paraformaldehyde for 10 minutes, treatment with 0.1% Triton X-100 for 15 minutes, and incubation overnight at 4°C with rabbit anti-col2α1 antibody (Servicebio). Then, incubation with Cy3-labeled goat anti-rabbit IgG for 1 hour, followed by DAPI staining for 5 minutes, and the results were acquired and analyzed using LSCM (ZEISS, Germany) and ImageJ.

[0073] Table 1 Primer sequences involved in real-time PCR

[0074]

[0075]

[0076] 8. Inducing osteoclast differentiation

[0077] Mouse bone marrow-derived macrophages (BMMs) and RAW264.7 cells are commonly used to induce osteoclast differentiation. The medullary cavities of the tibia and femur were flushed clean and cultured overnight in α-MEM containing a double antibody (1%) and fetal bovine serum (10%), under 5% CO2 at 37°C. Adherent cells were removed, and non-adherent cells were collected by centrifugation at 2500 rpm for 5 min. The collected cells were cultured for 48 hours in medium containing 30 ng / mL M-CSF (R&D Systems) to produce pure monocytes / macrophages. Next, after 2 days of culture, the normal medium was replaced with fresh medium containing RANKL (40 ng / mL) and M-CSF (20 ng / mL). The cells were then incubated with this medium for 7 days, and their activity was assessed by tartrate-resistant acid phosphatase (TRAP) staining (Sigma-Aldrich).

[0078] In addition, AHAMA, CQ-HA, and SCT-HA were supplemented in RANKL to investigate their effects on osteoclastogenesis. RAW264.7 cells were used to induce osteoclast differentiation. After incubation with RANKL (40 ng / mL) and M-CSF (20 ng / mL) for 3 days, total RNA was extracted from RAW264.7 cells, reverse transcribed, and detected by RT-qPCR. Primer sequences for PDGF-BB, NFATC1, Cathepsin K, and TRAP are shown in Table 1. Results were processed using the 2-ΔΔCT method, and each experiment was repeated three times.

[0079] 9. Mouse OA model

[0080] All animal experiments were approved by the Animal Research Committee of Ruijin Hospital, Shanghai Jiao Tong University. Ten-week-old male wild-type C57BL / 6 mice were used as animal models. First, the mice were anesthetized and prepared for surgery. The anterior cruciate ligament (ACL) was incised in the joint cavity to induce mechanical instability, and then the incision was sutured. The sham surgery involved anesthesia and skin surgery without ligament damage. Next, five groups of OA mice were classified. On days 14 and 35, they were injected with equal volumes of PBS, AHAMA, CQ-HA, and SCT-HA (100 μL), respectively. The PBS-injected group served as the control group.

[0081] 10. Histochemistry, immunohistochemistry, and histomorphometrics

[0082] Ten weeks post-surgery, mice were euthanized, and knee joint specimens were collected. Samples were fixed in 4% paraformaldehyde, decalcified, and embedded in paraffin. Sagittal histopathological features of the knee joint were assessed using O-fast green, toluidine blue, H&E, and TRAP staining. The samples were then evaluated and scored according to the OARSI criteria. Immunohistochemistry was performed on paraffin sections using standard methods. The following antibodies were used: MMP13 (Abcam, 1:100) and Col2α1 (Abcam, 1:100). Sections were stained with secondary antibody and DAB substrate.

[0083] Tissue sections were immunofluorescence stained and air-dried. They were then infiltrated with 0.3% Triton X-100 for 10 minutes, blocked with 5% donkey serum for 30 minutes at room temperature, and incubated overnight at 4°C. The following primary antibodies were used: Endomucin (Santa Cruz Biotechnology, Santa Cruz, 1:100) and CD31 (R&D Systems, Minneapolis, 1:100). After hybridization with the specific primary antibodies, sections were washed three times with PBS and incubated for one hour with a suitable Alexa fluorescently conjugated secondary antibody (molecular probe, 1:400), followed by nucleus staining with DAPI.

[0084] To label the SB mineralization deposit, calcein green (30 mg / kg, Sigma, USA) and alizarin red (30 mg / kg, Sigma, USA) were subcutaneously injected after dissolution in sodium bicarbonate (2%). Calcein green was injected 10 days before euthanasia, and alizarin red was injected 2 days before euthanasia. After removing the muscles and soft tissue around the knee joint, the knee joint was fixed with 70% alcohol. Next, the knee was dehydrated using a gradient of alcohols (70%, 80%, 90%, and 100%), applied for two days at each concentration (8 days in total). After embedding in polymethyl methacrylate (PMMA), 100 μm thick mouse tibia sections were obtained by sagittal cutting using the EXAKT cutting and grinding system. These sections were further ground to a thickness of 50 μm. Subsequently, the dual-labeled fluorescence of calcein green and alizarin red was observed under a fluorescence microscope to obtain mineral adhesion ratio (MAR).

[0085] 11. Micro-computed tomography (CT) and CT-based microangiography

[0086] Ten weeks post-surgery, knee joint specimens were collected from euthanized mice. Arthrography was performed using a high-resolution micro-CT imaging system (SkyScan1172, Bruker BioSpin, Belgium). Micro-CT scans and reconstructions were used to assess the following parameters: relative osteophyte volume, bone mineral density (BMD) of the SB plate, trabecular bone pattern factor (Tb.Pf), trabecular volume per tissue volume (BV / TV), and SB plate thickness (SBP.Th).

[0087] Angiography revealed micromembranes perfused into the long bone vessels. Mice in each group were euthanized 10 weeks post-surgery, the thoracic cavity was opened, and blood was expelled through a right atrial incision. The vascular system was flushed by injecting heparinized saline (100 U / mL) into the left ventricle via a needle. Specimens were fixed by perfusing the heart with 10% neutral formalin. After flushing the vessels with heparinized saline to remove excess formalin, the vascular system was labeled by injecting an opaque silicone rubber compound containing lead chromate (MicrofilMV-122, Flow Tech, USA). Overnight incubation at 4°C resulted in contrast agent polymerization in the samples. Mouse knee joints were harvested, immersed in 10% neutral buffered formalin for 4 days to ensure complete tissue fixation, then decalcified, and scanned using a SkyScan 1172. The number (VN) and volume (VV / YV) of the SB vessels within the knee joint were determined by micro-CT scanning and reconstruction.

[0088] 12. Transcriptome sequencing and data analysis

[0089] Ten weeks post-surgery, mice were euthanized, and sclerotherapy (SB) samples were collected from the knee joints. The samples were then divided into three groups: a sham-operated group, a PBS group (PBS injection group), and a SCT-HA group (SCT-HA injection group). In the PBS and SCT-HA groups, 100 μL of PBS and SCT-HA microspheres were injected into the knee joint at weeks 2 and 5 post-surgery, respectively, with three replicates per group. SB samples were rapidly frozen in liquid nitrogen, and total RNA was extracted using Trizol. Libraries were constructed using the NEB Next Ultra II DNA Library Preparation Kit (#E7490). RNA sequencing (RNA-seq) analysis was performed by Shanghai Yuansheng Biopharmaceutical Technology Co., Ltd. (Shanghai, China).

[0090] 13. Statistical Analysis

[0091] All data are recorded as mean ± standard deviation (SD). Student's t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons between more than two groups. P < 0.05 indicates significance.

[0092] II. Experimental Results

[0093] 1. Regulation of the preparation of bone / chondrocyte SCT-HA

[0094] SCT was obtained through an amide reaction between the -COOH group of TPP and the -NH2 group of SC, using EDC / NHS as an activator. An injectable hyaluronic acid hydrogel (AHAMA) modified with aldehyde methacrylate was synthesized. Sodium periodate was used to oxidize and modify hyaluronic acid, increasing the number of aldehyde groups in the material. These aldehyde groups can effectively bind to cartilage tissue, forming good tissue adhesion, and form Schiff base bonds with the -NH2 group of SC. These bonds break under weakly acidic conditions associated with arthritis. The methacrylate-modified hyaluronic acid exhibits inherent photocuring properties, allowing for rapid gelation. The corresponding preparation process is described in [link to preparation procedure]. Figure 1 .

[0095] Compared with traditional hydrogel biomaterials, the hydrogel microspheres prepared in this invention have several advantages, including similarity to the natural extracellular matrix (ECM), high processability, high water content and designability, and large specific surface area, thereby enabling the controlled delivery of various cells, drugs and nanoparticles.

[0096] 2. Physicochemical properties and cartilage permeability characterization of highly permeable micro / nano hydrogel microspheres

[0097] TEM results showed that the SCT was spherical with an average diameter of approximately 5 nm. Figure 2 Part A and Figure 3 The surface charge of SC and SCT is blocked by the Zeta potential. Figure 2 (Part C). SC carries a negative charge with a potential of -16mV, while SCT has a potential of +10.88mV, further indicating that the positively charged TPP adheres to the SC surface. Figure 2 (Part C). It is noteworthy that the covalent attachment of TPP cations to the SC surface did not significantly affect its morphology and size. Figure 3 ).

[0098] XPS was performed to examine the elemental composition of SCT and to confirm the presence of Se and P. Figure 2 (Part E). The UV spectrum of SCT has a characteristic peak at 260 nm. When excited at 350 nm, the photoluminescence results show a strong peak at 440 nm. Figure 2 Part B). At 5.7 ppm and 6.1 ppm, AHAMA's 1 The 1H NMR spectrum showed two newly appearing peaks, corresponding to the protons within the C=C bond of the methacrylate. Figure 2 The average particle size of the hydrogel microspheres is 185 μm. Figure 2 The N-part (part N) can be uniformly dispersed in ultrapure water, and injection with a syringe is sufficient to meet injection requirements. Figure 2 (Part F in the middle). Under conventional fluorescence and laser confocal microscopy, SCT-HA appears green, indicating the presence of a large amount of SCT (green) within the AHAMA microspheres. Figure 2 (GJ part).

[0099] Furthermore, scanning electron microscopy revealed numerous wrinkles on the surface of the hydrogel microspheres, indicating a large specific surface area. Energy dispersive spectroscopy (EDS) results confirmed the presence of O, N, P, and Se on the surface of the hydrogel microspheres, providing further evidence for the successful introduction of SCT. Figure 2 (K portion). SCT-HA microspheres were placed in PBS at 37℃, and the Se ion concentration was detected using inductively coupled plasma atomic emission spectrometry. SCT-HA released a large amount of SCT in the first 3 days, and then the release rate slowed down. The total concentration of SCT released on the 7th day was 51.12 μg / mL ( Figure 2 (Middle L section).

[0100] Furthermore, fresh cartilage explants obtained from porcine joints were used to evaluate the cartilage penetration properties of SCT-HA. We observed that SCT penetrated deep into the cartilage, with strong fluorescence signals throughout the entire cartilage layer, including the SB layer. The positively charged ultrasmall nanostructured SCT could be released from AHAMA hydrogel microspheres and efficiently penetrated the cartilage matrix under the influence of charge. In contrast, CQDs and SCs showed significantly different penetration ranges and capabilities compared to SCT at the same time point. Figure 4 (AC section). Therefore, the micro / nano hydrogel microspheres prepared in this invention can penetrate cartilage more effectively.

[0101] To further investigate the penetrability of SCT, SCT-HA and CQ-HA were injected into the knee joints of mice. Seven days later, the mice were euthanized, and the knee cartilage specimens were examined using a laser confocal microscope. We observed significant green fluorescence in the knee joints treated with SCT-HA, indicating that SCT penetrated into the cartilage. SCT can penetrate the dense surface layer and reach the cells of the deep cartilage zone, exhibiting a greater penetration range and ability than CQ-HA. Figure 4 (Parts D and E). Furthermore, positive green signals were detected in cells of the meniscus, SB, and bone marrow cavity. Figure 4 (Parts D and E).

[0102] 3. Highly permeable micro / nano hydrogel microspheres for protecting chondrocytes.

[0103] To further evaluate the biosafety of AHAMA, CQ-HA, and SCT-HA to chondrocytes, we used the CCK-8 and Live / Dead assays. After 1 and 3 days of culture, the number of live and dead cells was similar in all three groups. Figure 5 (Parts A and C). Similarly, there were no significant differences in chondrocyte proliferation activity and viability among the groups at different time points. Figure 5 Part D).

[0104] Furthermore, to assess the mitochondrial targeting of SCT-HA, the autofluorescence properties of SCT were utilized, and chondrocyte mitochondria were labeled with a red fluorescent dye (Mito-Tracker Deep red FM). LSCM was used to observe the co-localization (yellow) fluorescence signal between SCT and mitochondria, indicating that TPP-modified SCT effectively localized to cellular mitochondria. Figure 6 (Part A). In summary, these results indicate that SCT-HA is biocompatible and that its loaded SCT has an effective mitochondrial targeting effect on chondrocytes.

[0105] Chondrocytes affected by osteoarthritis (OA) produce excessive amounts of reactive oxygen species (ROS) in their mitochondria, which may also be sensitive targets for ROS. In chondrocytes, ROS and oxidative stress can damage mitochondrial DNA (mtDNA), leading to apoptosis, cellular senescence, and matrix degradation, ultimately resulting in OA. Next, we evaluated the reactive oxygen species scavenging capacity of different groups (…). Figure 6 Part B). Compared with the H2O2, AHAMA, and CQ-HA groups, the SCT-HA group showed a significant reduction in intracellular ROS, indicating a significant anti-ROS effect. Figure 6 (Part F of the middle section).

[0106] Furthermore, we used JC-1 to monitor mitochondrial membrane potential (MMP) to reveal early apoptosis of chondrocytes under ROS-induced injury conditions. JC-1 aggregates (red fluorescence) and monomers (green fluorescence) showed normal MMP, early apoptosis, and mitochondrial depolarization, respectively. The MMP levels in articular chondrocytes in the SCT-HA treatment group were similar to those in the control group. Figure 6 Part C). This indicates that SCT-HA treatment can effectively protect the mitochondrial membrane and significantly inhibit the decrease of MMPs and early apoptosis in chondrocytes under oxidative stress. Figure 6 (Part G of China).

[0107] We used a Transwell-based chondrocyte co-culture system to evaluate the effects of SCT-HA on chondrocyte inflammation and degeneration by enhancing mitochondrial antioxidant function. Chondrocytes were exposed to H2O2 to simulate oxidative stress, and qRT-PCR and immunofluorescence techniques were used to assess the protective effects of SCT-HA against inflammatory responses and extracellular matrix catabolism in H2O2-treated chondrocytes. Compared with the control group, after 12 hours of H2O2 treatment, IL-6, ADAMTs, and MMP-13 were significantly upregulated, while Aggrecan and Col2a1 were downregulated. Figure 6 (HK section). However, there were no differences in mRNA levels among the H2O2, AHAMA, and CQ-HA microsphere treatment groups. In addition, the addition of SCT-HA significantly downregulated the mRNA levels of ADAMTs, MMP-13, and IL-6, while gradually upregulating Aggrecan and Col2a1, similar to the results in the control group.

[0108] Col2α1, a major component of the extracellular matrix of chondrocytes, is widely considered a key marker of chondrocyte differentiation. After H2O2 intervention, chondrocytes were co-cultured with AHAMA, CQ-HA, and SCT-HA. Immunofluorescence staining showed that, compared with the H2O2 group, the Col2α1 protein staining level in the H2O2-free control group was significantly increased. Figure 6 Part D shows that H2O2 treatment significantly reduced the level of type II collagen (Col2α1) protein. Meanwhile, compared to the H2O2 group, SCT-HA effectively restored Col2α1 protein levels. Figure 6 (Middle DM section). These findings suggest that SCT-HA has a protective effect on chondrocytes and can alleviate oxidative stress.

[0109] 4. SCT-HA effectively inhibits osteoclast differentiation and function.

[0110] We evaluated the biosafety of AHAMA, CQ-HA, and SCT-HA in RAW246.7 cells using CCK-8 assays and live / dead cell viability assays. After 1 and 3 days of culture, all groups treated with AHAMA, CQ-HA, or SCT-HA showed nearly identical ratios of dead to live cells. Figure 5 (Parts B and C). Furthermore, these groups exhibited similar proliferative activity and cell viability at different time points (…). Figure 5 Part D).

[0111] Furthermore, to assess the mitochondrial targeting of SCT-HA, the mitochondria of RAW246.7 cells were labeled with a red fluorescent dye (Mito-Tracker Deep red FM) utilizing the inherent autofluorescence of SCT. Laser confocal microscopy revealed co-localization fluorescence signals (yellow), indicating co-localization of SCT and RAW246.7 mitochondria. The results demonstrate that SCT is effectively localized in the mitochondria of RAW246.7 cells. Figure 7 Part C).

[0112] We also investigated whether RANKL treatment could induce detectable ROS production in RAW246.7 cells. Intracellular ROS production was assessed using fluorescence microscopy and the cell-permeable oxidation-sensitive dye DCFH-DA. After 30 min of RANKL intervention, the intracellular DCF fluorescence intensity increased (…). Figure 7 Part B of the study indicates that RANKL stimulates ROS production in RAW246.7 cells. There were no differences in intracellular ROS levels between the RANKL group, RANKL+AHAMA group, RANKL+CQ-HA group, SCT-HA group, and Blank group. However, compared to the other three groups, the SCT-HA group showed a significantly lower intracellular ROS level, exhibiting a significant anti-ROS effect. Figure 8 ).

[0113] Furthermore, we established a Transwell co-culture system with mouse bone marrow-derived macrophages (BMMs) to investigate the intervention effect of SCT-HA on osteoclastogenesis. Six days after RANKL induction, mouse BMM cells increased in size, underwent pseudopodia fusion, and showed pink cytoplasmic TRAP staining (…). Figure 7 Part A of the middle group), while the SCT-HA group had fewer TRAP-positive cells ( Figure 7 Part E of the middle section).

[0114] RAW246.7 mouse macrophages, as osteoclast precursor cells, can differentiate into mature osteoclasts upon RANKL stimulation. Therefore, we investigated the effect of SCT-HA on RANKL-induced osteoclast differentiation of RAW264.7 mouse macrophages. Subsequently, qRT-PCR was used to analyze key transcription factors involved in osteoclast differentiation, including activating T cell nuclear factor-1 (NFATc1), platelet-derived growth factor-BB (PDGF-BB) which specifically regulates h-vascularity, and osteoclast-specific genes such as tartrate phosphatase (TRAP) and histone proteinase K (cathepsin K). These factors were significantly downregulated in the SCT-HA group. This indicates that the internal loading of SCT in SCT-HA can inhibit osteoclast differentiation and maturation. Figure 7 (Part F of the middle section).

[0115] 5. Radiological evaluation of SCT-HA treatment for OA

[0116] To investigate the therapeutic effect of SCT-HA on osteoarthritis (OA), we established a mouse OA model through ACL transection and surgery-induced joint biomechanical instability, which was used to assess the biological effects of the treatment. To evaluate changes in the spondylolisthesis (SB) and knee joint, mice were euthanized 10 weeks after OA-inducing surgery and analyzed using micro-CT. Reconstructed images showed that osteophyte volume was reduced in both the AHAMA and CQ-HAA groups compared to the PBS treatment group, but remained significantly higher than in the Sham group. Compared to the Sham group, the SCT-HA group showed improved joint morphology and reduced osteophyte volume, but this difference was not statistically significant. Figure 9 (Parts A and C).

[0117] Two-dimensional images showed that the trabeculae were widened and denser, exhibiting a "fusion" phenomenon. Some trabeculae showed disordered organization and uneven arrangement, resulting in a loss of the network structure. In the Sham and SCT-HA groups, the SB trabeculae were more uniform in shape, regular, and more orderly arranged. Figure 9 (Part B). Compared to the Sham group, the PBS group (SB) had higher bone mineralization density. However, compared to the PBS group, the AHAMA and CQ-HA groups had lower bone mineralization density, but their bone mineralization density was significantly higher than that of the Sham group. Quantitative results showed that the BV / TV values ​​of the PBS and Sham groups were 63.81% and 44.85%, respectively. The BV / TV values ​​of the AHAMA and CQ-HA groups were 55.45% and 55.17%, respectively. In contrast, the BV / TV value of the SCT-HA group was 42.94%, similar to that of the Sham group. Figure 9(Part E). Furthermore, the trends in SB trabecular thickness (SBP.Th) measurements across all five groups were consistent with the BV / TV results. The trabecular type factor (Tb.Pf) was -5.35 in the Sham group and 5.78 in the PBS group. These values ​​indicate increased bone remodeling activity in osteoarthritis SB (BV / TV, SBP.Th). However, the increased Tb.Pf suggests that the bone remodeling process is heterogeneous. There was no difference in Tb.Pf between the SCT-HA group and the Sham group ( Figure 9 (Part F in the middle), indicating that SCT-HA effectively prevents the development of SB remodeling. Furthermore, while OA progression was observed to be very slowed after AHAMA or CQ-HA treatment, the micro / nano hydrogel microspheres of this invention exhibited a very significant therapeutic effect.

[0118] 6. SCT-HA protects cartilage and inhibits abnormal SB remodeling.

[0119] Next, we performed H&E staining, special staining, immunohistochemistry, and fluorescent double labeling on knee joint specimens collected 10 weeks post-surgery to investigate the impact of SCT-HA on SB remodeling and comprehensive articular cartilage treatment. H&E and toluidine blue staining showed that cracks and deformities were common in the PBS group, representing the two most severe forms of articular cartilage erosion. The AHAMA and CQ-HA groups also showed significant erosion, while the SCT-HA group exhibited reduced cartilage surface damage and better morphological integrity, more similar to the Sham group. Figure 10 Part A of the middle section.

[0120] Subsequently, we assessed the severity of OA using the OARSI scale. Compared to the PBS group, the OARSI score decreased by 37.5% in the AHAMA group and by 41.7% in the CQ-HA group. The SCT-HA group showed the largest reduction, with an OARSI score decrease of 85.2%. Figure 10 (Parts E and F). These results indicate that SCT-HA microspheres effectively preserve the thickness of articular cartilage.

[0121] In addition, we assessed the levels of two key proteins involved in the synthesis and catabolism of articular cartilage: Col2α1 and MMP13. Figure 10 (Part B). Compared with the Sham group, the PBS, AHAMA, and CQ-HA treatment groups showed significantly lower Col2α1 levels. Meanwhile, there was no difference between the Sham group and the SCT-HA group. Figure 10 (Middle H portion). Conversely, compared to the Sham group, the PBS, AHAMA, and CQ-HA groups showed significantly elevated levels of MMP13 (a key protease involved in cartilage degradation). However, there was no difference between the SCT-HA and Sham groups. Figure 10(Part G in the middle). These results demonstrate that SCT-HA microspheres can target and attach to the surface of damaged cartilage, releasing SCT through the cartilage matrix and improving the delivery efficiency of biopharmaceuticals to cartilage.

[0122] In addition, the positively charged ultra-small SCT structure enables it to penetrate cartilage and selectively target chondrocytes within the cartilage matrix, effectively protecting chondrocytes from oxidative damage and reducing the generation of in-situ ROS in chondrocytes, thereby protecting chondrocytes and the cartilage matrix.

[0123] Abnormal osteoclast remodeling is a pathological feature of osteoarthritis (OA). Excessive activation of osteoclasts triggers the abnormal bone remodeling process in osteoarthritis. Therefore, inhibiting osteoclast activity can effectively alleviate osteoclast pathology. TRAP staining showed a significant increase in osteoclasts in the PBS group compared to the Sham group, indicating abnormal osteoclast activation in the PBS group under OA conditions. In ACLT mice treated with AHAMA and CQ-HA, high TRAP... + The number of osteoclasts is reduced. However, only SCT-HA will abnormal TRAP. + osteoclasts recovered to the level of the Sham group ( Figure 10 (Parts D and J). Aberrant activation of osteoclasts can lead to abnormal SB metabolism. Therefore, we used a fluorescent double labeling method to assess the MAR of SB. Compared with the Sham group, PBS mice showed increased SB formation (green and red), while AHAMA and CQ-HA treatments reduced the formation of abnormal SB ( Figure 10 (Parts C and I). SCT-HA treatment restored MAR to the levels of the Sham group.

[0124] In conclusion, SCT-HA can comprehensively improve abnormal articular cartilage and SB metabolism, and has a comprehensive and holistic therapeutic effect on arthritis.

[0125] 7. SCT-HA inhibits SB angiogenesis.

[0126] The effect of SCT-HA on stenotic vessel (SB) angiogenesis was investigated. Micro-CT angiography revealed that the number (VN) and volume (VV / YV) of SB vessels in the PBS group were significantly increased compared to the Sham group. Furthermore, compared to the AHAMA and CQ-HAA groups, the SCT-HA group inhibited the increase in both the number (VN) and volume (VV / YV) of vessels in the SB, with values ​​similar to those in the Sham group. Figure 11 (Parts B, E, and F).

[0127] The vascular types inhibited by SCT-HA on CD31 and Emcn in SB mice were analyzed using dual immunofluorescence staining. In PBS-treated mouse SB, the number of CD31hi and Emcnhi vessels was significantly reduced in the AHAMA and CQ-HA groups compared to the PBS group. However, only SCT-HA restored CD31hi and Emcnhi vessels similar to those in the sham-operated control group. Figure 11 (A and D). SCT-HA inhibits CD31hiEmcnhi vessels in OA SB, suppressing the "executors" of abnormal SB remodeling ( Figure 11 Part C).

[0128] 8. Transcriptional profiling of SCT-HA and regulation of SB function

[0129] Ten weeks post-surgery, knee joint specimens were isolated from mice, and RNA sequencing was performed on the osteoclast-related signaling pathways (SBs) in the Sham, PBS, and SCT-HA groups. The results showed that, compared to the Sham group, the PBS group exhibited alterations in signaling pathways related to osteoclast differentiation, such as AMPK, TNF, IL-17, and p53 signaling pathways. Figure 12 Part A). The PBS group showed a significant increase in inflammatory response and osteoclast activation. Figure 12 Parts B and C). Gene set enrichment analysis (GSEA) showed downregulation of osteoclast-related genes, the IL-17 signaling pathway, and the angiogenesis signaling pathway. Figure 12 (Medium GI section). Compared to the PBS group, the SCT-HA group showed altered signaling pathways including the IL-17 signaling pathway, the NF-κB signaling pathway, and pathways related to osteoclasts and inflammatory responses, such as ferroptosis. Figure 12 Part D). Considering osteoclast-related genes, those genes that showed significant changes in the SCT-HA group were investigated, and significant downregulation of most of these genes was observed. Figure 12 (Part E). To further understand the changes in individual signaling pathways compared to the PBS group, GSEA analysis was used to analyze the changes in weighted scores of each signaling pathway. Results showed that all inflammation-related signaling pathways were downregulated, including angiogenesis, IL-17, and osteoclast differentiation signaling pathways (…). Figure 12 (Part F of the middle section).

[0130] Therefore, the alleviation of angiogenesis, inflammation, and osteoclast-related signaling pathways in the SCT-HA group indicates the anti-inflammatory, osteoclast-inhibiting, and angiogenesis functions of this material.

[0131] In summary, SCT-HA hydrogel microspheres anchor to the surface of damaged cartilage through various mechanisms, including dynamic Schiff base reactions involving the formation of imine and hydrogen bonds, and physical interpenetration. Furthermore, in response to a weakly acidic environment, SCT-HA hydrogel microspheres release SCT to penetrate cartilage and reach the sclerosing cartilage (SB), inhibiting oxidative damage to chondrocytes and reducing the differentiation and maturation of monocytes / macrophages into osteoclasts. The effects of SCT on chondrocytes were further validated in a mouse osteoarthritis (OA) model. SCT released after injection of SCT-HA effectively penetrates the SB, significantly inhibiting abnormal cartilage metabolism and SB remodeling, and slowing the progression of osteoarthritis. Injectable SCT-HA microspheres possess excellent SB permeability and represent a novel SB intervention method with promising potential in the treatment of arthritis.

[0132] Comparative Example 1

[0133] By replacing TPP in this invention with the mitochondrial targeting peptide SS-31 (see CN 114042147 B), micro / nano hydrogels were prepared according to the method of Example 1, and their subchondral bone permeability was examined. The results showed that the micro / nano hydrogel prepared with the SS-31 targeting peptide had a fluorescence intensity of only about 1.02 in subchondral bone, while the micro / nano hydrogel prepared in this invention achieved a fluorescence intensity of 10.72 in in vitro experiments and 15.65 in in vivo experiments (see...). Figure 4 (Part E). This indicates that the micro / nano hydrogel prepared using the mitochondrial targeting peptide SS-31 has a much lower permeability to subchondral bone than the micro / nano hydrogel prepared in this invention.

[0134] Comparative Example 2

[0135] The mitochondrial targeting peptide Wyrgrl (see CN 114042147 B) was used to replace TPP in this invention, and micro / nano hydrogels were prepared according to the method of Example 1. The permeability of these hydrogels to subchondral bone was then investigated. The results showed that the micro / nano hydrogels prepared with the Wyrgrl targeting peptide had a fluorescence intensity of only about 1.21 in subchondral bone, while the micro / nano hydrogels prepared in this invention achieved a fluorescence intensity of 10.72 in in vitro experiments and 15.65 in in vivo experiments (see Example 1). Figure 4 (Part E). This indicates that the micro / nano hydrogel prepared using the mitochondrial-targeting peptide Wyrgrl has significantly lower permeability to subchondral bone than the micro / nano hydrogel prepared in this invention.

Claims

1. A method for preparing highly permeable micro / nano hydrogel microspheres, characterized in that, Includes the following steps: (1) Triphenylphosphine was activated by EDC / NHS and then reacted with selenium-doped carbon quantum dots to prepare triphenylphosphine-functionalized selenium-doped carbon quantum dots; (2) Sodium periodate was used to oxidize hyaluronic acid to obtain aldehyde hyaluronic acid, and then the aldehyde hyaluronic acid was mixed with methacrylic anhydride to prepare double bond modified aldehyde hyaluronic acid hydrogel. (3) Using the triphenylphosphine-functionalized selenium-doped carbon quantum dots prepared in step (1), the double bond-modified aldehyde hyaluronic acid hydrogel prepared in step (2), and the photoinitiator as the aqueous phase, and a mixture of Span 80 and paraffin oil as the oil phase, hydrogel microspheres were prepared by microfluidic method. The microspheres were cross-linked under ultraviolet light to obtain highly permeable micro-nano hydrogel microspheres.

2. The preparation method according to claim 1, characterized in that, The weight ratio of triphenylphosphine to selenium-doped carbon quantum dots in step (1) is 1:17.

5.

3. The preparation method according to claim 1, characterized in that, The reaction time described in step (1) is 12 hours.

4. The preparation method according to claim 1, characterized in that, The preparation steps of selenium-doped carbon quantum dots in step (1) are as follows: L-selenocysteine ​​is mixed with water to form an aqueous dispersion, the pH is adjusted to 10 with sodium hydroxide, and then the reaction is carried out at 60°C for 20 hours under nitrogen protection. The resulting product is centrifuged and dialyzed to obtain selenium-doped carbon quantum dots.

5. The preparation method according to claim 1, characterized in that, The preparation steps of the aldehyde-based hyaluronic acid in step (2) are as follows: dissolve hyaluronic acid in water, add sodium periodate solution to it, stir and react at room temperature for 2 hours, and freeze-dry the resulting product after dialysis to obtain aldehyde-based hyaluronic acid.

6. The preparation method according to claim 1, characterized in that, The preparation steps of the double bond modified aldehyde hyaluronic acid hydrogel in step (2) are as follows: dissolve aldehyde hyaluronic acid, add methacrylic anhydride, and then keep the mixture in an ice bath at pH=8-8.5 for 12 h. After the reaction is completed, dialyze and then freeze dry to obtain the double bond modified aldehyde hyaluronic acid hydrogel.

7. The preparation method according to claim 1, characterized in that, The weight ratio of the triphenylphosphine-functionalized selenium-doped carbon quantum dots, the double-bond modified aldehyde-based hyaluronic acid hydrogel, and the photoinitiator in step (3) is 1:5:

1.

8. The preparation method according to claim 1, characterized in that, The flow rate ratio of the water phase to the oil phase in step (3) is 1:

30.

9. A highly permeable micro / nano hydrogel microsphere prepared by the method according to any one of claims 1-8.

10. The use of the highly permeable micro / nano hydrogel microspheres according to claim 9 in the preparation of a medicament for treating osteoarthritis.

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