DSH loaded medicine as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and discloses a DSH loaded medicine as well as a preparation method and application thereof. The DSH loading type medicine is DNA (Deoxyribonucleic Acid) supramolecular hydrogel loaded with 2, 2, 6, 6-tetramethylpiperidine-1-oxide, and the DSH loading type medicine is a DNA supramolecular hydrogel loaded with the 2, 2, 6, 6- Wherein the DNA supramolecular hydrogel is formed by assembling a Y type DNA monomer and an L type DNA monomer. The DSH loaded medicine has good anti-inflammatory and peroxy free radical removing effects, and can achieve the purpose of treating knee osteoarthritis. In addition, the DSH loaded medicine can be used for reflecting the content of peroxy free radicals in knee joints by detecting the contrast ratio of knee joint images through magnetic resonance, and early diagnosis of knee osteoarthritis is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a DSH-loaded drug and a preparation method and application thereof. Background Art

[0002] Knee osteoarthritis (OA) is an age-related, low-grade, chronic inflammatory disease with a high probability of developing symptomatic knee OA. Cartilage degeneration is the main feature of the progression of knee OA, and chronic inflammation and oxidative stress play an important role in cartilage degeneration. Currently, the diagnosis of knee OA is mainly based on clinical manifestations, physical examinations, and imaging examinations. Conventional clinical treatment drugs for knee OA mainly include acetaminophen, nonsteroidal anti-inflammatory drugs, glucocorticoids, hyaluronic acid, glucosamine, chondroitin sulfate, etc. In the late stage of knee OA, joint replacement is required.

[0003] Currently, there are no medications that can slow the progression of knee OA. Acetaminophen, nonsteroidal anti-inflammatory drugs, and glucocorticoids can only alleviate symptoms, while the efficacy of hyaluronic acid, glucosamine, and chondroitin sulfate is uncertain. Currently, there is a lack of early diagnostic methods for knee OA, and by the time diagnosis is made, irreversible structural changes have already occurred in the joint.

[0004] Therefore, there is an urgent need to provide a drug / reagent for early diagnosis and treatment of knee osteoarthritis. Summary of the Invention

[0005] The present invention aims to address at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a DSH-loaded drug, its preparation method, and its use. This DSH-loaded drug has excellent anti-inflammatory and peroxyl radical scavenging effects, and can achieve the purpose of treating knee osteoarthritis. In addition, the DSH-loaded drug can reflect the peroxyl radical content in the knee joint through magnetic resonance imaging (MRI) to detect the contrast of knee joint images, thereby achieving early diagnosis of knee osteoarthritis.

[0006] Specifically, the present invention provides a DSH-loaded drug (TEMPO@DSH), which is a DNA supramolecular hydrogel (DSH) loaded with 2,2,6,6-tetramethylpiperidine-1-oxide (TEMPO); the DNA supramolecular hydrogel is assembled from Y-shaped DNA monomers and L-shaped DNA monomers.

[0007] The mixing of Y- and L-shaped DNA monomers triggers rapid gelation, transforming the mixture from a solution to a gel within 5 seconds, forming a DNA supramolecular hydrogel. DNA supramolecular hydrogel (DSH) is composed of a polymer network of cross-linked hydrophilic DNA biomacromolecules and is biocompatible and biodegradable. As a drug carrier, DSH enables sustained release of loaded drugs, prolonging the duration of drug action and exhibiting anti-inflammatory properties. 2,2,6,6-Tetramethylpiperidine-1-oxide (TEMPO) is a piperidine nitroxide free radical that scavenges peroxyl free radicals. The nitroxide free radical (·NO) contained in TEMPO imparts paramagnetic properties, which can affect the magnetic resonance signal of surrounding water molecules, thereby altering the contrast of magnetic resonance images. This study first discovered the therapeutic effect of TEMPO on knee OA and used intra-articular injection of TEMPO-loaded DSH (TEMPO@DSH) to achieve early diagnosis and treatment of knee OA.

[0008] Preferably, the Y-shaped DNA monomer consists of Y1, Y2 and Y3 chains, and the L-shaped DNA monomer consists of L1 and L2 chains; the nucleotide sequences of the Y1, Y2, Y3, L1 and L2 chains are shown in SEQ ID NO.1-5.

[0009] More preferably, the preparation method of the Y-shaped DNA monomer is: Y1, Y2, and Y3 chains are mixed with Mg 2+ The DNA was mixed with PBS buffer, heated for denaturation and annealing to form Y-shaped DNA monomers.

[0010] More preferably, the preparation method of the L-type DNA monomer is: L1 and L2 chains are mixed with Mg 2+ The DNA was mixed with PBS buffer, heated for denaturation and annealing to form L-type DNA monomers.

[0011] More preferably, the temperature of the heat denaturation is about 95° C., and the temperature of the annealing is about 4° C. The range represented by “about” is ±5%.

[0012] The present invention also provides a method for preparing the above-mentioned DSH-loaded drug, comprising the following steps: mixing 2,2,6,6-tetramethylpiperidine-1-oxide, Y-shaped DNA monomer, L-shaped DNA monomer with Mg-containing 2+ PBS buffer was mixed to prepare DSH-loaded drug.

[0013] The present invention also provides use of the DSH-loaded drug in preparing a drug for treating knee osteoarthritis.

[0014] The present invention also provides the use of the DSH-loaded drug in preparing a reagent for diagnosing knee osteoarthritis.

[0015] Test results show that DSH, as a carrier, can achieve a sustained release of TEMPO and prolong the duration of TEMPO's action. DSH also has anti-inflammatory effects, while TEMPO can scavenge excess peroxyl radicals within OA knee joints. The two can synergistically exert a therapeutic effect on knee OA. Knee OA produces a large number of peroxyl radicals in the early stages of articular cartilage, even before structural changes occur. TEMPO injected into the joint cavity reacts with peroxyl radicals while also being consumed. TEMPO can also alter the contrast of magnetic resonance imaging. Therefore, after injecting TEMPO@DSH into the joint cavity, the contrast of the knee joint image can be detected by magnetic resonance imaging to reflect the content of peroxyl radicals within the knee joint, enabling early diagnosis of knee OA.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) The DSH-loaded drug proposed in this invention uses DNA supramolecular hydrogel (DSH) as a carrier for 2,2,6,6-tetramethylpiperidine-1-oxide (TEMPO), effectively achieving sustained release of TEMPO and prolonging its duration of action within the joint cavity. Simultaneously, the anti-inflammatory effects of DSH and the antioxidant effects of TEMPO combine to create a synergistic therapeutic effect for knee OA.

[0018] (2) In the early stages of knee OA, before structural changes in the articular cartilage occur, a large number of peroxyl radicals are generated. TEMPO injected into the joint cavity reacts with peroxyl radicals and is also consumed. TEMPO can change the contrast of magnetic resonance images. Therefore, after injecting DSH-loaded drugs into the joint cavity, the contrast of the knee joint image can be detected by magnetic resonance imaging to reflect the content of peroxyl radicals in the knee joint, thereby achieving early diagnosis of knee OA.

[0019] (3) Mechanistic studies have shown that the DSH-loaded drug proposed in the present invention can reduce chondrocyte ROS and maintain the homeostasis of chondrocyte synthesis and catabolism, inhibit chondrocyte aging and related pathway activation in vitro, and inhibit chondrocyte aging and related pathway activation in vivo, thereby playing a protective role against osteoarthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the synthesis of Y-shaped and L-shaped DNA monomers and their characterization by polyacrylamide gel electrophoresis;

[0021] Figure 2 The schematic diagram of the synthesis of DSH and the actual picture of the gel state;

[0022] Figure 3 The rheological analysis diagram and scanning electron microscope image of DSH;

[0023] Figure 4 Schematic diagram of the synthesis and fluorescence imaging of TEMPO@DSH;

[0024] Figure 5 Figure 2 shows the uptake of TEMPO by cells after treatment with Cy5.5-labeled TEMPO and TEMPO@DSH for 24 hours (scale bar: 50 μm);

[0025] Figure 6 The activity of chondrocytes was detected by CCK8 assay after treatment with different concentrations of TEMPO for 24 hours;

[0026] Figure 7 The activity of chondrocytes was detected by CCK8 assay after treatment with different concentrations of DSH for 24 hours;

[0027] Figure 8 The activity of chondrocytes was detected by CCK8 assay after treatment with different concentrations of TEMPO@DSH for 24 hours;

[0028] Figure 9 The chondrocyte toxicity was assessed by acetoxymethyl ester / propidium iodide (AM / PI) after 24 h of drug treatment (scale bar: 100 μm);

[0029] Figure 10 Figure 2 shows the detection results of reactive oxygen species (ROS) levels and mitochondrial membrane potential (red / green fluorescence intensity ratio) in chondrocytes of different treatment groups;

[0030] Figure 11 for Figure 10 Quantitative data graph of (n=3);

[0031] Figure 12 Schematic diagram of the antioxidant mechanism of TEMPO@DSH on chondrocytes;

[0032] Figure 13 Figure 2 is the qRT-PCR analysis of MMP13, MMP3, ADAMTS5, COL2A1, ACAN, and SOX9 mRNA levels in chondrocytes of different treatment groups;

[0033] Figure 14 Western blot analysis of MMP13, MMP3, ADAMTS5, COL2A1, ACAN, and SOX9 mRNA in chondrocytes of different treatment groups;

[0034] Figure 15 for Figure 14 Quantitative data graph of (n=3);

[0035] Figure 16 Toluidine blue and alcian blue staining images of chondrocytes in different treatment groups;

[0036] Figure 17 Safranin O staining of human cartilage tissue blocks after different treatments (scale bar: 60 μm);

[0037] Figure 18 This is the heat map of differentially expressed genes in chondrocytes between the DSH@TEMPO treatment group and the OA model group;

[0038] Figure 19 Volcano plot of differentially expressed genes in transcriptome sequencing of chondrocytes from osteoarthritis models treated with TEMPO@DSH and those without.

[0039] Figure 20 for Figure 18 KEGG pathway enrichment analysis of differentially expressed genes;

[0040] Figure 21 Gene set enrichment analysis (GSEA) for cellular senescence and p53 signaling pathways;

[0041] Figure 22 Gene set enrichment analysis for related pathways;

[0042] Figure 23 Western blot analysis and quantitative data of p16 and p21 protein levels in chondrocytes of different treatment groups (n=3);

[0043] Figure 24 for γ-H2AX immunofluorescence staining and SA-β-gal staining;

[0044] Figure 25 for Figure 24 γ-H2AX fluorescence intensity and quantification of SA-β-gal positive cells (n=3);

[0045] Figure 26 Western blot analysis and quantitative data of proteins related to the mTORC signaling pathway (n=3);

[0046] Figure 27 Western blot analysis of proteins related to the NF-κB signaling pathway;

[0047] Figure 28 for Figure 27 Quantitative data of (n=3);

[0048] Figure 29 Schematic diagram of the mechanism of TEMPO@DSH as an MRI contrast agent for the diagnosis of early osteoarthritis;

[0049] Figure 30 This is T1-weighted imaging of the joint cavity using TEMPO@DSH as a contrast agent;

[0050] Figure 31 In vivo imaging of mice at different time points after TEMPO and TEMPO@DSH injection;

[0051] Figure 32 Agarose gel electrophoresis analysis of TEMPO@DSH placed in joint fluid for different times

[0052] Figure 33 Schematic diagram of the design of the drug administration experiment for mice;

[0053] Figure 34 Safranin O / Fast Green staining of the knee joints of mice in different treatment groups (top: low-power image of cartilage, the dotted box is the magnified area below; middle: high-power image; bottom: synovium image; scale bars are 60 μm and 100 μm, respectively);

[0054] Figure 35 OARSI scores for different treatment groups (n=5, expressed as mean ± 95% confidence interval, Mann-Whitney U test);

[0055] Figure 36 The synovitis scores of different treatment groups were calculated (n=5, expressed as mean ± 95% confidence interval, Mann-Whitney U test);

[0056] Figure 37 Immunohistochemical staining of MMP13 and COL2A1 in articular cartilage (scale bar: 60 μm);

[0057] Figure 38 for Figure 37 Quantification of MMP13-positive cells (n=5);

[0058] Figure 39 for Figure 37 Quantification of COL2A1 relative integrated optical density (IOD) (n=5);

[0059] Figure 40 Toluidine blue staining of cartilage tissues in different treatment groups (scale bar: 60 μm);

[0060] Figure 41 Hematoxylin-eosin (H&E) staining of cartilage tissues in different treatment groups (scale bar: 60 μm);

[0061] Figure 42 The histological morphology of the main organs of mice in different treatment groups (scale bar: 100 μm).

[0062] Figure 43Immunohistochemical staining of p16 and p21 in articular cartilage (scale bar: 60 μm);

[0063] Figure 44 for Figure 43 Quantification of p16 and p21-positive cells (n=5);

[0064] Figure 45 Immunohistochemical staining of p-p65 and NF-κB in articular cartilage (scale bar: 60 μm);

[0065] Figure 46 for Figure 45 Quantification of p-p65 and NF-κB positive cells (n=5);

[0066] Figure 47 Immunohistochemical staining of p-s6 and p-mTORC in articular cartilage (scale bar: 60 μm);

[0067] Figure 48 for Figure 47 Quantification of p-s6 and p-mTORC positive cells (n=5). DETAILED DESCRIPTION

[0068] In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the following examples are given for illustration. It should be noted that the following examples do not limit the scope of protection claimed in the present invention. Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial channels or can be obtained by existing known methods.

[0069] Example 1: Preparation of DSH-loaded drug (TEMPO@DSH)

[0070] The preparation method of DSH-loaded drug (TEMPO@DSH) specifically includes the following steps:

[0071] First, partially complementary single-stranded DNA sequences (as shown in Table 1) were designed for the preparation of Y- and L-type DNA monomers. For the Y-type DNA monomer, the Y1, Y2, and Y3 chains (12.5 μL each, 2 mM) were mixed with 62.5 μL PBS (pH 7.2, 2 mM MgCl2), denatured at 95°C for 5 minutes, and then cooled to 4°C for annealing. Similarly, the L1 and L2 chains (18.75 μL each, 2 mM) were mixed with 62.5 μL PBS (pH 7.2, 2 mM MgCl2) and treated in the same manner to form L-type DNA monomers. The schematic diagram of the synthesis of Y-type DNA monomers and L-type DNA monomers is shown in FIG. Figure 1 As shown in A. The molecular weight of single-stranded DNA (Y1, Y2, Y3, L1, L2) and Y / L DNA monomers was analyzed by polyacrylamide gel electrophoresis. Figure 1 As shown in B. Polyacrylamide gel electrophoresis (PAGE) showed that the gel migration rates of Y-DNA and L-DNA were slower than their corresponding complementary ssDNA, and the molecular weight of each ssDNA and DNA monomer was consistent with our design, indicating the successful formation of Y and L monomers.

[0072] Then DSH (DNA supramolecular hydrogel) is assembled, and its synthesis diagram is shown in Figure 2 As shown in Figure A, Y-type monomer (32 μL, 250 μM) and L-type monomer (32 μL, 375 μM) were mixed with 36 μL PBS (pH 7.2, 2 mM MgCl2) to induce rapid gelation (<5 seconds). Figure 2 As shown in Figure B, after a simple mixture of Y and L DNA monomers, DSH was immediately assembled at room temperature, and the mixture transitioned from a solution state to a gel state within 5 seconds. The rheological properties of DSH were measured using an ARES viscometer at a fixed frequency of 1 Hz and a strain of 1%. The oscillatory shear stress experiment of the rheological analysis showed that the shear loss modulus (G″) of the synthesized DSH was about 15 times lower than the shear storage modulus (G′). Figure 3 As shown in Figure 5A, the red curve is the storage modulus G' and the blue curve is the loss modulus G"), which shows obvious viscoelastic state characteristics, indicating the successful synthesis of DSH.

[0073] Finally, TEMPO (36 μL, 25 μg / mL) was premixed with Y-type and L-type DNA monomers, which could achieve gelation within 2 minutes at room temperature, and a sol-to-gel transition occurred simultaneously, thereby preparing TEMPO@DSH. The CAS registration number of TEMPO is 2564-83-2, and the chemical formula is C9H 18 NO.

[0074] In order to evaluate the encapsulation efficiency of TEMPO in DSH, NHS-Cy5.5 labeled TEMPO was mixed with DNA supramolecular hydrogel and fluorescence imaging was performed using an inverted microscope to verify whether the encapsulation was successful. The full scanning electron microscope (SEM) showed that DSH had a three-dimensional porous microstructure (such as Figure 3 TEMPO@DSH (shown in Figure B), indicating that it has good drug loading and release potential, which is considered to be an important advantage of DNA hydrogels. TEMPO can be mixed with Y and L-type DNA monomers to form TEMPO@DSH ( Figure 4 In Figure A), in order to investigate whether TEMPO can be uniformly encapsulated in DSH, TEMPO@DSH was synthesized using NHS-Cy5.5 labeled TEMPO. Figure 4 As shown in B, the fluorescence signal of Cy5.5 is evenly distributed and overlaps with that of DSH, indicating that DSH uniformly encapsulates TEMPO.

[0075] Table 1 Specific sequences of single-stranded DNA

[0076]

[0077] Example 2: Efficacy study of DSH-loaded drugs (TEMPO@DSH)

[0078] 1. Experimental Procedure

[0079] (1) Isolation and culture of chondrocytes

[0080] Knee cartilage tissue was obtained from patients with osteoarthritis undergoing knee replacement surgery in the Department of Orthopedics at Zhujiang Hospital, Southern Medical University. This study was approved by the Institutional Review Board of Zhujiang Hospital (approval number: 2019KY02203), and written informed consent was obtained from all participants before tissue collection.

[0081] Primary chondrocytes were isolated by sequential enzymatic digestion. Briefly, cartilage tissue was washed three times with PBS containing 10% penicillin-streptomycin (100 U / mL and 100 μg / mL, respectively) and then cut into 0.5 mm slices using a sterile scalpel. 3 The minced cartilage was digested with 0.25% trypsin (containing 0.02% EDTA) at 37°C for 30 minutes. Subsequently, the tissue fragments were digested with 0.2% type II collagenase on a thermostatic shaker (37°C, 90 rpm) for 16-24 hours. The isolated chondrocytes were cultured at a concentration of 1×10 4 cells / cm 2 Cells were seeded at a density of 100 μg / mL in a culture flask containing DMEM / F12 medium (10% fetal bovine serum and 1% penicillin-streptomycin). The cells were cultured at 37°C in a 5% CO2 atmosphere, with the medium changed every 48 hours. When the cells reached 80% confluency, they were subcultured using 0.25% trypsin (containing 0.02% EDTA).

[0082] (2) Cellular uptake analysis

[0083] NHS-Cy5-labeled TEMPO and TEMPO@DSH were added to chondrocytes and incubated at 37°C, 5% CO₂ for 24 hours. Following incubation, the cytoskeleton was labeled with β-actin, and the nuclei were stained with DAPI. Cellular uptake was imaged using an inverted fluorescence microscope.

[0084] (3) Cell viability assay

[0085] To evaluate the effect of drugs on cell viability, CCK-8 assay was performed. Human primary chondrocytes were cultured at 5 × 10 3Cells were seeded in 96-well plates at a density of 100 μL and incubated overnight at 37°C, 5% CO2 to allow attachment. After 24 hours of drug treatment, the culture medium was aspirated and each well was gently rinsed with PBS. Subsequently, 100 μL of fresh culture medium containing 10% CCK-8 reagent (Biyuntian Biotechnology) was added and incubated at 37°C, 5% CO2 for 2 hours. Cell viability was assessed by measuring absorbance at 450 nm using a microplate reader (BioTek).

[0086] (4) Cytotoxicity assay

[0087] Chondrocytes were incubated with 500 μl of calcein-AM / propidium iodide (AM / PI) cell dye for 15 minutes. In live cells, calcein-AM is hydrolyzed by intracellular esterases to calcein, which emits bright green fluorescence. In contrast, propidium iodide selectively enters dead cells through damaged cell membranes and intercalates into nuclear DNA, emitting red fluorescence. Fluorescent images were captured using a confocal microscope (Leika) to assess cell viability and death.

[0088] (5) Treatment of chondrocytes

[0089] Chondrocytes were cultured at 1×10 5 Cells were seeded at a density of 100 μg / mL in six-well plates and divided into five experimental groups: (1) PBS (control group), (2) IL-1β, (3) IL-1β+TEMPO, (4) IL-1β+DSH, and (5) IL-1β+TEMPO@DSH. The five groups were pretreated with PBS, PBS, TEMPO (25 μg / mL), DSH (2.5 mM), and TEMPO@DSH (containing 25 μg / mL TEMPO and 2.5 mM DSH) for 24 hours, respectively. Except for the control group, the cells in the other groups were stimulated with 10 ng / mL IL-1β for 24 hours.

[0090] (6) Determination of intracellular reactive oxygen species (ROS)

[0091] The medium from each well was aspirated, and the chondrocytes were gently rinsed three times with sterile phosphate-buffered saline (PBS). The cells were incubated with a 10 μM DCFH-DA working solution for 20 minutes at 37°C in the dark. After incubation, the cells were rinsed twice with PBS to remove excess probe, and then 2 mL of serum-free medium was added to terminate the reaction. Fluorescence images were captured using an inverted fluorescence microscope (Nikon), and fluorescence intensity was quantified using Image J software.

[0092] (7) Mitochondrial membrane potential analysis

[0093] Prepare an appropriate amount of JC-1 (200×) and dilute 50 μL of JC-1 with 8 mL of ultrapure water. Stir to ensure complete dissolution and mixing. Then, dilute the mixture with JC-1 staining buffer (5×) to prepare a JC-1 staining working solution. Wash the chondrocytes with PBS, then add the working solution and incubate at 37°C for 30 minutes. After washing, the chondrocytes were cultured in serum-free medium and images were acquired using an inverted fluorescence microscope (Nikon). The percentage of mitochondrial depolarization was determined by measuring the relative red-green fluorescence ratio.

[0094] (8) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis

[0095] Total RNA was extracted using TRIzol reagent. Purified RNA was reverse transcribed using a high-capacity cDNA reverse transcription kit. mRNA expression levels were quantified using SYBR Premix Ex Taq Master Mix (2×) (Takara), and relative target gene expression was calculated by the comparative Ct (ΔΔCt) method. Validated primer sequences for each gene are listed in Table S2. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control for data normalization.

[0096] (9) Western blot analysis

[0097] Chondrocytes were rinsed with ice-cold phosphate-buffered saline (PBS). Excess PBS was removed from the six-well plate, and radioimmunoprecipitation assay (RIPA) buffer containing 1% phenylmethylsulfonyl fluoride was added to each well to lyse the cells. The collected proteins were quantified and then denatured by boiling at 100°C. The denatured proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked with 5% bovine serum albumin (BSA). The membranes were incubated with the following primary antibodies for 12 h at 4°C: ACAN (1:2000; Abcam, ab315486), COL2A1 (1:2000; Abcam, ab34712), SOX9 (1:1000; Proteintech, Cat#67439-1-1g), MMP13 (1:2000; Abcam, ab39012), MMP3 (1:2000; Abcam, ab52915), ADAMTS5 (1:2000; Abcam, ab41037), p-mTORC (1:2000; Abcam, ab109268), mTOR (1:2000; Abcam, ab134903), p-S6K (1:2000; Cell Signaling Technology, 4858S), S6K (1:3000; Santa Cruz Biotechnology, Inc. Biotechnology, sc-74459), NFκB1 (1:2000; Proteintech, 14220-1-AP), p-p65 (1:1000; Proteintech, 82335-1-RR), p65 (1:1000; Proteintech, 10745-1-AP), GAPDH (1:5000; Proteintech, 60004-1-1g). The membrane was then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 hours at room temperature. Immunoreactivity was detected using an ultrasensitive chemiluminescent reagent. The intensity of each protein band was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or total protein (for phosphorylated proteins).

[0098] Table 2 Primer sequences for qRT-PCR

[0099]

[0100] GAPDH: glyceraldehyde-3-phosphate dehydrogenase; MMP13: matrix metallopeptidase 13; MMP3: matrix metallopeptidase 3; ADAMTS5: A Disintegrin AndMetalloproteinase with Thrombospondin Motifs 5; COL2A1: collagen type II alpha1chain; ACAN: aggrecan; SOX9: SRY-box transcription factor 9.

[0101] (10) Alcian blue and toluidine blue staining

[0102] Chondrocytes were seeded in 12-well plates and treated with the indicated drugs. After drug treatment, cells were cultured in culture medium for 14 days, with medium changes every 3 days. After fixation in 4% paraformaldehyde and three washes with PBS, proteoglycans were assessed by staining with toluidine blue or alcian blue for 15 minutes (Solebol). Excess dye was removed by gentle rinsing with distilled water, and the stained matrix was then observed under a light microscope (Nikon).

[0103] (11) Cartilage tissue block

[0104] Cartilage was isolated from fresh human knee joint specimens and cut into small pieces (5 mm × 5 mm × 2 mm). The pieces were then treated with the appropriate drugs according to the experimental group. The pieces were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin-streptomycin (Sigma-Aldrich). After 10 days of culture, the pieces were harvested for histological examination.

[0105] (12) SA-β-Gal staining

[0106] Chondrocytes were fixed with SA-β-Gal staining solution (Biyuntian) for 15 minutes at room temperature and then washed three times with PBS. The cells were then incubated in SA-β-Gal staining solution at 37°C for 12 hours. SA-β-Gal-positive cells were observed and photographed under an inverted microscope (Leica).

[0107] (13) Immunofluorescence staining

[0108] Chondrocytes were seeded onto confocal microplates. After treatment, cells were washed three times with sterile PBS and fixed with 4% paraformaldehyde. After permeabilization with 0.1% Triton X-100, samples were blocked with 5% bovine serum albumin (MedChemExpress) for 1 hour at room temperature. Primary antibody against γ-H2AX (1:200; Abcam, ab11175) was diluted to completely cover the samples and incubated with shaking at 4°C for 12 hours. After washing with PBS, fluorescent secondary antibody was diluted and applied to the samples, and incubated in the dark at room temperature for 1 hour. After washing again with PBS, cell nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) containing an anti-fluorescence quencher. A coverslip was carefully placed upside down on the mounting medium to seal the sample. Fluorescence signals were observed using an inverted fluorescence microscope (Nikon), and images were analyzed using Image J software.

[0109] (14) RNA sequencing analysis

[0110] After chondrocytes from osteoarthritis models were treated with TEMPO@DSH and PBS, total RNA was extracted using TRIzol reagent (TakaraBio). The samples were sequenced using the Illumina platform of Lianchuan Biotechnology Co., Ltd. Each transcriptome analysis used a 2×10 5 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and gene set enrichment analysis (GSEA) were used to identify pathways associated with therapeutic targets for osteoarthritis chondrocytes.

[0111] (15) OA model

[0112] All experiments were approved by the Animal Care and Use Committee of Zhujiang Hospital of Southern Medical University (LAEC-2022-122). Ten-week-old male C57BL / 6J mice and Sprague-Dawley (SD) rats were used to establish an OA model. Experimental OA was induced by surgical resection of the medial meniscotibial ligament (DMM) of the right knee joint, while a sham control group underwent only joint capsulotomy without disrupting the ligament. After successful modeling, the mice were randomly divided into four groups: PBS, TEMPO, DSH, and TEMPO@DSH. Each group received a 6 μL intra-articular injection of the respective treatment drug: PBS, TEMPO (500 μg / mL), DSH (50 mM), or TEMPO@DSH (containing 500 μg / mL TEMPO and 50 mM DSH). The injection was performed slowly to ensure uniform distribution of the drug within the joint cavity. Eight weeks after surgery, the mice were sacrificed, and knee joint specimens were obtained for histological evaluation.

[0113] (16) Magnetic resonance imaging of rat knee joints

[0114] Magnetic resonance imaging (MRI) examinations were performed using a 7.0T preclinical MRI system (PharmScan 70 / 16US; Bruker Biospin MRI; 200 mm horizontal aperture). The acquisition parameters for the T1-weighted imaging (T1WI) sequence were as follows: echo time (TE) = 7.31 ms, repetition time (TR) = 350 ms, slice thickness = 0.7 mm, and field of view (FOV) = 25.6 mm. Before imaging, rats were anesthetized with isoflurane, and 50 μL of TEMPO@DSH (containing 500 μg / mL TEMPO and 50 mM DSH) was injected into the intra-articular cavity of the knee joint. Images were acquired after injection. Images were processed and pseudo-colored using Paravision software for enhanced visualization. Signal intensity in regions of interest was quantified using Image J software.

[0115] (17) TEMPO retention in the knee joint

[0116] Both TEMPO and TEMPO@DSH were fluorescently labeled via covalent binding of NHS-Cy5.5. An equimolar dose of the labeled compound (6 μL TEMPO, 500 μg / mL) was injected intra-articularly into the knee joint of mice. Following injection, in vivo imaging was performed at pre-determined time points using a multispectral in vivo imaging system (Perkin Elmer) to monitor the distribution and retention of the labeled compound within the joint cavity.

[0117] (18) Stability of TEMPO@DSH in synovial fluid

[0118] TEMPO@DSH was incubated with synovial fluid for 0, 1, 2, 3, 5, 9, and 14 days. The mixture was then diluted with an equal volume of PBS and 5× DNA loading buffer was added. The samples were analyzed by agarose gel electrophoresis to assess the degradation of TEMPO@DSH.

[0119] (19) Histological evaluation

[0120] Mice were euthanized 8 weeks after surgery. Muscle and fascia tissue were removed from the knee joint specimens and fixed in 4% paraformaldehyde for 24 hours. Subsequently, the specimens were decalcified with 4% EDTA for two weeks. After fixation and decalcification, the specimens were embedded in paraffin and sectioned at pre-defined anatomical locations for subsequent histological analysis. Paraffin tissue sections were stained with Safranin O / Fast Green (Solarbio), Toluidine Blue (Solarbio), and Hematoxylin-Eosin (Sigma-Aldrich) to assess cartilage morphology, proteoglycan content, and overall tissue structure. Safranin O / Fast Green Staining: Paraffin sections were stained with 0.2% Safranin O solution for 3 minutes (to visualize proteoglycans) and counterstained with 0.2% Fast Green solution for 1 minute (to label the collagen matrix). Toluidine Blue Staining: Sections were stained with toluidine blue for 1 minute (to assess the distribution of metachromatic proteoglycans). Hematoxylin-eosin Staining: Sections were first stained with hematoxylin for 3 minutes (to label cell nuclei), rinsed with PBS to remove excess dye, and counterstained with eosin (to visualize cytoplasm and extracellular matrix components). Histological evaluation was based on the Osteoarthritis Research Society International (OARSI) scoring system and the Krenn synovitis scoring system.

[0121] (20) Immunohistochemical analysis

[0122] After the sections were baked at 68°C, they were immersed in xylene and gradient ethanol for dewaxing and hydration. Subsequently, antigen retrieval was performed by incubating in diluted sodium citrate-EDTA buffer for 12 hours in a 62°C water bath. Endogenous peroxidase activity was blocked with 3% HO, and the sections were blocked with immunohistochemistry blocking solution for 1 hour. The following primary antibodies were used to incubate the sections at 4°C for 12 hours: MMP-13 antibody (1:200; Abcam, cat. no. ab39012), COL2A1 antibody (1:200; Abcam, cat. no. ab34712), p-mTORC antibody (1:2000; Abcam, cat. no. ab109268), p-S6K antibody (1:2000; CST, cat. no. 4858s), p-p65 antibody (1:200; Proteintech, cat. no. 82 335-1-RR), NFκB1 antibody (1:2000; Proteintech, Cat. No. 14220-1-AP), p16INK4a antibody (1:200; Proteintech, Cat. No. 10883-1-AP), and p21 antibody (1:200; Proteintech, Cat. No. 10355-1-AP). After washing with PBS, HRP-conjugated secondary antibody (1:200; ABclonal, Cat. No. AS014) was added and incubated at room temperature for 1 hour. Images were acquired using an inverted light microscope (Leica).

[0123] (21) Statistical analysis

[0124] Statistical analyses were performed using GraphPad Prism 8.0.2. Error bars represent standard deviations or 95% confidence intervals (see figure legends for details). Intergroup comparisons were performed using the unpaired two-tailed Student's t-test or the Mann-Whitney U test, depending on the data distribution. Statistical significance was defined as P < 0.05.

[0125] 2. Experimental Results and Analysis

[0126] (1) TEMPO@DSH reduces chondrocyte ROS and maintains chondrocyte synthesis and catabolism homeostasis

[0127] Through cell uptake analysis, it was found that DSH encapsulation did not affect the uptake of TEMPO by chondrocytes. Figure 5 As shown ( Figure 5 The blue color represents the cell nucleus stained with DAPI, and the green color represents the cytoskeleton displayed by β-actin immunofluorescence staining). At the same time, an appropriate amount of TEMPO@DSH has no obvious cytotoxicity to chondrocytes (such as Figures 6 to 9 In order to study the antioxidant effect of TEMPO@DSH in chondrocytes, the ROS content and mitochondrial membrane potential of chondrocytes were detected. It was found that TEMPO and DSH could inhibit the production of ROS and the downregulation of mitochondrial membrane potential in OA model chondrocytes to a certain extent, while TEMPO@DSH treatment had the most significant inhibitory effect on oxidative stress and mitochondrial damage in chondrocytes ( Figure 10 、 11 This result suggests that TEMPO@DSH may play a cartilage-protective role by reducing ROS production and mitochondrial damage in chondrocytes ( Figure 12 ). The effects of TEMPO@DSH on chondrocyte decomposition and anabolism were further evaluated. The experimental results showed that TEMPO@DSH treatment of OA model chondrocytes can significantly reduce the expression of their decomposition metabolic markers (MMP-13, MMP-3 and ADAMTS5), while increasing the expression of anabolic markers (COL2A1, Aggrecan and SOX9), and its effect is better than the effect of TEMPO or DSH alone ( Figure 13-15 ). Staining of the extracellular matrix of chondrocytes revealed that TEMPO@DSH treatment significantly increased the levels of glycosaminoglycans in chondrocytes and human cartilage explants ( Figure 16 、 Figure 17 ). The above research results show that TEMPO@DSH may play a cartilage protection role in OA by reducing mitochondrial damage caused by oxidative stress. Figure 11 、 13The data in , 14, and 15 are expressed as mean ± standard deviation, with *P<0.05, ***P<0.001, and ****P<0.0001.

[0128] (2) TEMPO@DSH inhibits chondrocyte senescence and related pathway activation in vitro

[0129] To investigate the mechanism by which TEMPO@DSH protects chondrocytes, transcriptome sequencing was performed using OA in vitro model chondrocytes treated with and without TEMPO@DSH ( Figure 18 、 Figure 19 Gene enrichment analysis found that two groups of genes were significantly enriched in aging-related pathways ( Figure 20 、 Figure 21 、 Figure 22 Further testing of the effect of TEMPO@DSH on aging indicators of OA model chondrocytes revealed that aging of IL-1β model chondrocytes was significantly increased. Treatment with TEMPO or DSH could reduce cell aging to a certain extent, and TEMPO@DSH treatment had the most significant inhibitory effect on cartilage aging ( Figure 23-Figure 25 To explore the potential mechanism by which TEMPO@DSH affects aging, the expression of aging-related signaling pathways mTORC and NF-κB was detected. It was found that TEMPO and DSH could inhibit the upregulation of mTORC and NF-κB pathways induced by IL-1β stimulation, while TEMPO@DSH had the most significant inhibitory effect on mTORC and NF-κB pathways, suggesting that TEMPO@DSH may delay chondrocyte aging by inhibiting mTORC and NF-κB pathways. Figure 26-Figure 28 It should be noted that the above quantitative data are expressed as mean ± standard deviation, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0130] (3) TEMPO@DSH as a contrast agent for early diagnosis of knee OA

[0131] The nitrogen oxide free radical (·NO) contained in TEMPO makes it paramagnetic, and this feature enables it to affect the magnetic resonance signal of the surrounding water molecules, thereby changing the contrast of the magnetic resonance image. When TEMPO reacts with ROS to generate TEMPO-H, its paramagnetism disappears. In the early stages of knee OA, when the articular cartilage has not yet been obviously damaged, a large amount of ROS can be produced. TEMPO injected into the knee OA joint may react with ROS and be consumed, resulting in the magnetic resonance imaging signal intensity it produces being weaker than that of TEMPO injected into a normal knee joint. At this time, early diagnosis of knee OA can be achieved through magnetic resonance imaging ( Figure 29To verify this hypothesis, TEMPO@DSH was injected into the joint cavity of rats with early-stage knee OA model and sham-operated control rats, and T1-weighted magnetic resonance imaging was performed. The imaging results showed that the magnetic resonance signal intensity of the joint cavity of early-stage knee OA model mice was significantly lower than that of sham-operated mice. In addition, the magnetic resonance signal intensity of the knee joints of mice with two weeks of knee OA modeling was significantly lower than that of mice with one week of modeling ( Figure 30 This result suggests that TEMPO@DSH can reflect the level of local ROS in the joint under magnetic resonance imaging, making it a promising contrast agent for early diagnosis of knee OA.

[0132] (4) TEMPO@DSH has a protective effect on osteoarthritis

[0133] DSH has the function of sustained drug release as a drug carrier. To test whether DSH can prolong the retention time of TEMPO in the joint cavity, in vivo imaging was used to detect the retention of TEMPO and TEMPO@DSH in the knee joint of mice. The results showed that DSH did prolong the retention time of TEMPO in the joint cavity to a certain extent, suggesting that DSH as a carrier has a sustained release effect on TEMPO ( Figure 31 ). At the same time, the stability of TEMPO@DSH in human joint fluid was tested, and it was found that TEMPO@DSH could still be detected in the joint fluid after 14 days ( Figure 32 ), suggesting that TEMPO@DSH has good stability in synovial fluid.

[0134] To clarify the potential therapeutic value of TEMPO@DSH for knee OA, mice with knee OA model for one week were injected intra-articularly once a week ( Figure 33 ). After 8 weeks of modeling, it was found that single treatment with TEMPO or DSH could alleviate knee cartilage damage, imbalance between synthesis and catabolism, and synovial inflammation to a certain extent, while TEMPO@DSH could better play an OA protective role compared with TEMPO and DSH ( Figures 34-41 Further investigation into whether TEMPO@DSH has visceral toxicity revealed that no significant morphological abnormalities were observed in the major visceral organs of mice in each group ( Figure 42 These results suggest that TEMPO@DSH has good efficacy and safety as a treatment for knee OA.

[0135] (5) TEMPO@DSH inhibits chondrocyte senescence and related pathway activation in vivo

[0136] To further verify whether TEMPO@DSH protects against OA by inhibiting chondrocyte senescence, the expression of aging marker proteins in the knee cartilage of mice was detected. The results showed that TEMPO and DSH could reduce chondrocyte senescence caused by OA modeling to a certain extent, while TEMPO@DSH could more significantly reduce the production of senescent chondrocytes ( Figure 43-44 ). In addition, the expression of aging-related signaling pathways in mice was detected, and it was found that TEMPO@DSH could also inhibit the activation of NF-κB and mTORC signaling pathways in OA chondrocytes better than TEMPO and DSH ( Figures 45-48 These results suggest that TEMPO@DSH may play a protective role against knee OA by delaying chondrocyte senescence through inhibiting NF-κB and mTORC pathways.

[0137] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A DSH-loaded drug, characterized in that: The DSH-loaded drug is a DNA supramolecular hydrogel loaded with 2,2,6,6-tetramethylpiperidine-1-oxide; the DNA supramolecular hydrogel is assembled from Y-shaped DNA monomers and L-shaped DNA monomers.

2. The DSH-loaded drug according to claim 1, characterized in that The Y-shaped DNA monomer consists of Y1, Y2 and Y3 chains, and the L-shaped DNA monomer consists of L1 and L2 chains; the nucleotide sequences of the Y1, Y2, Y3, L1 and L2 chains are shown in SEQ ID NO.1-5.

3. The DSH-loaded drug according to claim 2, characterized in that The preparation method of the Y-shaped DNA monomer is as follows: Y1, Y2, and Y3 chains are mixed with Mg 2+ The DNA was mixed with PBS buffer, heated for denaturation and annealing to form Y-shaped DNA monomers.

4. The DSH-loaded drug according to claim 2, characterized in that The preparation method of the L-type DNA monomer is as follows: L1 and L2 chains are mixed with Mg 2+ The DNA was mixed with PBS buffer, heated for denaturation and annealing to form L-type DNA monomers.

5. The DSH-loaded drug according to claim 3 or 4, characterized in that The temperature of the heat denaturation is about 95°C, and the temperature of the annealing is about 4°C.

6. The method for preparing the DSH-loaded drug according to any one of claims 1 to 5, characterized in that: The following steps are involved: 2,2,6,6-tetramethylpiperidine-1-oxide, Y-type DNA monomer, L-type DNA monomer were mixed with Mg 2+ PBS buffer was mixed to prepare DSH-loaded drug.

7. Use of the DSH-loaded drug according to any one of claims 1 to 5 in the preparation of a drug for treating knee osteoarthritis.

8. Use of the DSH-loaded drug according to any one of claims 1 to 5 in the preparation of a reagent for diagnosing knee osteoarthritis.

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