Preparation method and application of osteoarticular chondrocyte aptamer and nanoparticles

Abstract

The present application relates to the technical field of biotechnology, in particular to a kind of bone joint chondrocyte aptamer, the preparation method and application of nanoparticle.The preparation method of bone joint chondrocyte aptamer nanoparticle provided by the present application includes the following steps: S1, the nucleotide sequence of chondrocyte aptamer is enriched using SELEX technology, obtain several candidate aptamer nucleotide sequences;Wherein, target cell is the chondrocyte cultured to the third generation, and the subtractive cell is the chondrocyte cultured to the seventh generation;S2, candidate aptamer nucleotide sequence is respectively incubated with normal rat chondrocyte and incubated, and the aptamer C22 that is specifically combined with chondrocyte is selected;S3, the nanoparticle of aptamer C22 selected in step S2 is prepared.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for preparing and applying bone and joint chondrocyte aptamers and nanoparticles. Background Technology

[0002] Osteoarthritis (OA) is a chronic degenerative joint disease characterized by degenerative changes in articular cartilage, chondrocyte apoptosis, synovial inflammation, and bone hyperplasia. It is most common in middle-aged and elderly people, and clinically manifests as joint pain, stiffness, and limited mobility, severely impacting patients' quality of life and being a significant cause of disability in this population. The pathological process of OA is complex, with core components including excessive chondrocyte apoptosis, extracellular matrix degradation, massive release of inflammatory factors, and abnormal subchondral bone remodeling. Currently, there is a lack of radical cures in clinical practice, and traditional treatments mainly focus on anti-inflammatory and analgesic therapies, which are insufficient to reverse cartilage degeneration and protect chondrocytes.

[0003] Current OA treatments generally suffer from drawbacks such as poor targeting, weak cartilage penetration, low bioavailability, and significant systemic toxicity. Small molecule drugs are prone to diffusion and loss after intra-articular injection, have short local retention times, and are difficult to accumulate in the deep cartilage layers. Systemic administration can easily cause liver and kidney toxicity, gastrointestinal adverse reactions, and is difficult to penetrate the dense cartilage matrix to reach the lesion site. In recent years, nanodelivery systems have shown potential in targeted OA therapy, but existing carriers lack chondrocyte-specific recognition capabilities, making precise targeting difficult. They are also easily taken up non-specifically by non-target cells such as synovium and osteoblasts, resulting in low treatment efficiency and insufficient safety.

[0004] However, there is currently no aptamer-modified nanodelivery system that specifically targets chondrocytes, does not bind to synovial fibroblasts / osteoblasts, and can co-deliver therapeutic drugs and AIE fluorescent molecules, leaving a significant gap in OA targeted therapy. Summary of the Invention

[0005] This invention provides a method for preparing osteoarthritis chondrocyte aptamer nanoparticles to address the problem in related technologies where OA treatment drugs lack chondrocyte-specific targeting capabilities.

[0006] In a first aspect, the present invention provides a method for preparing osteoarthritis chondrocyte aptamer nanoparticles, comprising the following steps: S1. Chondrocyte aptamer sequences were enriched using SELEX technology to obtain several candidate aptamer sequences; among them, the target cells were chondrocytes cultured to the third generation, and the subtractive cells were chondrocytes cultured to the seventh generation. S2, candidate aptamer sequences were incubated with normal rat chondrocytes to select aptamer C22 that specifically binds to chondrocytes; S3, preparation of nanoparticles modified with aptamer C22 selected in step S2.

[0007] In some embodiments, the nucleotide sequence of the aptamer C22 is as shown in SEQ ID NO.1, specifically: 5'-GCAATGGTACGGTACTTCCGGCGACTTTCTCGTCTCACGCAGCGGCGTATTCGGTGTTACAAAAGTGCACGCTACTTTGCTAA-3'; In some embodiments, the specific process of step S1 is as follows: prepare buffer solution → incubate target cells → amplify binding nucleotide sequences → amplify secondary libraries → identify enriched libraries → perform single-clone sequencing on enriched libraries → analyze and obtain nucleotide sequences.

[0008] In some embodiments, the prepared buffers include: binding buffer: pH 7.4 PBS containing 5 mmol / L MgCl2, 0.1 μg / μL yeast tRNA and 0.1 μg / μL salmon sperm DNA; washing buffer: pH 7.4 PBS containing 5 mmol / L MgCl2; gel elution buffer: pH 8.0 containing 0.5 mol / L NH4Ac, 0.1% SDS and 1 mmol / L EDTA.

[0009] In some embodiments, the process of incubating target cells is as follows: P3 CC target cells quantified using a cell counting chamber are washed with washing buffer and set aside; the aptamer library is denatured and pretreated in an ice-water bath using binding buffer; yeast tRNA and salmon sperm DNA are added to the pretreatment system to prepare an incubation system, and then the incubation system is mixed with the target cells for incubation; the denatured aptamer library is mixed with the subtracted cells for incubation, and the supernatant is collected after centrifugation, and then the supernatant is mixed with the pretreated target cells for incubation; after incubation, the cells are washed and centrifuged multiple times using washing buffer, the supernatant is removed, and DEPC water is added for heating treatment to obtain the PCR amplification template.

[0010] In some embodiments, the process of amplifying the binding nucleotide sequence is as follows: amplification is performed according to the standard PCR reaction system, 100 μL / tube, and the optimal number of PCR amplification cycles is determined by denaturing gel electrophoresis with 7 M urea and 8% polyacrylamide.

[0011] In some embodiments, the process of amplifying the secondary library is as follows: the amplification product is denatured and then subjected to denaturing polyacrylamide gel electrophoresis. After electrophoresis, nucleic acid staining is performed, the target nucleic acid band is located under ultraviolet light and the corresponding gel is cut. After the gel is crushed, gel elution buffer is added and the gel is eluted. After collecting the elution buffer, magnesium ion solution, sodium acetate solution and anhydrous ethanol are added in sequence and mixed to precipitate nucleic acid. After centrifugation, the supernatant is discarded. The precipitate is washed with pre-cooled ethanol and centrifuged again. After removing the supernatant, the precipitate is allowed to stand and dry. Pure water is added to dissolve the single-stranded DNA, which is then quantified and stored at low temperature for later use.

[0012] In some embodiments, the process of identifying enriched libraries is as follows: CY3 fluorescent labeling is applied to aptamer secondary libraries recovered in a specific round; using these secondary libraries as templates, PCR amplification is performed using CY3-labeled primers to prepare a fluorescent library, which is then stored in the dark; P3 CC cells are seeded in confocal microscopy dishes for culture; the fluorescent library is dissolved in buffer, denatured, and pretreated in an ice-water bath; yeast tRNA and salmon sperm DNA are added to prepare an incubation system; P3 CC cells in the confocal microscopy dishes are washed with washing buffer, then added to the incubation system for incubation; the CY3 fluorescence intensity is observed using a confocal microscope to complete the identification of the enriched library.

[0013] In some embodiments, the process of single-clone sequencing of enriched libraries is as follows: the enriched aptamer library is amplified by PCR, and the dsDNA library is prepared by polyacrylamide gel electrophoresis; the dsDNA library is ligated with a T vector, mixed with competent cells, and the bacterial culture is spread on LB medium containing ampicillin. After culturing, single colonies are picked for sequencing.

[0014] In some embodiments, the process of analyzing and obtaining nucleotide sequences is as follows: DNAMAN is used to perform primary sequence analysis on the single-clone sequencing results, MEME online software is used for family analysis, and RNA structure is used for secondary structure analysis, and the lowest energy level is selected as the candidate sequence.

[0015] In some embodiments, step S3 is specifically performed as follows: First, amino-modified chondrocyte aptamers were combined with COOH-PEG. 2000 -DSPE connection, forming C22-PEG 2000 -DSPE, followed by C22-PEG 2000 -DSPE and NH2-PEG 2000 -DSPE was dissolved in PBS, and a tetrahydrofuran solution containing 4BC fluorescent molecules and the therapeutic drug pelicin was added to prepare C22-PEG@4BC-PA nanoparticles by co-precipitation.

[0016] In some embodiments, DSPE-PEG 2000 The preparation process of -C22 is as follows: COOH-PEG2000-DSPE is dissolved in MES buffer, EDC and Sulfo-NHS are added to the solution sequentially, and after mixing, it is incubated in the dark to activate the terminal carboxyl groups of the polymer; the activated COOH-PEG 2000 - The DSPE solution was transferred to HEPES buffer pre-dissolved with amino-modified aptamer C22-NH2, mixed well, and reacted in the dark. After the reaction was completed, Tris-HCl buffer was added to quench unreacted active esters. The mixed reaction solution was transferred to an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa. Small molecule byproducts and unreacted free substances were removed by centrifugation and ultrafiltration. The purified coupling product was collected to obtain DSPE-PEG2000-C22.

[0017] In some embodiments, the preparation process of C22-PEG@4BC-PA nanoparticles is as follows: Targeting polymer DSPE-PEG2000-C22 and conventional polymer NH2-PEG2000-DSPE are weighed, dissolved in ultrapure water, and ultrasonically treated to form a polymer micelle aqueous phase; the hydrophobic drug palicin (PA) and fluorescent molecule 4BC are dissolved in tetrahydrofuran to prepare an organic phase; under magnetic stirring, the organic phase is uniformly added dropwise to the polymer micelle aqueous phase to induce preliminary self-assembly of the nanoparticles; the mixed solution is ultrasonically broken to form a uniform nanoemulsion, and open-mouth magnetic stirring is used to completely evaporate the organic solvent; free drug and large aggregates are removed by filtration using an aqueous microporous membrane; the filtrate is concentrated by ultrafiltration centrifugation, and the purified targeting nanoparticle ANP solution is collected and stored in the dark.

[0018] Secondly, the present invention also provides a pharmaceutical formulation comprising the above-mentioned nanoparticles.

[0019] In some embodiments, the pharmaceutical preparation is used for targeted therapy of osteoarthritis, targeting chondrocytes.

[0020] The beneficial effects of the technical solution provided by this invention include: 1. This invention uses SELEX technology to screen and obtain the aptamer C22 that specifically targets chondrocytes and does not bind to synovial fibroblasts / osteoblasts / mesenchymal stem cells. After being modified onto the surface of nanoparticles, it can accurately recognize Fas protein on the surface of chondrocyte membranes, achieve efficient targeted enrichment of diseased cartilage tissue, and significantly increase the local concentration of the drug. 2. The nanoparticles provided by this invention can respond to the weakly acidic microenvironment of the OA joint cavity, and the surface charge reversal promotes penetration into the dense cartilage matrix, reaching the deep lesion site of the cartilage, overcoming the defects of traditional drugs with weak penetration and short retention. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying 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.

[0022] Figure 1 This is a schematic diagram of chondrocyte-specific aptamer screening.

[0023] Figure 2 This is a schematic diagram of the analysis of the aptamer C22 target protein.

[0024] Figure 3 This is a schematic diagram of the construction and characterization of nanomaterials.

[0025] Figure 4 A schematic diagram illustrating the use of nanomaterials to repair an OA animal model. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] This invention provides a method for preparing osteoarthritis chondrocyte aptamer nanoparticles, which can solve the problem that existing OA treatment drugs lack chondrocyte-specific targeting ability.

[0028] This invention provides a method for preparing osteoarthritis chondrocyte aptamer nanoparticles, comprising the following steps: Step S1: Enrich chondrocyte aptamer sequences using SELEX technology; the target cells are chondrocytes cultured to the third generation (cultured in DMEM medium containing 10% fetal bovine serum and 1% streptomycin / penicillin antibiotics in an incubator (37℃, 5% CO2), and the subtractive cells are chondrocytes cultured to the seventh generation (culture conditions are the same as for the third generation chondrocytes); the specific process is as follows: Step S101, prepare the buffers: Binding buffer: pH 7.4 PBS, containing 5 mmol / L MgCl2, 0.1 μg / μL yeast tRNA (commercially available) and 0.1 μg / μL salmon sperm DNA; Washing buffer: pH 7.4 PBS, containing 5 mmol / L MgCl2; Gel elution buffer: pH 8.0, containing 0.5 mol / L NH4Ac, 0.1% SDS and 1 mmol / L EDTA; Step S102, incubation of the library with target cells: Count the cells using a cell counting chamber to obtain 1×10⁻⁶ cells. 6 Target cells P3 CC (third-generation chondrocytes) were washed twice with washing buffer and set aside. In the first round, a 5 nmol aptamer (ssDNA) library was added, and binding buffer was added. The cells were denatured at 100°C for 5 min, followed by an ice-water bath for 5 min. Yeast tRNA and salmon sperm DNA were added under 4°C ice-water bath conditions, mixed thoroughly, and an incubation system with a final concentration of 1×PBS-5 mg / L MgCl2-0.1 μg / μL yeast tRNA-0.1 μg / μL salmon sperm DNA was added to the target cells and incubated at 37°C for 30 min. In rounds 4, 5, 6, and 7, a subtraction selection was performed first. The denatured aptamer library was added to subtracted cells P7 CC (seventh-generation chondrocytes), mixed thoroughly, and incubated at 37°C for 30 min. The cells were then centrifuged at 1400 rpm for 5 min, and the supernatant was collected and added to the washed target cells P3 CC. The mixture was mixed thoroughly and incubated at 37°C for 30 min. min; after incubation, add 1 mL of washing buffer and gently pipette, centrifuge at 1400 rpm × 5 min, remove the supernatant, repeat washing 3-5 times, remove the supernatant, add 100 μl of DEPC water and boil for 5 min, and use as a PCR amplification template for later use. Step S103, Amplifying the binding nucleotide sequence: This step is to determine the optimal number of PCR cycles required. This operation must be performed in every step of SELEX. Unequal-length PCR is used to amplify the binding sequence. First, a small amount of template is amplified according to the standard PCR reaction system, 100 μL / tube, aliquoted into 10 tubes, and labeled as 12 rounds, 15 rounds, 18 rounds, 21 rounds, 24 rounds, 27 rounds, and 30 rounds respectively. The optimal number of PCR amplification cycles is determined by denaturing gel electrophoresis with 7 M urea and 8% polyacrylamide. Step S104, Amplification and Preparation of Secondary Library: After determining the optimal number of PCR cycles, the remaining template was amplified in large quantities using the same procedure. After amplification, the product was denatured at 100℃ for 5 min, then in an ice-water bath for 5 min, and electrophoresed using a 7 M urea 8% polyacrylamide denaturing gel at 140 V. The gel was then stained with Goldview nucleic acid staining solution and placed on a UV lamp platform lined with plastic wrap. The target band position was confirmed, and the gel at the target band was cut off with a blade. The gel strip was cut into 1 cubic millimeter pieces and transferred to 1.5 mL centrifuge tubes. 400 μL (more than twice the volume of the gel strip) of gel elution buffer was added to each centrifuge tube, and the mixture was eluted at 37℃ on a shaker for 8 h. The centrifuge tubes were briefly centrifuged, and the elution buffer was carefully transferred to a new 1.5 mL centrifuge tube to prevent gel spillage. 1 / 100 volume of 1 mol / L MgCl2 and 1 / 10 volume of 3 mol / L NaAc (pH 10.5) were added to the centrifuge tubes. 5.2) Add 2.5 times the volume of anhydrous ethanol sequentially, vortex to mix, and let stand at -80℃ for 8 h to precipitate; centrifuge at 13000 rpm for 30 min at 4℃; discard the supernatant, add 1 mL of pre-cooled 75% ethanol to wash, centrifuge at 13000 rpm for 30 min; discard the supernatant, dry at room temperature for about 30 min (ethanol evaporates), add 100 μL of ddH2O to dissolve ssDNA, quantify using a UV spectrophotometer, and store at -20℃ for later use; Step S105, Confocal Identification of Enriched Libraries: The secondary libraries recovered in rounds 5, 6, and 7 were identified as enriched libraries. The aptamer secondary libraries were labeled with CY3 fluorescence; that is, using the ssDNA secondary library as a template, primers were labeled with CY3, and amplification was performed according to the above PCR procedure to prepare a fluorescent library, which was then stored at -20℃ in the dark. P3 CC was seeded in confocal dishes and cultured until 70% confluence. The CY3-labeled ssDNA secondary library or GP40 library was dissolved in 1×PBS-5mmol / L MgCl2 solution (freshly prepared), denatured at 100℃ for 5 min, and then incubated in an ice-water bath for 5 min. The previously prepared yeast tRNA and salmon sperm DNA were added and mixed thoroughly to achieve a final incubation concentration of 1×PBS-5mmol / L MgCl2. 2- 0.1 μg / μL yeast tRNA-0.1 μg / μL salmon sperm DNA, total volume 200 μL; P3 CC in confocal microscopy dish was rinsed twice with washing buffer, the above library was added, and incubated at 37℃ for 30 min; CY3 fluorescence intensity was observed under a confocal microscope; simultaneously, in addition to confocal microscopy, 1×10⁻⁶ samples were obtained. 6 The fluorescence intensity of the secondary library was detected using flow cytometry for each P3 CC.

[0029] Step S106, Single-clone sequencing of enriched library: After confirming library enrichment, further sequencing is required to obtain aptamer nucleotide sequences. First, the enriched library is amplified and recovered by PCR to prepare a dsDNA library. Isolength PCR is used, and the standard PCR reaction system is followed by 10% polyacrylamide gel electrophoresis for recovery. The recovered dsDNA library is ligated with a T vector and mixed with competent cells. 100 μL of bacterial culture is taken in a clean bench, spread on LB medium containing ampicillin, dried, and then incubated upside down overnight in a 5% CO2, 20% O2, 37℃ incubator. Single colonies are picked for sequencing. Step S107, nucleotide sequence analysis: Primary sequence analysis of the single-clone sequencing results was performed using DNAMAN, and family analysis and secondary structure analysis of RNA structure were performed using the MEME online software (http: / / meme.sdsc.edu / meme / cgi-bin / meme.cgi); (Reference) Figure 1 (in, Figure 1 a and Figure 1 b. Confocal microscopy and flow cytometry were used to detect the enrichment of the libraries in rounds 5, 6, and 7, respectively. Figure 1 c represents the flow cytometry analysis of the binding affinity of the four candidate sequences to chondrocytes; Figure 1 d represents the flow cytometry comparison of the specificity of aptamer C22 for osteoblasts and synovial cells, ultimately yielding 44 aptamers enriched in seven rounds. Figure 1 a, Figure 1 b), and selected the four lowest energy levels (C14, C22, C26, C46) as candidate sequences; Step S2: To detect the binding affinity and specificity of the aptamers, the four candidate aptamers were first incubated with chondrocytes. Flow cytometry analysis showed that C22 had the strongest binding affinity to chondrocytes. Figure 1 c); C22 was then incubated with synovial fibroblasts, osteoblasts, and chondrocytes, respectively. Flow cytometry analysis showed that C22 did not bind to synovial fibroblasts and osteoblasts, exhibiting specificity for binding to chondrocytes. Figure 1 d) The nucleotide sequence of aptamer C22 is: 5'-GCAATGGTACGGTACTTCCGGCGACTTTCTCGTCTCACGCAGCGGCGTATTCGGTGTTACAAAAGTGCACGCTACTTTGCTAA-3'; refer to Figure 2 (in, Figure 2 a) To detect the colocalization of aptamer C22 with the cytoskeletal protein phalloidin using confocal microscopy; Figure 2b is a flow cytometry assay to determine whether C22 still binds to chondrocytes after trypsin and proteinase K digest cell membrane proteins; Figure 2 c represents the analysis of the C22 target protein band using polyacrylamide gel electrophoresis and Coomassie brilliant blue staining. Figure 2 d represents the co-localization of aptamer C22 and target protein Fas as detected by confocal microscopy. Figure 2 e represents the use of polyacrylamide gel to detect the pull-down of Fas by aptamer C22; Figure 2 f represents the confocal assay used to detect whether C22 still binds to chondrocytes after si-Fas downregulates Fas expression in chondrocytes. Figure 2 g represents flow cytometry analysis of the cytotoxicity of aptamer C22 on cells. To further understand the targeting mechanism of C22, flow cytometry analysis after cell digestion with protein K and trypsin showed that C22 binding was significantly weakened. Figure 2 a, Figure 2 b). Therefore, cell membrane proteins may be an important factor in C22 binding to chondrocytes. Next, chondrocyte membrane proteins were extracted for a pull-down assay. Coomassie blue staining showed that the aptamer C22 group exhibited a specific band near 35 kDa, distinct from the IgG group ( Figure 2 c). The Fas peptide chain showed the highest intensity and reliability in the LC-MS mass spectrometry results. Immunofluorescence showed co-localization of C22 and Fas fluorescence after cell incubation. Figure 2 d). Western blot results confirmed the binding of Fas to C22 ( Figure 2 e). Furthermore, knocking down Fas in OA cells showed a significant decrease in the binding of C22 to chondrocytes (e). Figure 2 f). Fas is an important apoptosis receptor in cells. To investigate whether C22 affects apoptosis, flow cytometry analysis showed that C22 did not induce significant apoptosis in chondrocytes (f). Figure 2 g). Therefore, C22 can recognize and bind to chondrocytes via Fas, thus ensuring targeted drug delivery.

[0030] Step S3: Prepare nanoparticles modified with aptamer C22 selected in step S2: Preparation of DSPE-PEG 2000 -C22: In order to synthesize the targeted polymer DSPE-PEG 2000 -C22, this invention employs an EDC / NHS chemical coupling method to specifically link an amino-modified aptamer to the free carboxyl terminus of a polymer. First, accurately weigh 1 mg of COOH-PEG. 2000-DSPE was dissolved in 500 μL of MES buffer (0.1 M, pH 5.0), and the solution was gently heated to 40 °C if necessary to aid dissolution. Then, 6.4 mg of EDC ((1-ethyl-(3-dimethylamino)carbodiimide hydrochloride)) and 3.6 mg of Sulfo-NHS (sulfonyl-N-hydroxysuccinimide) were added sequentially to the solution. After thorough vortexing, the mixture was incubated at room temperature in the dark for 30 minutes to activate the carboxyl groups at the polymer ends. After activation, the activated COOH-PEG was... 2000 The DSPE solution was rapidly transferred to a centrifuge tube pre-filled with 0.43 nmol of the aptamer (C22-NH2) (which had been dissolved in 500 μL of 0.1 M HEPES buffer at pH 8.0). After vortexing again, the reaction mixture was incubated at room temperature in the dark for 4 hours (or overnight at 4 °C to further improve the coupling yield). After the coupling reaction was complete, 50 μL of 1 M Tris-HCl buffer (pH 7.5) was added to quench any unreacted active esters in the system. Finally, the mixture was transferred to an ultrafiltration centrifuge tube with a molecular weight cutoff (MWCO) of 10 kDa and centrifuged at 14,000 × g for 15 minutes. Ultrafiltration effectively removed small molecule byproducts and unreacted free substances from the system, and the purified coupling product was collected for later use.

[0031] Preparation of C22-PEG@4BC-PA nanoparticles (ANP): To prepare targeted nanoparticles (C22-PEG@4BC-PA, abbreviated as ANP) co-encapsulating the drug penicillin (PA) and the AIE fluorescent molecule 4BC, this invention employs an antisolvent precipitation combined with ultrasonic emulsification. First, the coupled targeted polymer DSPE-PEG was weighed at a mass ratio of 1:40. 2000 -C22 and conventional polymer NH2-PEG 2000-DSPE (approximately 5 mg in total) was added to 10 mL of ultrapure water and dissolved completely by brief sonication in a water bath, forming a clear polymer micelle aqueous phase. Subsequently, an appropriate amount of the hydrophobic drug PA and the fluorescent molecule 4BC were completely dissolved in 2 mL of tetrahydrofuran (THF) as the organic phase. Under room temperature and moderate magnetic stirring, the THF organic phase containing PA and 4BC was rapidly and uniformly added dropwise to the above polymer aqueous solution using a pipette to induce the initial self-assembly of nanoparticles. Immediately afterwards, the mixed solution was transferred to a probe-type ultrasonic disruptor and continuously sonicated at 45% output power for 2 minutes in an ice bath environment to form a nanoemulsion with uniform particle size. After sonication, the emulsion was placed in a fume hood and continuously magnetically stirred at room temperature for 4 to 6 hours or overnight to ensure complete evaporation of the organic solvent THF. After complete solvent evaporation, the solution was filtered through a 0.22 μm aqueous microporous membrane to remove unencapsulated and free precipitated drug molecules and any large polymer aggregates that may be present. Finally, the collected filtrate was transferred to an ultrafiltration centrifuge tube for centrifugation and concentration to achieve the required final volume and concentration. The resulting purified targeted nanomedicine carrier ANP solution was stored at 4 °C in the dark for use in subsequent experiments.

[0032] refer to Figure 3 (in, Figure 3 a) Dynamic light scattering (DLS) and transmission electron microscopy (TEM) are used to analyze the particle size of nanomaterials; Figure 3 b represents the potential of nanomaterials detected by the Zeta potential; Figure 3 c represents the drug encapsulation efficiency test; Figure 3 d and Figure 3 e represents drug stability testing; Figure 3 f represents the nanomaterial cartilage assembly penetration experiment; Figure 3 g represents the detection of cellular uptake of nanomaterials; Figure 3 h represents the in vivo stability test of the drug; Figure 3 i represents cytotoxicity detection; Figure 3 j and Figure 3 k represents lysosomal colocalization analysis and Pearson correlation coefficient. Characterization results show that the hydrated particle size distribution of ANPs, as determined by dynamic light scattering (DLS) and transmission electron microscopy (TEM), is between 40 and 60 nm. Figure 3 a). Due to the protonation of the amino group (-NH2) in acidic environments, ANP exhibits excellent pH responsiveness: when the pH value is less than 6.5, its Zeta potential changes from negative to positive, reaching +5 to +10 mV ( Figure 3 b). Regarding drug encapsulation, the encapsulation efficiency of ANP is optimal at a carrier-to-drug ratio of 1:10, approximately 80%. Figure 3c). Stability testing showed that ANP maintained morphological stability after 36 days of incubation with synovial fluid from OA patients. Figure 3 d); no significant drug release was observed within 12 days, with only slight leakage occurring at 24 and 36 days. Figure 3 e). In terms of biological function and in vivo behavior evaluation, in vivo tissue penetration experiments showed that the weakly acidic environment within the OA knee joint induced a positive charge effect of surface amino groups, enabling ANPs to effectively penetrate into the deep cartilage layer, significantly superior to the control group without modified amino groups (e). Figure 3 f). Cell uptake experiments showed that after co-incubating nanoparticles with chondrocytes for 1, 3, and 6 hours, the intracellular fluorescence intensity of the ANP group was significantly higher than that of the control group without aptamer modification (f). Figure 3 g), confirming the highly efficient targeting mediated by the aptamer. In vivo imaging results in mice further demonstrated that ANP maintained fluorescence in vivo for up to 35 days after intra-articular injection, with a significantly longer local retention time in the joint compared to the control group without aptamers. Figure 3 Furthermore, CCK-8 assays confirmed the good biocompatibility of ANP, showing no significant toxicity when co-incubated with chondrocytes at a concentration of 20 μM for 72 hours. Figure 3 i). Regarding intracellular delivery mechanisms, lysosomal colocalization analysis ( Figure 3 j) and Pearson correlation coefficient (PCC) quantitative ( Figure 3 k) showed that with increasing incubation time (1 to 6 hours), the colocalization signal of fluorescence and lysosomal probes in the ANP group was significantly weaker than that in the control group. This strongly demonstrates that 4BC sensitization produces [the desired signal] under light excitation. 1 O2 successfully disrupted the lysosomal barrier, enabling ANPs to achieve spatiotemporally controlled lysosomal escape and rapidly deliver therapeutic drugs into the cytoplasm. In summary, ANPs can intelligently respond to the weakly acidic microenvironment of OA to penetrate the cartilage matrix, precisely target chondrocytes under aptamer guidance to deliver PA drugs, and achieve lysosomal escape through AIE molecule-mediated photochemical internalization (PCI).

[0033] ANP testing: Singlet oxygen detection: To evaluate the performance of ANP under photoexcitation 1 O2 production capacity was detected using an SOSG fluorescent probe. After cell adhesion, cells were incubated in culture medium containing ANP for 6 hours. Subsequently, 5 μM of SOSG working solution (Beyotime, S0067) was added to the wells, and the cells were equilibrated at 37°C in the dark for 30 minutes. O2 production capacity was measured using a 660 nm laser (power density 100 mW / cm²). 2 Irradiate for 5 minutes to stimulate 1O2 was generated. After irradiation, the cells were washed three times with cold PBS to remove the free probes. They were then fixed with 4% paraformaldehyde for 15 minutes, stained with DAPI solution in the dark for 10 minutes, and observed under a fluorescence microscope.

[0034] Cellular uptake and lysosomal escape: Cells were seeded in confocal microplates, and when the density reached 80%, experimental and control nanoparticles were added and incubated for 1 h, 3 h, and 6 h, respectively. Lysosome staining was performed according to the kit instructions (Beyotime, C1047S). After staining, fluorescence observation and imaging were quickly performed using a laser confocal microscope (Leica). To quantify lysosome escape, the Pearson coefficient (PCC) of positive cells (red and green) was calculated using ImageJ software.

[0035] Drug loading and encapsulation efficiency assessment: The compound was placed in an ultrafiltration centrifuge tube and centrifuged at 10,000 rpm for 30 minutes. After diluting the filtrate, the fluorescence intensity was measured at 295 nm using a fluorescence spectrophotometer, and the amount of potent drug was calculated. The drug loading and encapsulation efficiency were calculated using the following formulas.

[0036] DL (%) = Total drug dosage - Free drug dose / Total drug dosage + Nanoparticle dose * 100% EF (%) = (Total drug administered - Free drug dose) / (Total drug administered) * 100% Bioinformatics analysis: ATAC sequencing data (GSE108301) and RNA seq sequencing data (GSE114007, GSE129147) were downloaded from the GEO database. After quality control and alignment, the ATAC-seq data were used for peak identification using MACS2, differential analysis was performed using DiffBind, and the bigWig signal was extracted and plotted using deepTools. Finally, chromatin accessibility changes were visualized using Integrative GenomicsViewer. After data cleaning, the RNA seq data were used for differential and enrichment analysis in Rstudio (version 4.5.1), and ggplot was applied for plotting.

[0037] Nanomaterials for repairing OA animal models: refer to Figure 4 (in, Figure 4 a represents the therapeutic effect; Figure 4 b、 Figure 4 c and Figure 4 d is a schematic diagram of ANP treatment for knee osteoarthritis; Figure 4 e represents cartilage tissue degeneration; Figure 4f and Figure 4 g represents the immunohistochemical result; Figure 4 h represents the pain-related behavioral score; Figure 4 i represents the TUNEL staining result; Figure 4 j represents the fluorescence imaging result; Figure 4 (k represents the HE staining analysis results of organs). To verify the in vivo therapeutic effect of ANP, an anterior cruciate ligament (OA) animal model was created by transection of the anterior cruciate ligament in wild-type SD rats. One week post-surgery, 20 mM nanoparticles were injected into the knee joint of the rats once a week for four weeks. The therapeutic effect was observed after eight weeks. Figure 4 a). Gross analysis revealed that the knee cartilage damage in the ANP treatment group was less severe than that in other control groups. Figure 4 b, above). MicroCT analysis of the knee joint showed that the ANP treatment group had significantly fewer osteophytes in the knee joint than the other three control groups ( Figure 4 (b, lower), the number and thickness of trabeculae in the subchondral bone, as well as the hardness of the subchondral bone plates, are also significantly lower. Figure 4 c. Figure 4 d). These results demonstrate that ANP can effectively alleviate bone degeneration during the course of OA.

[0038] HE and SO / FG histological staining of the knee joint showed significantly milder degeneration of cartilage tissue in the ANP group (Figure 4e). Immunohistochemical results indicated higher expression of col2 and lower expression of mmp13 in the cartilage tissue of the ANP group. Figure 4 f). The ANP treatment group had a lower Mankin score for the knee joint compared to the other control groups. Figure 4 g), showing a significantly better overall treatment effect. Immunofluorescence results indicated that IGFBP3 expression was lower after ANP treatment compared to the control group, while pIGF1R expression was significantly increased (g). Figure 4 e). This suggests that the ANP treatment mechanism improves OA through the IGFBP3 / IGF1-AKT signaling pathway. In pain-related behavioral scores, the withdrawal thresholds in the ANP and PA groups were significantly higher than those in the control group. Figure 4 The results (h) indicate that ANP treatment can significantly alleviate the pain symptoms of OA. Furthermore, TUNEL staining results showed a significant reduction in cell apoptosis in the cartilage tissue of the treatment group (h). Figure 4 i). Regarding in vivo targeting, organ fluorescence imaging showed that fluorescence signals were only present in the knee joint within 48 and 72 hours after ANP treatment (i). Figure 4 j). Organ HE staining analysis also indicated that ANP treatment did not cause significant toxic side effects in other organs. Figure 4 k).

[0039] Experimental method description: (1) Animal model: An OA animal model is established by performing anterior cruciate ligament transection on SD rats. In the sham operation group, only the knee joint capsule is incised and then sutured. During the intervention with drugs and nanoparticles, a 10-gauge needle syringe is used to inject 0.3 ml of PBS, PA (20 μM), C22-PEG@4BC (20 μM) and C22-PEG@4BC-PA (20 μM) into the knee joints of rats in the corresponding groups respectively. One week after the operation, the injection is performed once a week for a total of 4 times. 8 weeks after the operation, all rats are euthanized, and the knee joints are quickly isolated and placed in 4% paraformaldehyde for fixation for histopathological and imaging analysis. The animal experiment protocol is approved by the Animal Ethics Committee of the Third Hospital Affiliated to Wuhan University and implemented in accordance with the agreement (Ethics Approval No.: Wusan Yishi Lun 25-002). The entire process complies with the relevant ethical regulations for animal experiments and research.

[0040] (2) Cell culture and OA model establishment: After the rats are sacrificed, the bilateral knee joints are quickly isolated, the subchondral bone is avoided from the medial and lateral femoral condyles, and the hyaline cartilage is carefully separated to extract primary rat chondrocytes. Cartilage slices are selected from the OA lesion area of the medial femoral condyle in the tissue excised after human joint replacement to extract human OA chondrocytes. The excised cartilage slices are washed repeatedly with PBS, and other tissues are carefully inspected and removed. The cartilage slices are cut into small pieces and placed in a 15 ml centrifuge tube. 5 ml of 0.2% type II collagenase solution prepared with low-glucose DMEM is added. After mixing evenly, the mixture is placed in a shaker at 37°C for 10 hours at a speed of 55 rpm. Cells are extracted by centrifugation and filtration. The cells are cultured in an incubator, and the cell culture medium is prepared with Dulbecco's Modified Eagle Medium containing 10% fetal bovine serum and 1% streptomycin / penicillin double antibody. The medium is replaced every 2-3 days. When the cells grow to a density of about 80%, subculture is performed. During cell subculture, the medium is first discarded, and then the cells are washed 3 times with PBS. 0.25% trypsin is added for digestion for 2 minutes, then complete medium is added to terminate the digestion, and the cells are pipetted to disperse evenly and then transferred into a 15 ml centrifuge tube. After centrifugation at 1200 rpm for 5 minutes, the supernatant is discarded, and complete medium is added. After the cells are pipetted evenly, they are transferred into 2 cell culture flasks respectively for further culture. All experiments in the present invention use primary chondrocytes at passages 1-2. When the cells grow to about 60%, 10 ng / ml IL-1β is added for 48 hours to establish the OA cell model.

[0041] (3) Flow cytometry analysis: Apoptosis was detected according to the instructions of the apoptosis detection kit (Beyotime, C1062S). After cell digestion, the cells were centrifuged, the supernatant was discarded, and the cells were washed with PBS and resuspended. 5 μL of Annexin V-FITC staining was added for 20 minutes, followed by 10 μL of PI staining for 10 minutes. Cells that were Annexin V-FITC positive but PI negative were apoptotic cells. For aptamer-bound cells, the cells were gently pipetted to resuspend them, centrifuged, and the supernatant was discarded. 1 ml of aptamer incubation system (1xPBS-5 mg Cl2-0.1 μg / μL yeast tRNA, and 1% BSA) was added for resuspending, followed by incubation with 100 pmol of aptamer for 2 hours. The cells were then centrifuged, the supernatant was discarded, and flow cytometry detection solution was added. Fluorescence intensity was detected using a CytoFlex (BECKMAN) instrument. Data analysis was performed using FlowJo (Tree Star) software.

[0042] (4) Mass spectrometry analysis: The protein eluted from the nucleic acid aptamer experiment was subjected to 10% SDS-PAGE gel electrophoresis, and the gel was stained with silver or Coomassie blue. After observing the differences in bands, gel blocks from the experimental and control groups were excised and digested with trypsin. The extracted proteins were reduced and alkylated, and then digested into peptides using trypsin. Subsequently, peptide separation was performed using an Easy-nLC 1200 ultra-high performance liquid chromatography system (Thermo Fisher), which was then directly connected to a Q Exactive HF-X mass spectrometer (Thermo Fisher). Mass spectrometry acquisition adopted data-dependent scanning (DDA) mode: a first-stage full scan (300-1800 m / z, resolution 60,000) was performed in Orbitrap, followed by high-energy collisional dissociation (HCD) lysis of the top 20 intensity precursor ions (normalized collision energy of 28%), and a second-stage MS / MS scan. Raw data were imported into MaxQuant (v2.1.0) software and compared with the UniProt human protein database using the built-in Andromeda search engine. Trypsin was set as the digestive enzyme, allowing two missed cleavage sites; cysteine ​​aminomethylation was a fixed modification, while methionine oxidation and protein N-terminal acetylation were variable modifications. Quality calibration and LFQ label-free quantification were performed using the built-in algorithm, and the false positive rate (FDR) for both peptide and protein levels was strictly controlled to be less than 1%.

[0043] (5) TUNEL staining: Following the instructions for the Click-iT 488 TUNEL Cell Apoptosis Detection Kit (Servicebio, G1504), tissue sections were dewaxed and hydrated. Proteinase K was added for 20 min, followed by washing three times with PBS. After permeabilization, equilibration buffer was added and incubated for 10 min, then washed. TDT incubation solution was added for labeling for 1 h, followed by washing and then click reaction solution, incubated in the dark for 30 min. After washing, dapi solution was added for nuclear staining for 10 min. The sections were then mounted and observed and photographed under a microscope.

[0044] (6) Nucleic acid aptamers: Biotin-labeled aptamer C22 was synthesized and incubated with Streptavidin magnetic beads (Beyotime, P2151). Chondrocyte membrane proteins were extracted using a membrane protein extraction kit (Beyotime, P0033). The extracted protein solution was then added to aptamer binding buffer (NaCl, MgCl) and C22-bound magnetic beads, and incubated by rotation for 5 hours. After incubation, the magnetic beads were washed four times with aptamer binding buffer, and then boiled at 60°C for 30 minutes with 1% protein loading buffer. The resulting protein solution was then analyzed by SDS-PAGE, Coomassie blue staining (Servicebio, G2012), Western blot, and LC-MS.

[0045] (7) Miniature CT scan After euthanasia, the knee joint was separated, surface muscle tissue was removed, and the joint was fixed in 4% paraformaldehyde. Scanning was performed using a miniature CT scanner (Bruker). Image reconstruction was performed using CTvox software, and measurement data were analyzed using CT Analyzer software.

[0046] (8) Histopathological evaluation Tissue sections from rat knee joints were stained with hematoxylin-eosin (H&E) and safranin-fast green (SO / FG). Cartilage degeneration, synovial inflammation, and osteophyte maturity were quantitatively assessed using the OARSI grading system. Histological images were obtained using an Aperio slide scanner (Leica).

[0047] In the description of this specification, the references to terms such as "one embodiment / mode," "some embodiments / modes," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment / mode or example is included in at least one embodiment / mode or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment / mode or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments / modes or examples. Furthermore, without contradiction, those skilled in the art can combine and integrate the different embodiments / modes or examples described in this specification, as well as the features of different embodiments / modes or examples.

[0048] It should be noted that in this invention, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. In this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise expressly specified.

[0049] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A bone and articular chondrocyte aptamer, characterized in that, The nucleotide sequence of the aptamer is shown in SEQ ID NO.1, specifically: 5'-GCAATGGTACGGTACTTCCGGCGACTTTCTCGTCTCACGCAGCGGCGTATTCGGTGTTACAAAAGTGCACGCTACTTTGCTAA-3'.

2. A method for preparing osteoarthritis chondrocyte aptamer nanoparticles, characterized in that, Includes the following steps: S1 provides an aptamer C22 that specifically binds to chondrocytes; the nucleotide sequence of said aptamer C22 is shown in SEQ ID NO. 1; S2, Preparation of aptamer C22 modified nanoparticles: First, amino-modified chondrocyte aptamers were combined with COOH-PEG. 2000 -DSPE connection, forming DSPE-PEG 2000 -C22, then DSPE-PEG 2000 -C22 and NH2-PEG 2000 -DSPE was dissolved in PBS, and a tetrahydrofuran solution containing 4BC fluorescent molecules and the therapeutic drug pelicin was added to prepare C22-PEG@4BC-PA nanoparticles by co-precipitation.

3. The method for preparing osteoarthritis chondrocyte aptamer nanoparticles according to claim 2, characterized in that, DSPE-PEG 2000 The preparation process of -C22 is as follows: COOH-PEG 2000 -DSPE is dissolved in MES buffer, and EDC and Sulfo-NHS are added to activate the carboxyl groups; the activated COOH-PEG 2000 The DSPE solution was mixed with HEPES solution of C22-NH2 and reacted; the reaction was quenched by adding Tris-HCl buffer, and impurities were removed by ultrafiltration to purify and obtain DSPE-PEG. 2000 -C22.

4. The method for preparing osteoarthritis chondrocyte aptamer nanoparticles according to claim 3, characterized in that, The preparation process of C22-PEG@4BC-PA nanoparticles is as follows: Weigh out DSPE-PEG 2000 -C22 and NH2-PEG 2000 -DSPE was dissolved in ultrapure water and ultrasonically treated to form an aqueous phase; the hydrophobic drug paclobutrazol and the fluorescent molecule 4BC were dissolved in tetrahydrofuran to prepare an organic phase; under stirring conditions, the organic phase was uniformly added to the aqueous phase and ultrasonically treated to form a uniform nanoemulsion, which was then filtered and ultrafiltered to concentrate the nanoparticles.

5. The use of the nanoparticles prepared by the method according to any one of claims 2 to 4 in the preparation of targeted therapeutic drugs for osteoarthritis.

6. The application according to claim 5, characterized in that, The drug targets chondrocytes.

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