Use of an ankrd1 inhibitor for the preparation of a medicament for the treatment of cartilage inflammation

Abstract

The application belongs to the technical field of biomedicine, and specifically discloses application of an Ankrd1 inhibitor in preparation of a drug for treating cartilage inflammation. It is found that the Ankrd1 inhibitor has a key regulating effect on cartilage inflammation. Further, an AAV vector containing the Ankrd1 inhibitor is self-assembled with a liposome and modified, thereby providing a high-efficiency and stable nano delivery system with bone targeting and in-vivo visual tracing functions. The delivery system has good in-vivo safety, can effectively improve mechanical pain sensitivity of a DMM model mouse, relieve subchondral bone sclerosis, reduce joint cartilage damage and reduce OARSI score; while effectively silencing the Ankrd1 gene, the delivery system can also inhibit downstream RUNX2 expression, thereby playing a role in delaying pathological progression of osteoarthritis. The Ankrd1 inhibitor and the delivery system provided by the application can be used for preparing a drug for preventing and treating cartilage inflammation including osteoarthritis, and have a wide clinical conversion prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of Ankrd1 inhibitors in the preparation of drugs for treating cartilage inflammation. Background Technology

[0002] For cartilage inflammation such as arthritis, current clinical treatments mainly include drug therapy and surgery. Commonly used drugs include nonsteroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids; however, drug therapy is often accompanied by significant side effects and adverse reactions. Although surgery can intervene in severe cases, it is highly invasive, has a long postoperative recovery period, carries the risk of infection, and some patients still experience pain recurrence after surgery.

[0003] Gene therapy has become a hot research topic in the biomedical field in recent years, with the key being the development of safe and efficient gene delivery vectors. Adeno-associated virus (AAV) has become one of the most promising viral vectors for gene therapy due to its low immunogenicity, ability to mediate long-term gene expression, and non-pathogenicity. However, AAV viruses suffer from limited tissue targeting and insufficient specificity in vivo distribution, which restricts their application in precision gene therapy.

[0004] Cationic liposomes, as a non-viral vector, possess advantages such as high encapsulation efficiency, low immunogenicity, good safety, and mass production capability. They can encapsulate negatively charged biomolecules through electrostatic interactions and have been widely used in gene transfer. However, traditional cationic liposome / AAV complexes still suffer from drawbacks such as insufficient targeting, difficulties in in vivo tracking, room for improvement in encapsulation efficiency, and poor stability.

[0005] In targeted therapy for cartilage inflammation such as arthritis, traditional vectors cannot accurately target bone marrow mesenchymal stem cells, making it difficult to achieve specific silencing of pathogenic genes and effectively intervene in core pathological processes such as cartilage degeneration and subchondral bone sclerosis. At the same time, they lack visualization and tracking capabilities, making it difficult to accurately monitor the distribution and effects of the vector in vivo, which greatly limits the efficacy and application of gene therapy in the treatment of arthritis. Summary of the Invention

[0006] To address the aforementioned shortcomings, this invention provides an application of Ankrd1 inhibitors in the preparation of drugs for treating cartilage inflammation. This invention creatively discovers that Ankrd1 inhibitors play a crucial regulatory role in cartilage inflammation. Our research confirms that silencing the Ankrd1 gene can alleviate pain caused by osteoarthritis, delay its progression, and alleviate subchondral bone sclerosis caused by DMM surgery. Based on these research results, this invention provides the application of Ankrd1 inhibitors in the preparation of drugs for treating cartilage inflammation, offering a new strategy for clinical intervention in cartilage inflammation.

[0007] To achieve the above-mentioned objectives, the embodiments of the present invention employ the following technical solutions: In a first aspect, the present invention provides the use of Ankrd1 inhibitors in the preparation of medicaments for treating cartilage inflammation.

[0008] This invention creatively discovers that silencing the Ankrd1 gene can effectively alleviate pain caused by osteoarthritis, delay the progression of osteoarthritis, and improve DMM-induced subchondral bone sclerosis. Based on the above findings, this invention provides the application of Ankrd1 inhibitors in the preparation of drugs for treating cartilage inflammation, offering a new strategy for clinical intervention in the treatment of cartilage inflammation.

[0009] As a first limitation of the above application, the cartilage inflammation includes at least one of osteoarthritis or patellar chondromalacia.

[0010] As a further limitation of the first limitation of the above application, the cartilage inflammation is osteoarthritis.

[0011] As a second limitation on the above application, the Ankrd1 inhibitor includes at least one of Ankrd1 protein function inhibitors or Ankrd1 gene expression inhibitors.

[0012] As a further limitation of the second limitation of the above application, the Ankrd1 gene expression inhibitor includes at least one of shRNA targeting the Ankrd1 gene or siRNA targeting the Ankrd1 gene.

[0013] As a further limitation of the second limitation of the above application, the target gene sequence of the shRNA targeting the Ankrd1 gene is shown in SEQ ID No. 1.

[0014] For example, the shRNA targeting the Ankrd1 gene includes a 5′ adapter sequence, a shRNA stem region sense strand sequence, a loop sequence, a shRNA stem region antisense strand sequence, and a 3′ termination sequence.

[0015] In a second aspect, the present invention provides a delivery system for targeting and inhibiting bone marrow mesenchymal stem cells Ankrd1, comprising an adeno-associated virus loaded with an Ankrd1 inhibitor and liposomes with surface-modified EPLQLKM peptides.

[0016] As a first limitation of the above applications, the liposomes are also labeled with CY5.5 fluorescent marker.

[0017] As a second limitation of the above application, the adeno-associated virus loaded with the Ankrd1 inhibitor contains a vector of shRNA targeting the Ankrd1 gene.

[0018] For example, the preparation method of the above-mentioned delivery system for targeting and inhibiting bone marrow mesenchymal stem cells Ankrd1 includes the following steps: S1. Mix 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), cholesterol, CY5.5-labeled distearylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG-CY5.5), and surface-modified EPLQLKM peptide distearylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG-EPLQLKM), and dissolve in a short-chain alcohol to obtain a lipid mixed solution; S2. Adeno-associated virus (referred to as recombinant adeno-associated virus AAV-U6-shRNA-CMV-mScarlet-WPRE) carrying the shRNA interference fragment of the Ankrd1 gene is placed in a buffer solution to obtain a recombinant virus solution. The solution is then added to the lipid mixture and mixed to allow the cationic liposomes and the virus to self-assemble through electrostatic interaction to form a liposome / AAV complex. S3. The liposome / AAV complex is extruded through a liposome extruder to obtain a delivery system that targets and inhibits bone marrow mesenchymal stem cells Ankrd1.

[0019] This invention modifies the liposome-AAV composite carrier to combine stem cell targeting, in vivo visualization and tracing, and efficient and stable gene delivery capabilities. This allows for precise targeting and regulation of key pathogenic genes in osteoarthritis, helping to overcome the technical bottlenecks in gene therapy for osteoarthritis and improve the pathological damage caused by the disease. Specifically, the delivery system for targeting and inhibiting bone marrow mesenchymal stem cells Ankrd1 provided by this invention has the following advantages: (1) Targeted delivery capability: This invention modifies the surface of liposomes with the targeting peptide EPLQLKM peptide, thereby endowing the complex with the ability to actively target specific tissues or cells, and significantly improving the enrichment efficiency of AAV virus at the target site. (2) In vivo tracking function: By incorporating CY5.5 fluorescently labeled DSPE-PEG-CY5.5, the complex is equipped with near-infrared fluorescence imaging capability, which can realize real-time in vivo tracking and monitor the in vivo distribution, metabolic process and targeting efficiency of the carrier. (3) High encapsulation rate: The AAV virus is self-assembled with cationic lipid DOTAP through electrostatic interaction, combined with the homogenization treatment of liposome extruder, and the encapsulation rate of AAV virus can reach more than 65%, which is significantly better than the existing methods; (4) Excellent particle size and potential characteristics: The average particle size of the obtained composite is 99~120nm, PDI is 0.23~0.25, and Zeta potential is 40.7~43.7mV, which shows good dispersion stability and uniformity; (5) Maintaining viral activity: FTIR characterization showed that the structure of the AAV capsid protein was not destroyed during encapsulation, and the vector genome copy of the AAV virus in the complex could reach 4.70 × 10⁻⁶. 11 vg / mL indicates that the AAV particle structure was well preserved; (6) Excellent delivery system stability: The solid dry powder preparation is made by freeze drying and can be stored for 3 months at -20℃ in the dark, which is convenient for transportation and long-term storage.

[0020] For example, the mass ratio of DOTAP, DOPE, cholesterol, DSPE-PEG-CY5.5 and DSPE-PEG-EPLQLKM is 12~16:3~7:2~4:1~2:1~2.

[0021] In this invention, DOTAP is the main cationic lipid that provides positive charge; DOPE assists in lipid membrane fusion; cholesterol enhances vesicle structure stability; and the two PEG lipids are in approximately equal proportions to balance the ratio of fluorescent labeling to targeted peptide modification, thus avoiding fluorescence self-quenching or targeting competition caused by excessive levels of a single modification component.

[0022] For example, the short-chain alcohol is at least one of methanol or ethanol.

[0023] For example, the molecular weight of PEG in DSPE-PEG-CY5.5 and DSPE-PEG-EPLQLKM is 2000 Da.

[0024] For example, the volume ratio of the recombinant virus solution to the lipid mixture is 2.5:1 to 4:1.

[0025] For example, the buffer solution is a 50 mM citrate buffer adjusted to pH 4 for dispersing recombinant adeno-associated virus.

[0026] A weakly acidic environment reduces the negative electrostatic repulsion of the AAV capsid, enhances the electrostatic adsorption of cationic lipids, and significantly improves the viral encapsulation efficiency; too high a pH will weaken electrostatic binding, and too low a pH will cause viral protein denaturation and inactivation.

[0027] For example, the recombinant virus solution is slowly dripped into the lipid mixture, mixed well, and left to stand in the dark for 18 to 25 minutes.

[0028] 18-25 minutes is sufficient for lipids and recombinant viruses to complete electrostatic assembly; avoiding light is to prevent photodegradation of the CY5.5 fluorescent group; selective dropwise addition is to avoid excessive local concentration that could cause particle aggregation.

[0029] For example, the adeno-associated virus titer in the recombinant viral solution is 1×10⁻⁶. 10 ~1×1012 vg / mL.

[0030] For example, during the extrusion process, a pre-extrusion treatment is first performed using 500nm and 200nm filter membranes, followed by repeated extrusion 8 to 12 times using a polycarbonate membrane with a pore size of 80-120nm.

[0031] The hydrodynamic particle size of liposomes is controlled within the range of 80nm-120nm. This size is suitable for in vivo vascular circulation and is not easily intercepted by capillaries. If the pore size of the filter membrane is too large, the particles will be uneven, and if it is too small, it will easily clog the membrane pores and reduce the preparation efficiency.

[0032] For example, after extrusion, the liposome complex is purified through a 30 nm dialysis membrane. This dialysis membrane can retain liposome complexes at around 100 nm, removing impurities such as free lipids, free recombinant AAV, and small molecule ethanol, achieving efficient purification.

[0033] For example, the dialysate prepared above is freeze-dried to obtain a delivery system that targets and inhibits bone marrow mesenchymal stem cells Ankrd1.

[0034] Compared with the prior art, the present invention has the following advantages and positive effects: This invention creatively discovers that Ankrd1 inhibitors play a key regulatory role in cartilage inflammation. Further, by loading Ankrd1 inhibitors onto AAV and then self-assembling them with liposomes to improve their stability, the liposome-AAV composite carrier is modified to possess the capabilities of targeting bone marrow mesenchymal stem cells, in vivo visualization and tracing, and efficient and stable gene delivery, thereby achieving the goal of precisely targeting and regulating key pathogenic genes in osteoarthritis.

[0035] Experimental results demonstrate that the delivery system for targeting and inhibiting bone marrow mesenchymal stem cells (Ankrd1) provided by this invention can effectively alleviate pain in osteoarthritis model mice, slow the progression of arthritis, reduce subchondral bone sclerosis caused by DMM surgery, reduce articular cartilage damage, and lower OARSI scores. This targeted intervention strategy provides a novel molecular mechanism and therapeutic approach for clinical intervention in cartilage disorders, including osteoarthritis, and possesses significant application value and broad clinical translational prospects in the preparation of targeted drugs for the prevention and treatment of cartilage disorders. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1This is a schematic diagram of the plasmid pAAV-U6-spgRNAv2.0-CMV-mScarlet-WPRE in Example 1 of the present invention; Figure 2 This is a schematic diagram of the plasmid map of the recombinant plasmid pAAV-U6-shRNA-CMV-mScarlet-WPRE in Example 1 of the present invention; Figure 3 These are transmission electron microscope images of the delivery system CY5.5-Lipo@AAV(Ankrd1)-EPLQLKM at different fields of view and magnification in Embodiment 2 of the present invention; Figure 4 Transmission electron microscope images of the delivery system CY5.5-Lipo@AAV(Ankrd1) in Comparative Example 1 of this invention at different fields of view and magnification; Figure 5 The following are the ultraviolet absorption spectra of different groups in Example 1 of the present invention; Figure 6 The fluorescence spectra of different groups in Example 1 of the present invention are shown. Figure 7 The infrared spectra of different groups in Example 1 of the present invention are shown. Figure 8 The results of in vivo imaging of mice at different time points after drug administration using the delivery systems prepared in Example 2 and Comparative Example 1 in Example 2 of the present invention; Figure 9 The images show the results of in vitro tissue imaging of mice in each group 24 hours after drug administration in Example 2 of the present invention. Figure 10 This is the statistical result of fluorescence intensity in different isolated tissues of mice in each group 24 hours after drug administration in Example 2 of the present invention; Figure 11 The statistical results of the relative expression levels of Ankrd1 mRNA in different groups in Example 3 of the present invention; Figure 12 This is a flowchart of the animal experiment in Example 4 of the present invention; Figure 13 The results of measuring the body weight and mechanical withdrawal threshold of mice in each group in Example 4 of the present invention; Figure 14 The results of the detection of tibial plateau bone microstructure, bone mass and skeletal morphology-related quantitative indicators in each group of mice in Example 4 of the present invention; Figure 15 These are representative images of mice stained with safranin and fast green in each group in Example 4 of the present invention; Figure 16 The OARSI scores of mice in each group in Example 4 of the present invention are statistical results. Figure 17The images and statistical results of Ankrd1 immunohistochemical staining in each group of mice in Example 4 of the present invention are shown below. Figure 18 The images and statistical results of Runx2 immunohistochemical staining in each group of mice in Example 4 of the present invention are representative images. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0039] Example 1 This invention commissioned Shanghai Heyuan Biotechnology (Group) Co., Ltd. to construct a recombinant AAV virus containing an Ankrd1 shRNA interference fragment. An shRNA interference fragment targeting Mouse Ankrd1 was designed, and this interference fragment was constructed into a viral vector using molecular biology techniques. This vector can interfere with Ankrd1 gene expression. Besides being able to directly and transiently transfect cells for Ankrd1 gene interference, this vector can also interfere with Ankrd1 gene expression at the animal level.

[0040] shRNA, as a method of RNA interference, utilizes the promoters of human and mouse RNA polymerase III (polIII), such as U6 and H1, to transcribe short interfering RNA (shRNA) DNA molecules. The intracellularly transcribed shRNA is processed and incorporated into the RNA-induced silencing complex (RISC), which guides nucleases to degrade the target RNA. Viral vectors are used for shRNA expression, offering the advantage of directly and efficiently infecting cells for gene silencing studies, avoiding the inconveniences caused by low plasmid transfection efficiency, and providing more stable transfection results.

[0041] The method for constructing recombinant adeno-associated virus provided by this invention is as follows: shRNA targets were designed based on the transcript of the Mouse Ankrd1 gene, and primers were synthesized. Single-stranded primers were annealed to double-stranded oligo sequences and ligated into a double-digested linearized RNA interference vector, replacing the original ccdB virulence gene. Transformants were screened by colony PCR, and positive clones were sequenced for verification. For clones verified by sequencing, high-purity plasmids were extracted, packaged, and recombinant adeno-associated virus (AAV) was obtained. The specific method for constructing recombinant AAV containing the Ankrd1 gene shRNA interference fragment is as follows: 1. Interference target design and primer synthesis Based on the general principles and experience of shRNA design, shRNA targets were designed and primer synthesis was arranged. The target sequences of the shRNAs in this invention are shown in Table 1. Detailed information on the functional elements of the DNA primers used to construct the shRNA adeno-associated virus vector is shown in Table 2. By sequentially assembling the functional elements corresponding to the primers with the same number in Table 2 along the 5' to 3' direction, complete DNA primer fragments can be obtained. The full-length sequences of each complete primer are shown in Table 3.

[0042] Table 1 Table 2 The forward and reverse primer sequences used in constructing the shRNA adeno-associated virus vector in this invention are shown in Table 2. Each primer, from the 5′ end to the 3′ end, contains a 5′ adapter sequence, a shRNA stem region sense strand sequence, a loop sequence, a shRNA stem region antisense strand sequence, and a 3′ termination sequence. Among them, primers Y47090-F and Y47090-R are shRNA primers targeting the Ankrd1 gene; primers Y14305-F and Y14305-R are shRNA primers for the negative control NC2; the 3′ sequences of Y47090-F and Y14305-F contain the U6 promoter transcription termination signal TTTTTTTg.

[0043] Table 3 2. Primer annealing forms double-stranded fragments with sticky ends. The synthesized oligos were dissolved in oligo annealing buffer to a concentration of 20 μM, and 30 μl of each complementary single strand was mixed. The oligo mixture was then heated in a water bath at 95 °C for 5 min, and then allowed to cool naturally to room temperature with the lid off to form double-stranded oligo fragments. 1 μl was used for subsequent ligation reactions, and the remainder was stored at -20 °C.

[0044] 3. Preparation of linearized expression vectors The expression vector was digested with restriction endonucleases. The digestion reaction system consisted of 2 μL plasmid, 5 μL 10× reaction buffer, 1 μL each of restriction endonucleases, and deionized water to a final volume of 50 μL. The mixture was incubated in a water bath at 37°C for at least 2 hours. The digestion products were analyzed by agarose gel electrophoresis to assess the digestion efficiency. The target vector band was excised from the agarose gel and recovered using the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver. 3.0.

[0045] The plasmid is pAAV-U6-shRNA / spgRNA v2.0-CMV-mScarlet-WPRE, and its plasmid map is shown in the figure below. Figure 1 As shown; the restriction endonucleases are AgeI and NheI.

[0046] 4. Interference fragments are ligated into the expression vector. After preparing the ligation reaction system according to Table 4, the ligation was carried out overnight at 16°C to obtain the recombinant plasmid. It should be noted that the added annealed double-stranded oligos were synthesized in steps 2 and 3 above. The negative control group used double-stranded oligos prepared with primers Y14305-F / R, and the positive control group used double-stranded oligos prepared with primers Y47090-F / R.

[0047] Table 4 5. Transformation and Validation of Positive Plasmids The recombinant plasmid was transformed into DH5α competent cells, cultured, and the transformants grown on the plates were picked and resuspended in 10 µL of LB medium. 1 µL of this medium was used as a template for colony PCR identification. The PCR reaction system is shown in Table 5. The PCR reaction program consisted of 30 cycles of 94℃ for 30 s, 55℃ for 30 s, and 72℃ for 1 min.

[0048] Table 5 6. Positive clones are sent for sequencing. Positive clones obtained from colony identification were sent to a sequencing company for sequencing verification. The negative control group ultimately yielded recombinant plasmid Y14305; the experimental group containing interfering RNA (shRNA) ultimately yielded recombinant plasmid Y47090. Sequencing results were compared and analyzed.

[0049] Sequencing revealed the nucleotide sequence of the expression cassette for recombinant plasmid Y47090 as shown in SEQ ID No. 7. The nucleotide sequence of the expression cassette for recombinant plasmid Y14305 is shown in SEQ ID No. 8. Sequence analysis showed that the nucleotide sequence of recombinant plasmid Y47090 contains the nucleotide sequence shown in SEQ ID No. 3, which can be used for subsequent silencing of the Ankrd1 gene. The nucleotide sequence of recombinant plasmid Y14305 contains the nucleotide sequence shown in SEQ ID No. 5, and includes an irrelevant NC control plasmid.

[0050] The recombinant plasmid containing the Ankrd1 gene interference fragment is designated pAAV-U6-shRNA-CMV-mScarlet-WPRE, also known as recombinant plasmid Y47090; the control recombinant plasmid is designated pAAV-U6-NC-CMV-mScarlet-WPRE, also known as recombinant plasmid Y14305. A schematic diagram of the plasmid map for recombinant plasmid pAAV-U6-shRNA-CMV-mScarlet-WPRE is shown below. Figure 2 As shown.

[0051] 7. Plasmid extraction Once the positive clones were confirmed to be correct by sequencing, plasmid extraction was performed to obtain the positive plasmids.

[0052] The nucleotide sequence of the expression cassette of recombinant plasmid Y47090 is shown in SEQ ID No. 7, and the nucleotide sequence of the expression cassette of recombinant plasmid Y14305 is shown in SEQ ID No. 8.

[0053] 8. Packaging of recombinant AAV virus The recombinant plasmids Y47090 and Y14305 were co-transfected into 293T cells with helper and helper plasmids, respectively. After co-culturing for 72 hours, the cells were removed from the incubator, and the supernatant was collected for later use. The cell pellet was lysed by repeated freeze-thaw cycles, and the supernatant was collected by centrifugation. Finally, the supernatant was combined with the collected supernatant for later use. The virus was concentrated and coagulated overnight with a coagulation solution to obtain the viral fluid.

[0054] Among them, the recombinant adeno-associated virus AAV-U6-shRNA(Ankrd1)-CMV-mScarlet-WPRE containing the Ankrd1 shRNA interference fragment in the viral fluid obtained by transfection with recombinant plasmid Y47090 is referred to as viral fluid I. The recombinant adeno-associated virus AAV-U6-NC-CMV-mScarlet-WPRE containing the negative control fragment of shRNA in the viral fluid prepared by transfection with recombinant plasmid Y14305 is designated as viral fluid I.

[0055] The adeno-associated virus titer in both of the above-mentioned viral solutions was 1×10⁻⁶. 12 vg / mL.

[0056] Example 2 This invention provides a method for preparing a delivery system that targets and inhibits bone marrow mesenchymal stem cells (Ankrd1) using viral fluid I containing recombinant adeno-associated virus (AAV-U6-shRNA-CMV-mScarlet-WPRE prepared in Example 1 as a raw material. The preparation method includes the following steps: S1. Mix 14mg DOTAP, 5mg DOPE, 3mg cholesterol, 1.5mg DSPE-PEG-CY5.5, and 1.5mg DSPE-PEG-EPLQLKM and dissolve them in 1mL of anhydrous ethanol. Then, vortex thoroughly in a round-bottom flask to obtain the lipid ethanol phase. The preparation method of DSPE-PEG-CY5.5 is as follows: DSPE-PEG-CY5.5 was synthesized by directly coupling Cy5.5-NHS ester with commercially available DSPE-PEG-NH2. DSPE–PEG–NH2 and Cy5.5–NHS were dissolved separately in anhydrous DMSO, then mixed at a molar ratio of 1:1, and a small amount of TEA was added. The mixture was stirred overnight at 22°C in the dark. The solution was purified by gradient dialysis using a dialysis bag with a molecular cutoff of 3500 Da, concentrated, and freeze-dried to obtain powdered DSPE-PEG-CY5.5.

[0057] The preparation method of DSPE-PEG-EPLQLKM is as follows: DSPE-PEG-NHS and EPLQLKM peptide with Boc protection only on the Lys side chain amino group were dissolved separately in anhydrous DMSO. Then, the Boc-protected peptide (1.1 times the molar amount of DSPE-PEG-NHS) was slowly added dropwise to the DSPE-PEG-NHS solution. A trace amount of triethylamine (TEA) was added to adjust the system to weakly alkaline, and the mixture was stirred at room temperature in the dark for 6 hours. After the reaction was complete, the Boc protecting group was removed using a trifluoroacetic acid (TFA) system. The mixture was then purified by gradient dialysis using a dialysis bag with a molecular cutoff of 3500 Da, concentrated, and freeze-dried to obtain powdered DSPE-PEG-EPLQLKM.

[0058] S2. Adjust the pH to 4 with 50 mM citrate buffer, add the virus solution I prepared in Example 1, and obtain the recombinant virus solution; the adeno-associated virus titer in the recombinant virus solution was determined to be 1 × 10⁻⁶. 11 vg / mL; Take 3 mL of recombinant virus solution and slowly add it to 1 mL of the lipid ethanol phase obtained above. Mix well and let stand in the dark for 20 min to obtain liposome / AAV complex. S3. The liposome / AAV complex was first pretreated by extrusion using 500nm and 200nm filter membranes, and then extruded 10 times using a polycarbonate membrane with a pore size of 100nm. After that, it was dialyzed using a nanodialysis device (dialysis membrane: polycarbonate membrane, pore size 30nm). The dialysate was then freeze-dried to obtain a delivery system that targets and inhibits bone marrow mesenchymal stem cells Ankrd1. The obtained delivery system is denoted as CY5.5-Lipo@AAV(Ankrd1)-EPLQLKM.

[0059] The delivery system prepared in this embodiment was characterized, and its transmission electron microscope images at different fields of view and magnifications are shown below. Figure 3 As shown in the figure. Simultaneously, particle size, PDI, Zeta potential, viral titer, and encapsulation efficiency were measured. The results are as follows: particle size 111.44±10.35 nm, PDI 0.232±0.027, Zeta potential 45.20±0.93 mV, viral titer 4.70×10⁻⁶. 11 vg / mL, encapsulation efficiency 66.2%.

[0060] Example 3 This invention provides a method for preparing a delivery system that targets and inhibits bone marrow mesenchymal stem cells (Ankrd1) using viral fluid I containing recombinant adeno-associated virus (AAV-U6-shRNA-CMV-mScarlet-WPRE prepared in Example 1 as a raw material. The preparation method includes the following steps: S1. Mix 12mg DOTAP, 3mg DOPE, 2.5mg cholesterol, 1mg DSPE-PEG-CY5.5, and 1mg DSPE-PEG-EPLQLKM and dissolve them in 1mL of anhydrous ethanol. Then, vortex thoroughly in a round-bottom flask to obtain the lipid ethanol phase. The preparation methods of DSPE-PEG-CY5.5 and DSPE-PEG-EPLQLKM are the same as those in Example 1; S2. Adjust the pH to 4 with 50 mM citrate buffer, add the virus solution I prepared in Example 1, and obtain an adeno-associated virus titer of 8.3 × 10⁻⁶. 10 Recombinant virus solution at a concentration of vg / mL; Take 3.6 mL of recombinant virus solution and slowly add it to 1 mL of the lipid ethanol phase obtained above. Mix well and let stand in the dark for 20 min to obtain liposome / AAV complex. S3. The liposome / AAV complex was first pretreated by extrusion using 500nm and 200nm filter membranes, then extruded 12 times using a polycarbonate membrane with a pore size of 100nm, and then dialyzed using a nanodialysis device (dialysis membrane: polycarbonate membrane, pore size 30nm). The dialysate was then freeze-dried to obtain a delivery system that targets and inhibits bone marrow mesenchymal stem cells Ankrd1.

[0061] Example 4 This invention provides a method for preparing a delivery system that targets and inhibits bone marrow mesenchymal stem cells (Ankrd1) using viral fluid I containing recombinant adeno-associated virus (AAV-U6-shRNA-CMV-mScarlet-WPRE prepared in Example 1 as a raw material. The preparation method includes the following steps: S1. Take 16mg DOTAP, 6mg DOPE, 4mg cholesterol, 2mg DSPE-PEG-CY5.5, and 2mg DSPE-PEG-EPLQLKM and dissolve them in 1mL of anhydrous ethanol. Then, vortex the mixture thoroughly in a round-bottom flask to obtain the lipid ethanol phase. The preparation methods of DSPE-PEG-CY5.5 and DSPE-PEG-EPLQLKM are the same as those in Example 1; S2. Adjust the pH to 4 with 50 mM citrate buffer, add the virus solution I prepared in Example 1, and obtain an adeno-associated virus titer of 1.2 × 10⁻⁶. 11 Recombinant virus solution at a concentration of vg / mL; Take 2.5 mL of recombinant virus solution and slowly add it to 1 mL of the lipid ethanol phase obtained above. Mix well and let stand in the dark for 20 min to obtain liposome / AAV complex. S3. The liposome / AAV complex was first pretreated by extrusion using 500nm and 200nm filter membranes, then extruded 8 times using a polycarbonate membrane with a pore size of 100nm, and then dialyzed using a nanodialysis device (dialysis membrane: polycarbonate membrane, pore size 30nm). The dialysate was then freeze-dried to obtain a delivery system that targets and inhibits bone marrow mesenchymal stem cells Ankrd1.

[0062] Comparative Example 1 The present invention provides a comparative example of a method for preparing a delivery system, which is basically the same as that in Example 2, except that: DSPE-PEG-EPLQLKM in step S1 is replaced with an equal mass of DSPE-PEG, and the remaining steps and parameters are the same as in Example 2. The resulting delivery system is denoted as CY5.5-Lipo@AAV(Ankrd1).

[0063] The delivery system prepared in this comparative example was characterized, and its transmission electron microscope images at different fields of view and magnifications are shown below. Figure 4 As shown in the figure. Simultaneously, the particle size, PDI, Zeta potential, viral titer, and encapsulation efficiency were measured. The results are as follows: particle size 109.42±8.53 nm, PDI 0.238±0.013, Zeta potential 42.41±1.49 mV, viral titer 4.31×10⁻⁶. 11 vg / mL, encapsulation efficiency 60.75%.

[0064] Comparative Example 2 The present invention provides a comparative example of a method for preparing a delivery system, which is basically the same as that in Example 2, except that the “virus liquid I” in step S2 is replaced with “virus liquid pair I” containing an equal mass of control virus vector. The remaining steps and parameters are the same as in Example 2. The final delivery system is denoted as CY5.5-Lipo@AAV(NC)-EPLQLKM.

[0065] Example of effect 1 To investigate whether CY5.5, after encapsulation in liposomes, can generate a normal fluorescence signal to meet the requirements of in vivo imaging applications, and whether the EPLQLKM peptide was successfully modified into the delivery system, the following experiments were conducted: 1. Ultraviolet spectroscopy UV-Vis-NIR absorption scanning was performed on DSPE-PEG-CY5.5 and the different delivery systems prepared in Example 2 and Comparative Example 1. The UV absorption spectra are shown below. Figure 5 As shown.

[0066] Depend on Figure 5 It can be seen that the maximum characteristic absorption peak of CY5.5 in free DSPE-PEG-CY5.5 is located at 670 nm. The delivery systems prepared in Example 2 and Comparative Example 1 both retain the characteristic absorption signal corresponding to CY5.5, with the maximum absorption wavelength red-shifted to 690 nm. This shift in absorption peak position is presumably due to the change in the microenvironment of CY5.5 after encapsulation by liposomes, proving that the encapsulated CY5.5 still possesses the spectral basis for fluorescence excitation.

[0067] 2. Fluorescence spectrum The fluorescence emission spectra of the delivery systems prepared in Example 2 and Comparative Example 1 were measured, and their fluorescence spectra are shown below. Figure 6 As shown.

[0068] Depend on Figure 6 It can be seen that the fluorescence emission peak positions of the delivery systems prepared in Example 2 and Comparative Example 1 are basically the same, and the fluorescence signal intensity does not decrease significantly, indicating that liposome encapsulation and surface peptide modification do not damage the fluorescence emission performance of CY5.5.

[0069] 3. Infrared Spectroscopy Using DSPE-PEG-EPLQLKM prepared in Example 2 as a baseline control, Fourier transform infrared spectroscopy (FTIR) was used to characterize the different delivery systems prepared in Example 2 and Comparative Example 1. Their infrared spectra are shown below. Figure 7 As shown.

[0070] Depend on Figure 7 It can be seen that in the infrared spectrum of the DSPE-PEG-EPLQLKMD sample, at 3300 cm⁻¹... -1(NH / OH stretching vibration), 2930cm -1 / 2850cm -1 (CH stretching vibration), 1650cm -1 (Amide I band, C=O stretching vibration), 1540cm -1 (Amide II band, NH bending vibration), 1465cm -1 (CH bending vibration) and 1100cm -1 Both (COC ether bond stretching vibrations) exhibited characteristic strong absorption peaks, consistent with the characteristics of peptides and PEG segments. In the infrared spectrum of the CY5.5-Lipo@AAV(Ankrd1)-EPLQLKM group of the CY5.5-labeled delivery system prepared in Example 2, the peak at 3400 cm⁻¹ was... -1 The broad peak enhancement at 1740 cm⁻¹ is attributed to the increased hydroxyl and amino groups introduced by liposomes and AAV. -1 A strong peak appears at 1650 cm⁻¹, corresponding to the stretching vibration of the ester carbonyl group (C=O) in liposomes. -1 and 1540cm -1 The amide absorption at 1170 cm⁻¹ is significantly reduced, indicating that some vibrational signals of the AAV capsid protein are shielded by the lipid layer. -1 and 1060cm -1 The absorption peaks originated from the lipid components and the nucleic acid phosphate backbone. After surface modification with EPLQLKMD peptides, the overall peak shape of the material was similar to that before modification, indicating that the liposome and AAV structures were not damaged. Compared with the unmodified peaks, the peak shape at 1170 cm⁻¹ was significantly higher. -1 and 1060cm -1 The peak disappeared, replaced by 1157cm. -1 and 1100cm -1 The weak peak reflects the change in the surface chemical environment after peptide modification, which may be related to the overlap and intensity redistribution of CO and CN vibrational modes of PEG segments and peptide side chains.

[0071] In summary, in the delivery system for targeting and inhibiting bone marrow mesenchymal stem cells Ankrd1 provided by this invention, CY5.5 can still emit fluorescence normally after being encapsulated in liposomes, making it suitable for in vivo imaging; infrared spectroscopy results prove that the EPLQLKMD peptide was successfully modified into the delivery system.

[0072] Example 2 This invention uses BALB / c Nu athymic nude mice to conduct in vivo imaging experiments. The delivery system was prepared as a working solution of 2 mg / mL, and a single tail vein injection was administered to nude mice at a dose of 100 μL. In vivo fluorescence images of the small animals were acquired at 1 h, 2 h, 4 h, 8 h, and 24 h after injection. The mice were dissected 24 h after administration for in vitro tissue fluorescence imaging observation.

[0073] The in vivo imaging results of mice at different time points after drug administration using the delivery system prepared in Example 2 and Comparative Example 1 are as follows: Figure 8 As shown in the figure. Imaging results of isolated tissues from mice in each group 24 hours after drug administration are shown in the figure. Figure 9 As shown in the figure. The statistical results of fluorescence intensity in different isolated tissues of mice in each group 24 hours after drug administration are as follows. Figure 10 As shown.

[0074] Results 7-9 show that the fluorescence signals of both the delivery systems prepared in Example 2 and Comparative Example 1, after being introduced into the body via the tail vein, gradually decreased over time, indicating that both systems can be metabolically cleared in mice. In vitro tissue analysis after dissection showed that both delivery systems could be distributed in various tissues and organs, mainly accumulating in the liver, kidney, and femur; the fluorescence signals of both groups of samples in the heart, spleen, and lung tissues remained at low levels, with no significant differences between groups. Compared with Comparative Example 1, the total fluorescence count in the kidney and femur tissues of Example 2 was significantly increased. As the femur is a representative bone tissue, this quantitative result indicates that modification with the EPLQLKM peptide effectively promotes the accumulation of the delivery system in bone tissue, further confirming the bone-targeting capability of this delivery system; the increased fluorescence signal in the kidney also suggests that peptide modification alters the organ distribution behavior of the carrier in vivo.

[0075] Example 3 This invention measures the silencing efficiency of recombinant adeno-associated virus (AAV-U6-shRNA-CMV-mScarlet-WPRE) on the Ankrd1 gene in bone marrow mesenchymal stem cells. The specific details are as follows: Six-week-old wild-type mice were used to extract primary mesenchymal stem cells using the bone marrow flushing method. The cells were passaged to the third generation and used to target and silence the target gene using recombinant adeno-associated virus AAV-U6-shRNA-CMV-mScarlet-WPRE (denoted as shRNA-Ankrd1 group). At the same time, a negative control group (denoted as Con group) was set up using recombinant adeno-associated virus AAV-U6-NC-CMV-mScarlet-WPRE.

[0076] Third-generation mesenchymal stem cells in good growth condition were seeded into six-well plates, and the cell density was adjusted to 2.1 × 10⁻⁶. 4 cells / cm 2 Then add each to a final concentration of 5 × 10⁻⁶ titers. 9 After 72 hours of viral infection with either virus solution I or virus solution I (vg / mL), RNA was extracted and Ankrd1 expression was detected. The statistical results of the relative expression levels of Ankrd1 mRNA in different groups are as follows: Figure 11 As shown.

[0077] Depend on Figure 11It can be seen that, compared with the negative control group (Con group), the recombinant AAV virus AAV-U6-shRNA-CMV-mScarlet-WPRE provided by the present invention can achieve a silencing efficiency of up to 70% on the Ankrd1 gene of bone marrow mesenchymal stem cells.

[0078] Example of effect 4 The present invention investigates the efficacy of the delivery systems prepared in Example 2 and Comparative Example 2 in the prevention and treatment of osteoarthritis, as detailed below: Fifteen 12-week-old mice were randomly divided into three groups: Sham+Lipo@AAV(NC)-EPLQLKM, DMM+Lipo@AAV(NC)-EPLQLKM, and DMM+Lipo@AAV(Ankrd1)-EPLQLKM. Both hind limbs underwent DMM or Sham surgery. Two weeks post-surgery, at 14 weeks of age, the mice were given a single intravenous injection of the corresponding material according to their group. The injection regimen was 10 mg / kg. Groups labeled "DMM" underwent DMM surgery, while groups labeled "Sham" underwent sham surgery. The groups marked "NC" received tail vein injection of CY5.5-Lipo@AAV(NC)-EPLQLKM prepared in Comparative Example 2, while the groups marked "shAnkrd1" received tail vein injection of CY5.5-Lipo@AAV(Ankrd1)-EPLQLKM prepared in Example 2.

[0079] Eight weeks post-surgery, the Von Frey behavioral test was performed, and bone-related parameters such as body weight, BV / TV (bone volume fraction), Tb.Th (trabecular thickness), Tb.N (trabecular number), Tb.Sp (trabecular separation), BMD (bone mineral density), tibial plateau width, and subchondral bone thickness were measured in different groups of mice. Immunohistochemical staining of Runx2 and Ankrd1 was also performed, and the results were statistically analyzed.

[0080] The flowchart of animal experiments in this invention is as follows: Figure 12 As shown; the results of the measurement of mouse body weight and mechanical withdrawal threshold in each group are as follows. Figure 13 As shown; the detection results of quantitative indicators related to tibial plateau bone microstructure, bone mass, and skeletal morphology in each group of mice are as follows. Figure 14 As shown; representative images of mice stained with safranin and fast green in each group are shown below. Figure 15 As shown; the statistical results of OARSI scores for each group of mice are as follows. Figure 16 As shown; representative Ankrd1 immunohistochemical staining images of mice in each group and their statistical results are as follows. Figure 17 As shown; representative images and statistical results of Runx2 immunohistochemical staining in each group of mice are as follows. Figure 18 As shown.

[0081] in, Figure 14 A represents the microCT images of mice in each group. Figure 14 BC shows 3D reconstructions of the tibial plateau from different perspectives in each group of mice. Figure 14 D represents the statistical results of bone volume fraction in each group of mice. Figure 14 E represents the statistical results of trabecular bone thickness in each group of mice. Figure 14 F represents the statistical results of the number of trabeculae in each group of mice. Figure 14 G represents the statistical results of trabecular bone separation in each group of mice. Figure 14 H represents the bone mineral density statistics for each group of mice. Figure 14 I represents the statistical results of the tibial plateau width of mice in each group. Figure 14 J represents the statistical results of subchondral bone thickness in each group of mice. Figure 14 K represents the statistical results of the medial proximal tibia angle (MPTA angle) of each group of mice.

[0082] Depend on Figure 13 As shown in A, in the Von-Frey experiment, the withdrawal threshold of mice in the DMM+NC group after DMM modeling decreased significantly, and obvious mechanical pain sensitivity was observed. After intervention with the targeted silencing Ankrd1 delivery system provided in Example 2 of this invention, the withdrawal threshold of mice in the DMM+shAnkrd1 group increased significantly, proving that the delivery system can effectively alleviate the pain symptoms of DMM osteoarthritis model mice.

[0083] Depend on Figure 13 As shown in Figure B, there were no significant differences in baseline and postoperative body weight among the three groups of mice: Sham+NC, DMM+NC, and DMM+shAnkrd1. This indicates that the targeted nanodelivery system provided by this invention has no obvious systemic toxicity and will not affect the normal growth and survival of mice.

[0084] Depend on Figure 14 Micro-CT bone microstructure analysis revealed that DMM surgery induced subchondral bone sclerosis in mice. Compared with the Sham+NC sham-operated group, the DMM+NC model group showed significantly increased bone volume fraction (BV / TV), bone mineral density (BMD), trabecular bone thickness (Tb.Th), and subchondral bone thickness. Compared with the DMM+NC model group without Ankrd1 silencing, the DMM+shAnkrd1 group showed a significant decrease in all the aforementioned subchondral bone sclerosis-related indicators, and the trabecular bone microstructure was improved, indicating that the delivery system provided by this invention can effectively alleviate DMM-induced subchondral bone sclerosis.

[0085] Figure 15 , 16Safranin-Fix Green (SO-FG) staining and OARSI scores showed that the DMM+NC group had a large loss of articular cartilage matrix, severe damage to cartilage structure, and a significantly increased OARSI score; the DMM+shAnkrd1 group had relatively intact cartilage morphology and structure, and a significantly reduced OARSI score, further confirming that targeted silencing of Ankrd1 can reduce articular cartilage damage and delay the pathological progression of osteoarthritis.

[0086] Figure 17 , 18 The immunohistochemical results further showed that the number of ANKRD1 and RUNX2 positive cells in the joint tissue increased significantly after DMM modeling. After intervention with the delivery system provided by this invention, the proportion of ANKRD1 and RUNX2 positive cells decreased significantly, proving that the delivery system can effectively silence Ankrd1 and inhibit the expression of the downstream osteogenic-related gene RUNX2.

[0087] In summary, the EPLQLKM peptide-modified, Ankrd1-targeting nanodelivery system of this invention possesses both bone-targeting capability and good in vivo safety. After administration via tail vein, this delivery system is metabolized and cleared in mice, primarily accumulating in the liver, kidneys, and femur. EPLQLKM peptide modification significantly enhances the accumulation level of the carrier in femoral tissue. Animal experiments further confirmed that this delivery system has no significant systemic toxicity, effectively improves mechanical pain sensitivity in DMM model mice, alleviates subchondral bone sclerosis, reduces articular cartilage damage, and lowers OARSI scores; simultaneously, it effectively silences Ankrd1 and inhibits downstream RUNX2 expression, thus delaying the pathological progression of osteoarthritis. Therefore, Ankrd1 inhibitors can be used to prepare drugs for the prevention and treatment of cartilage inflammation, including osteoarthritis, and possess good clinical translational value.

[0088] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of Ankrd1 inhibitors in the preparation of drugs for treating cartilage inflammation.

2. The application as described in claim 1, characterized in that, The cartilage inflammation includes at least one of osteoarthritis or patellar chondromalacia.

3. The application as described in claim 2, characterized in that, The cartilage inflammation mentioned is osteoarthritis.

4. The application as described in claim 1, characterized in that, The Ankrd1 inhibitor includes at least one of Ankrd1 protein function inhibitors or Ankrd1 gene expression inhibitors.

5. The application as described in claim 4, characterized in that, The Ankrd1 gene expression inhibitor includes at least one of shRNA targeting the Ankrd1 gene or siRNA targeting the Ankrd1 gene.

6. The application as described in claim 5, characterized in that, The target gene sequence of the shRNA targeting the Ankrd1 gene is shown in SEQ ID No.

1.

7. A delivery system for targeting and inhibiting bone marrow mesenchymal stem cells Ankrd1, characterized in that: This includes adeno-associated viruses loaded with Ankrd1 inhibitors and liposomes with surface-modified EPLQLKM peptides.

8. The delivery system for targeting and inhibiting bone marrow mesenchymal stem cells Ankrd1 as described in claim 7, characterized in that: The liposomes are also labeled with CY5.5 fluorescent markers; and / or The adeno-associated virus loaded with an Ankrd1 inhibitor contains a vector of shRNA targeting the Ankrd1 gene.

9. The use of the delivery system for targeting and inhibiting bone marrow mesenchymal stem cells Ankrd1 as described in claim 7 or 8 in the preparation of a medicament for treating cartilage inflammation.

10. The application as described in claim 9, characterized in that, The cartilage inflammation includes at least one of osteoarthritis or patellar chondromalacia.

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