Intelligent drug delivery system for treating osteoarthritis as well as preparation method and application of intelligent drug delivery system

Abstract

The invention provides an intelligent drug delivery system for treating osteoarthritis as well as a preparation method and application thereof, the intelligent drug delivery system is a drug-loaded micelle of which the surface is covalently coupled with a virus glycoprotein mimic peptide, and the hydrodynamic diameter is 16 + / -5 nm; the sequence of the mimic peptide comprises a type II collagen adhesion domain and a matrix metalloproteinase 13 specific lysis domain, and the mimic peptide can simulate a dual action mechanism of a virus: firstly, the mimic peptide is non-specifically adhered to a cartilage matrix to realize long-acting retention, and then the mimic peptide is specifically activated in a microenvironment in which pathological cells overexpress MMP13, a cell penetrating peptide is exposed, and efficient intracellular delivery of a drug is driven. The technical bottlenecks of short drug residence time, poor cartilage penetrability and lack of lesion cell targeting in the traditional treatment method are solved, the breakthrough from cartilage targeting to precise lesion cartilage cell targeting is realized, cartilage degeneration can be obviously delayed, symptoms can be improved, and excellent curative effects and clinical transformation prospects are shown.

Description

Technical Field

[0001] This invention relates to a drug delivery system, its preparation method, and its application, and more particularly to an intelligent drug delivery system for treating osteoarthritis, its preparation method, and its application, belonging to the technical field of drug delivery systems, their preparation methods, and applications. Background Technology

[0002] Osteoarthritis (OA) is a joint disease caused by a variety of factors, including fibrosis, cracking, ulceration, and loss of articular cartilage. Its occurrence is related to age, obesity, inflammation, trauma, and genetic factors. It is a serious global health problem, and its impact on patients' health and the resulting medical costs are constantly increasing.

[0003] The goal of osteoarthritis treatment is to reduce or eliminate pain, correct deformities, improve or restore joint function, and improve the patient's quality of life. In terms of drug therapy, current treatments mainly focus on symptom relief, such as intra-articular injection of corticosteroids. However, because hormones can damage cartilage, long-term use can worsen symptoms and increase the risk of infection; therefore, indiscriminate use is discouraged, and repeated use is strongly discouraged. Systemic analgesics such as nonsteroidal anti-inflammatory drugs (NSAIDs) can be used, but their potential risks to the gastrointestinal tract, liver, kidneys, and cardiovascular system must be considered. Therefore, to date, no disease-modifying osteoarthritis drugs (DMOADs) have been approved by regulatory agencies. DMOADs are defined as drugs that can prevent further deterioration of joint structure and improve symptoms.

[0004] Intra-articular drug delivery is a potential way to improve the bioavailability of cartilage drugs and reduce systemic toxicity. However, free drugs are rapidly cleared from the constantly renewing synovial fluid via synovial vessels or lymphatic vessels, resulting in a very short intra-articular retention time, usually only a few hours. Repeated intra-articular injections greatly increase the risk of infection, thus limiting long-term treatment and leading to poor efficacy. While nanoparticles can prolong the intra-articular drug retention time, they face challenges in penetrating the cartilage matrix. This is because the extracellular matrix of articular cartilage has a dense structure, with the collagen network having a pore size of approximately 60 nm. This structure forms a physical barrier to nanoparticle penetration, preventing nanoparticles larger than this size from penetrating the cartilage. Ultra-small nanoparticles... While particles smaller than 60 nm can penetrate the entire cartilage matrix, they are easily removed from the cartilage through convection. Furthermore, the distribution of diseased chondrocytes in the cartilage tissue of osteoarthritis patients is not uniform; approximately 83% of OA patients have at least one joint compartment with normal cartilage. This necessitates that drug delivery systems be able to distinguish between diseased and healthy chondrocytes to achieve precision treatment. Some reported targeting ligands, such as TGG2 aptamers, chondrocyte homing peptides (with the amino acid sequence DWRVIIPPRPSA), and cRGD peptides, can achieve cartilage targeting, but their ability to distinguish between diseased and healthy chondrocytes remains limited, making it difficult to meet the therapeutic needs for precise intervention on diseased chondrocytes.

[0005] The process of viral invasion of host cells in nature is also highly valuable for reference. The delivery of viral genetic material (DNA or RNA) to a new host cell is a complex and precise process. Upon initial contact with the host cell, viral membrane glycoprotein molecules non-specifically bind to C-type lectins (TIM-1, LSECtin, and DC-SIGN) or integrins (αVβ3) on the host cell surface, enabling rapid viral attachment and retention. Subsequent environmental factors trigger conformational changes in these glycoproteins, promoting internalization within the host cell. For example, coronavirus surface glycoproteins non-specifically adhere to the host cell's ACE2 receptor, subsequently interacting with the TMPRSS2 protease, causing conformational changes that mediate viral fusion with the host cell membrane and delivery of genetic material into the host cell. Similarly, for human immunodeficiency virus (HIV-1), research has found that before attacking host cells, its viral envelope proteins attach to receptors on the host cell, enabling the virus to dock and invade. This glycoprotein "adhesion-response activation" is a necessary condition for viral invasion of host cells.

[0006] Many enveloped viruses may employ similar virus-host cell membrane fusion mechanisms. After viral surface glycoproteins bind to host cell receptors, a series of conformational changes in the viral fusion protein are initiated. For example, rubella virus (RV) envelope glycoproteins E2 and E1 are type I envelope glycoproteins that can form heterodimers to create spike structures on the viral surface, performing their main biological functions. The glycosylation sites and specific leucine sites on these proteins play crucial roles in virus-induced cell fusion. These viruses achieve effective cell invasion through the specific interactions between their surface components and host cells.

[0007] However, despite the aforementioned understanding of viral invasion mechanisms, the challenge remains of skillfully translating these efficient and specific mechanisms into targeted drug therapy for osteoarthritis. This challenge is primarily reflected in the following aspects:

[0008] First, existing nanodelivery systems struggle to penetrate dense cartilage matrix while achieving long-term drug retention at the lesion site.

[0009] Secondly, and more importantly, there is currently a lack of targeted head bases that can accurately distinguish between diseased and healthy chondrocytes, making it difficult for any advanced delivery system to achieve efficient drug delivery to diseased chondrocytes. Summary of the Invention

[0010] To overcome the technical shortcomings of existing osteoarthritis drug treatments, such as short intra-articular drug retention time, poor cartilage penetration, and lack of targeting of diseased cells, this invention provides an intelligent drug delivery system that can achieve long-term retention, high-efficiency penetration, and precise identification of diseased cells, as well as its preparation method and application.

[0011] To achieve the above objectives, the present invention adopts the following technical solution:

[0012] In a first aspect, the present invention provides an intelligent drug delivery system for treating osteoarthritis. The system is a drug-loaded micelle with a hydrodynamic diameter of 16±5 nm and a viral glycoprotein mimic peptide (CMP) covalently coupled to its surface. The sequence of the viral glycoprotein mimic peptide is WYRGRL-PEG3-e9-C6-(GPLGVRG)-r9-C6-Cys. This sequence can achieve non-specific adhesion to the cartilage matrix and be specifically activated in the diseased cell microenvironment.

[0013] Furthermore, the drug-loaded micelles comprise a disease-modifying drug for treating osteoarthritis, DSPE-PEG3400-maleimide, and DSPE-PEG2000, wherein the molar ratio of the disease-modifying drug to DSPE-PEG3400-maleimide and DSPE-PEG2000 is 5:1:9.

[0014] Furthermore, the disease-modifying drug is any one of the following: hypoxia-inducible factor-1α prolyl hydroxylase-2 inhibitor IOX4, Wnt pathway inhibitor, gene therapy drug, or metabolic regulator.

[0015] Furthermore, the viral glycoprotein mimic peptide is linked to the maleimide group on the surface of the drug-loaded micelle via a click chemistry reaction through the thiol group of its C-terminal cysteine ​​residue.

[0016] Secondly, the present invention provides a method for preparing the intelligent drug delivery system as described above, comprising the following steps:

[0017] First, the disease-modifying drug, DSPE-PEG3400-maleimide, and DSPE-PEG2000 are dissolved in an organic solvent in a certain proportion, and then the organic solvent is removed to form a lipid film.

[0018] The lipid film was hydrated with a buffer solution, and then the lipid film was dispersed and filtered to obtain drug-loaded micelles.

[0019] The viral glycoprotein mimic peptide is dissolved, and then the dissolved viral glycoprotein mimic peptide is mixed with the drug-loaded micelles. The mixture is reacted for 10 to 12 hours under conditions of pH 7.2 to 8.0, reaction temperature of 25°C and inert gas protection to obtain the reaction product.

[0020] The reaction product is purified to obtain the intelligent drug delivery system.

[0021] Furthermore:

[0022] The organic solvent is chloroform, the buffer solution is PBS buffer with a pH of 7.4, and the inert gas is nitrogen.

[0023] The solvent for dissolving the viral glycoprotein mimic peptide is 6M guanidine hydrochloride, and the dissolved viral glycoprotein mimic peptide is matched with the thiol group of its C-terminal cysteine ​​to the maleimide on the surface of the drug-loaded micelle in a molar ratio of 4:1.

[0024] The purification was performed using Sepharose CL-4B gel column chromatography.

[0025] Thirdly, the present invention provides the application of the intelligent drug delivery system as described above in the preparation of drugs for treating osteoarthritis or promoting cartilage damage repair, wherein the drugs are administered via intra-articular injection at a frequency of once a month or less.

[0026] Fourthly, the present invention provides a pharmaceutical composition comprising the intelligent drug delivery system as described above and a pharmaceutically acceptable carrier or excipient.

[0027] Fifthly, the present invention provides an application of a viral glycoprotein mimic peptide in the preparation of a targeted drug delivery system for degenerative joint diseases, wherein the sequence of the viral glycoprotein mimic peptide is WYRGRL-PEG3-e9-C6-(GPLGVRG)-r9-C6-Cys.

[0028] In a sixth aspect, the present invention also provides a reagent for the diagnosis or imaging of degenerative joint diseases, comprising a fluorescently labeled intelligent drug delivery system as described above, wherein the fluorescently labeled intelligent drug delivery system is prepared by the preparation method as described above, and in the step of forming the lipid film, 0.08% by weight of the fluorescent dye DiR or coumarin 6 is added. Compared with the prior art, the outstanding beneficial effects and significant progress of the present invention are as follows:

[0029] 1) The technical solution provided by this invention achieves precise identification and intelligent activation delivery of diseased chondrocytes, thus solving the core problem of lack of selectivity in existing technologies. Compared with ligands such as tgg2 aptamers and chondrocyte homing peptides used in existing technologies that cannot distinguish between diseased and healthy chondrocytes, this invention designs a specific cleavage sequence (GPLGVRG) of MMP-13 in a viral glycoprotein mimic peptide, so that the delivery system is activated only around diseased chondrocytes that overexpress MMP-13, thereby precisely limiting the drug action to the diseased area and achieving a major breakthrough from "cartilage targeting" to "disease chondrocyte targeting".

[0030] 2) The technical solution provided by this invention integrates the dual functions of "non-specific adhesion and capture" and "conditional activation endocytosis" in the viral invasion process into a single peptide sequence for the first time. That is, the WYRGRL sequence simulates the initial adhesion mechanism of the virus to ensure long-term retention of the drug in the cartilage matrix, while its MMP13-responsive charge reversal mechanism simulates the activation endocytosis process of the virus, realizing efficient and controllable delivery of the drug into the cell. This innovative dual action mechanism of the virus achieves a perfect combination of long-term retention and controllable endocytosis.

[0031] 3) Existing nanoparticles often fail to meet the requirements of penetrating the cartilage matrix and resisting synovial fluid clearance. The technical solution provided by this invention precisely controls the hydrodynamic diameter of the delivery system to 16±5nm. This size is smaller than the pore size of the cartilage matrix (60nm) to ensure effective penetration. At the same time, combined with the type II collagen adhesion of CMP peptide and the endocytosis function triggered by MMP13, it can achieve cartilage adhesion and retention of diseased chondrocytes, achieving an ideal balance between penetration and retention.

[0032] 4) The technical solution provided by this invention reveals the true therapeutic effect of disease modification and shows excellent prospects for clinical translation. Among them, the intelligent drug delivery system provided by this invention can effectively deliver disease modification drugs such as selective PHD2 inhibitors (IOX4). In animal models, it can simultaneously achieve the dual efficacy of delaying cartilage degeneration (structural protection) and improving functional symptoms. It fully meets the definition criteria of disease-modifying osteoarthritis drugs (DMOADs). Moreover, its monthly dosing regimen is significantly better than the regimens in the prior art that require frequent injections, which can greatly improve patient compliance and reduce the risk of infection.

[0033] 5) The delivery system provided by this invention adopts a modular design, which can not only carry IOX4, but also be applied to the delivery of various disease-modifying drugs such as Wnt inhibitors, gene drugs, and metabolic regulators. In addition, this technology platform can be further expanded to the treatment of cartilage damage repair and other degenerative joint diseases, showing broad application prospects, strong platform technology potential and wide applicability. Moreover, the system uses fully synthetic and biocompatible materials, and the preparation process is mature and stable, with good industrialization feasibility. Compared with the existing technology, it has outstanding beneficial effects and significant progress. Therefore, it has great promotion and application value. Attached Figure Description

[0034] To more clearly illustrate the technical solution of the present invention and the technical effects of implementing the present invention, the accompanying drawings used in the examples of the effects of the present invention will be briefly introduced below.

[0035] Obviously, the accompanying drawings described below are only a portion of the drawings used in the examples of the effects of this invention. For those skilled in the art, other drawings can be obtained from these drawings without any creative effort, but these other drawings also fall within the scope of the drawings required for the embodiments of this invention, wherein:

[0036] Figure 1 The HPLC chromatogram of the viral glycoprotein mimic peptide CMP (molecular weight: 4587.1) provided in Example 1 of the present invention;

[0037] Figure 2 The HPLC chromatogram of type II collagen adhesion peptide, CP (molecular weight: 4587.1), provided in Example 1 of the present invention;

[0038] Figure 3 The HPLC chromatogram of MMP13-activated cell-penetrating peptide, i.e., MP (molecular weight: 3552.0), provided as an example of the effectiveness of this invention (Example 1);

[0039] Figure 4 MS spectrum of CMP (molecular weight: 4587.1), a viral glycoprotein mimic peptide provided in Example 1 of the present invention;

[0040] Figure 5 MS spectrum of type II collagen adhesion peptide CP (molecular weight: 4587.1) provided in Example 1 of the present invention;

[0041] Figure 6 MS spectrum of MMP13-activated cell-penetrating peptide, i.e. MP (molecular weight: 3552.0), provided as an example of the efficacy of the present invention;

[0042] Figure 7 Dynamic light scattering (DLS) particle size distribution of the micelle formulation provided in Example 1 of the present invention;

[0043] Figure 8 Transmission electron microscope (TEM) image of CM-Ms provided for Example 1 of the present invention, wherein the scale bar is 100 nm;

[0044] Figure 9 Zeta potential distribution of CM-Ms after treatment with Ms, CM-Ms, and MMP13 is provided as an example of the effectiveness of the present invention;

[0045] Figure 10 The particle size variation curves of CM-Ms@IOX4 at 37°C or 4°C are provided in Example 1 of the present invention for evaluating the storage stability of CM-Ms@IOX4.

[0046] Figure 11 The cumulative release curves of 10X4 in different formulations provided for Example 1 of the present invention are shown, wherein the data are expressed as mean ± SD (n=3);

[0047] Figure 12 The expression level of MMP13 mRNA in chondrocytes after IL-1β stimulation at different time intervals is shown in Example 2 of the present invention.

[0048] Figure 13 A graph showing the expression level of MMP13 protein in normal or IL-1β-induced diseased chondrocytes, provided as an example of the effectiveness of this invention (Example 2).

[0049] Figure 14 The flow cytometry histogram provided for Example 2 of the effectiveness of the present invention;

[0050] Figure 15 Example 2 of the present invention provides a basis based on Figure 14 Calculated average fluorescence intensity of coumarin-6 (C6);

[0051] Figure 16The diagram shows the cell uptake of CM-Ms@C6 in the presence of an MMP13 inhibitor, as provided in Example 2 of the present invention. In the diagram, DB04760 is a selective MMP-13 inhibitor, GM6001 is a broad-spectrum MMP inhibitor, blue is DAP dye, green is C6 dye, and the scale bar is 50 µm.

[0052] Figure 17 The image provided in Example 2 of the present invention shows a cross-section of a human cartilage explant incubated with a C6-containing formulation for 2 hours and then rinsed for 24 hours. In the image, red represents MMP13, green represents C6, and blue represents DAP. The scale bar is 100µm.

[0053] Figure 18 The HPLC detection result of CMP solution treated with recombinant human MMP13 (rhMMP13) for 8 hours is shown in Example 2 of the present invention. The black circles represent the peaks of CMP peptide and its degradation products, respectively.

[0054] Figure 19 Example 2 of the effect of the present invention Figure 18 Mass spectrum of the degradation peak (WYRGRL-PEG3-(d-Glu)9-Acp-GPLG, molecular weight: 2652.96);

[0055] Figure 20 The graph showing the change in CMP degradation rate under different MMP13 concentrations is provided for Example 2 of the present invention.

[0056] Figure 21 Representative IVIS images at different time points after a single intra-articular injection of a DiR-labeled preparation into the OA knee joint, provided as an example of the effectiveness of the present invention, in Example 3.

[0057] Figure 22 Example 3 of the present invention provides a basis for... Figure 21 Semi-quantitative analysis of calculated fluorescence radiation efficiency of the knee joint in OA mice, where n=5;

[0058] Figure 23 Example 3 of the present invention provides a basis for... Figure 22 Calculate the area under the curve (AUC), where n=5;

[0059] Figure 24 Representative fluorescence images of mice after intra-articular injection of CM-Ms provided in Example 3 of the present invention;

[0060] Figure 25 A bar chart showing the ratio of fluorescence radiation efficiency of the liver and joints in the DiR and DiR-labeled CM-Ms groups at a specified time point, as provided in Example 3 of the present invention.

[0061] Figure 26The images show representative Safranin O / Fix Green stained knee joints of OA mice, a model of medial meniscus instability (DMM), provided in Example 3 of the present invention. The green box represents the normal area, the blue box represents the diseased area, and the scale bar is 200 µm.

[0062] Figure 27 After a single injection of the C6-labeled formulation provided in Example 3 of the present invention Figure 26 Fluorescence imaging of selected areas, where green represents C6, red represents MMP13, and blue represents DAPI, with a scale bar of 50 µm;

[0063] Figure 28 A bar chart showing the average fluorescence intensity analysis of C6 in diseased and normal regions provided in Example 3 of the present invention;

[0064] Figure 29 Example 3 of the present invention provides a basis for... Figure 27 The corresponding ratios are shown in the bar chart. Data are expressed as mean ± SD. *P < 0.001, ns indicates no significant difference between groups.

[0065] Figure 30 The results of the Von Frey test (n=5) during the treatment of the mouse osteoarthritis model with CM-Ms@IOX4 provided in Example 3 of the present invention, where P < 0.01, and ns indicates no significant difference between groups;

[0066] Figure 31 Representative images of the proximal tibial articular cartilage stained with Safranin O / Fix Green at weeks 4 and 8 of treatment, provided as an example of the efficacy of the present invention, wherein the scale bar is 100 µm;

[0067] Figure 32 Example 3 of the present invention provides a basis for... Figure 31 Bar chart analyzing OARSI scores for knee cartilage in week 4;

[0068] Figure 33 Example 3 of the present invention provides a basis for... Figure 31 Bar chart analyzing OARSI scores for knee cartilage at week 8;

[0069] Figure 34 A representative H&E staining image of the synovial membrane of a mouse joint after 8 weeks of treatment, provided as Example 3 of the efficacy of the present invention, wherein the scale bar is 400 µm;

[0070] Figure 35 The representative three-dimensional sagittal imaging image of the medial inferior tibial cartilage at the 8th week of treatment provided for Example 3 of the efficacy of the present invention, wherein the scale bar is 200 µm;

[0071] Figure 36A bar chart of bone volume fraction analysis (BV / TV%) based on three-dimensional reconstruction results provided for Example 3 of the present invention;

[0072] Figure 37 A bar chart analyzing the beam thickness (Tb.Th, mm) based on the three-dimensional reconstruction results is provided for Example 3 of the present invention.

[0073] Figure 38 Two-dimensional micro-CT coronal images of the medial inferior tibial cartilage at week 8 of treatment, provided as an example of the efficacy of the present invention, wherein white arrows indicate the location of osteophyte formation;

[0074] Figure 39 Example 3 of the present invention provides a basis for... Figure 38 A bar chart showing the number of osteophytes;

[0075] Figure 40 The mouse osteoarthritis model provided in Example 3 of the present invention uses representative images of HIF-1α immunofluorescence staining of tibial articular cartilage after 4 and 8 weeks of treatment with CM-Ms@IOX4.

[0076] Figure 41 The bar chart for the average fluorescence intensity analysis of HIF-1α provided in Example 3 of the present invention;

[0077] Figure 42 The mouse osteoarthritis model provided in Example 3 of the present invention uses representative immunohistochemical (IHC) images of type II collagen (COL2) in the tibial articular cartilage after CM-Ms@IOX4 treatment;

[0078] Figure 43 A bar chart showing the percentage of COL2 positive area provided in Example 3 of the present invention;

[0079] Figure 44 Immunohistochemical images of MMP13 in tibial articular cartilage after treatment with CM-Ms@IOX4 were used to illustrate the effect of the present invention in the mouse osteoarthritis model.

[0080] Figure 45 A bar chart showing the percentage of positive MMP13 area provided in Example 3 of the present invention;

[0081] Figure 46 Immunohistochemical images of tibial articular cartilage aggregate glycan (ACAN) in the mouse osteoarthritis model provided in Example 3 of the present invention were obtained after treatment with CM-Ms@IOX4.

[0082] Figure 47 A bar chart showing the percentage of positive ACAN area provided in Example 3 of the present invention;

[0083] Figure 48Immunohistochemical images of ADAMTS-5 in tibial articular cartilage after treatment with CM-Ms@IOX4 were obtained from the mouse osteoarthritis model provided in Example 3 of this invention.

[0084] Figure 49 The percentage of positive area of ​​ADAMTS-5 in the tibial articular cartilage after treatment with CM-Ms@IOX4 in the mouse osteoarthritis model provided in Example 3 of the present invention is shown in the bar chart.

[0085] Figure 50 A bar chart illustrating the regulatory effect of CM-Ms@IOX4 on downstream genes of HIF-1α, provided in Example 3 of the present invention.

[0086] Figure 51 The results of Safranin O / Fix Green staining of tibial articular cartilage in the negative control group NC and the Hif1a silent AAV group at week 8 after sham surgery or anterior cruciate ligament transection (ACLT) provided in Example 3 of the present invention are shown in Figure 3. The scale bar is 200 µm.

[0087] Figure 52 Example 3 of the present invention provides a basis for... Figure 50 A bar chart analyzing the OARSI score of knee cartilage.

[0088] Figure 53 A collection of representative photographs of sheep walking gait after treatment, provided as an example of the effectiveness of this invention, in Example 4.

[0089] Figure 54 The image shown is an X-ray image set of articular cartilage of a sheep osteoarthritis model provided in Example 4 of the present invention, wherein the yellow dashed circle indicates the bone marrow lesion area;

[0090] Figure 55 Example 4 of the present invention provides a basis based on Figure 54 MOCART score analysis bar chart;

[0091] Figure 56 This is a collection of representative photographs of osteoarthritis cartilage after treatment with saline, sodium hyaluronate injection, IOX4, and CM-Ms@IOX4, provided as an example of the effectiveness of this invention (Example 4).

[0092] Figure 57 Example 4 of the present invention provides a basis based on Figure 56 Macroscopic scoring bar chart of femoral condyle and tibial plateau joint surface injuries;

[0093] Figure 58 The image shown is a representative set of Safranin O / Fix Green staining images of femoral condyle tissue after 12 weeks of treatment, provided in Example 4 of the present invention.

[0094] Figure 59 Example 4 of the present invention provides a basis based on Figure 58 The OARSI score bar chart, with the upper scale bar at 500 µm and the lower scale bar at 2 cm;

[0095] Figure 60 A representative set of H&E staining images of femoral condyle tissue after 12 weeks of treatment, provided as an example of the effectiveness of the present invention (Example 4).

[0096] Figure 61 The bar chart showing the thickness ratio of hyaline cartilage to calcified cartilage provided in Example 4 of the present invention has an upper scale bar of 500 µm and a lower scale bar of 2 cm.

[0097] Figure 62 A bar chart for evaluating the cytotoxicity of CM-Ms against primary chondrocytes CCK-8 after 24 hours of incubation, provided as Example 5 of the present invention;

[0098] Figure 63 A bar chart evaluating the cytotoxicity of 10X4 on primary chondrocytes CCK-8 after 24 hours of incubation, as provided in Example 5 of the present invention.

[0099] Figure 64 The representative image set of H&E staining of the knee joint of sham-operated mice on days 0, 3 and 10 after intra-articular injection of different formulations provided in Example 5 of the present invention, wherein the scale bar is 200 µm;

[0100] Figure 65 The image set of representative H&E staining of the heart, liver, spleen, lungs and kidneys of OA mice after 8 weeks of treatment is provided for Example 5 of the present invention, wherein the scale bar is 200 µm;

[0101] Figure 66 The representative H&E staining image set of the heart, liver, spleen, lung and kidney of OA sheep after 8 weeks of treatment is provided for Example 5 of the present invention, wherein the scale bar is 200 µm;

[0102] Figure 67 The hematological index test results provided in Example 5 of the present invention are shown in which the orange dashed line represents the normal range of each index, and the data are expressed as mean ± standard deviation (n=3).

[0103] In the above bar charts, the data are expressed as mean ± SD standard deviation (n = 5). *P < 0.05 and **P < 0.01 indicate significant differences, and ns indicates no statistical significance. The statistical analysis was performed using nonparametric two-tailed ANOVA and Tukey post-hoc test. Detailed Implementation

[0104] To make the technical solution, beneficial effects and significant progress of the present invention clearer and more comprehensive, the technical solution provided by the present invention will be clearly and completely described below through specific embodiments and their effects. Obviously, all embodiments and their effects described below are only some embodiments and effects of the present invention, and not all of them.

[0105] Based on the embodiments and effects provided by this invention, all other embodiments and effects obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0106] It should be noted that:

[0107] The terms "firstly," "secondly," etc., used in the claims, description, and examples and effects of the embodiments of this invention are only used to distinguish different objects and not to describe a specific order; furthermore, the term "comprising" and any variations thereof are intended to cover non-exclusive inclusion, for example, including not only a series of listed steps or units of a process, method, system, product, or device, but also optionally steps or units not listed, or optionally other operational steps or units inherent to these processes, methods, products, or devices.

[0108] What needs to be understood is:

[0109] In the description of the embodiments of the present invention, some basic operational terms commonly used in the art are used, such as "dissolving" and "drying". These terms should be interpreted broadly, that is, they can refer to routine operations performed using various conventional equipment and instruments in the art, or operations performed using the latest equipment, such as programmed operations and unmanned automatic operations. Unless otherwise explicitly limited, those skilled in the art should understand the specific meaning of the above terms in the present invention according to the specific circumstances and adopt specific operating methods to achieve their operational objectives.

[0110] It should also be noted that:

[0111] The following specific embodiments can be combined with each other. The same or similar concepts or processes may not be repeated in some implementation cases and comparative examples. In addition, all kinds of instruments, equipment, raw materials, reagents and standards involved in the following specific embodiments are commercially available unless otherwise specified.

[0112] The technical solution of the present invention will now be described in detail with reference to specific embodiments. Example 1

[0113] This embodiment provides a 16 nm smart drug delivery system for treating osteoarthritis.

[0114] 1.1) Preparation of viral glycoprotein mimic peptide (CMP):

[0115] On an automated peptide synthesizer, a viral glycoprotein mimic peptide (CMP) with the sequence WYRGRL-PEG3-e9-C6-(GPLGVRG)-r9-C6-Cys was synthesized using a solid-phase peptide synthesis method combined with an Fmoc protection strategy. After synthesis, the CMP was cleaved from the resin using a cleavage buffer (TFA:TIS:H2O=95:2.5:2.5), and the peptide product was collected by precipitation with cold diethyl ether and centrifugation.

[0116] 1.2) Preparation of drug-loaded micelles:

[0117] Accurately weigh 5 µmol of IOX4, 1 µmol of DSPE-PEG3400-maleimide and 9 µmol of DSPE-PEG2000, dissolve them together in 10 mL of chloroform, remove the solvent using a rotary evaporator in a 35°C water bath to form a uniform lipid film, and place this lipid film in a vacuum drying oven overnight to completely remove residual solvent.

[0118] The lipid membrane was hydrated at 4°C using 10 mL of PBS buffer with a pH of 7.4. After vortexing, the membrane was filtered through a 0.22 µm polyethersulfone membrane to obtain drug-loaded micelles Ms@IOX4 with uniform particle size.

[0119] 1.3) Preparation of peptide-micelle conjugates:

[0120] CMP was dissolved in 6M guanidine hydrochloride solution and added to Ms@IOX4 micelle suspension at a molar ratio of 4:1 for thiol to maleimide groups. The pH was adjusted to 7.5 with 0.1M NaOH solution, and the reaction was carried out at 25°C under nitrogen protection for 10–12 hours. After the reaction was completed, the mixture was purified by chromatography using a Sepharose CL-4B gel column. The first blue opalescent fraction to elute was collected to obtain CM-Ms@IOX4.

[0121] The detailed functions of each unit in the CMP provided in this embodiment are as follows:

[0122] a) WYRGRL (6 amino acids): A type II collagen-specific adhesion sequence that can bind to type II collagen synthesized by chondrocytes and anchored on the chondrocyte plasma membrane, serving as a "capture-hold" mechanism on the surface of cartilage and chondrocytes.

[0123] b) PEG3: polyethylene glycol spacer group, providing flexibility and hydrophilicity;

[0124] c) e9 (9 D-glutamates): a negatively charged polyanionic sequence used to neutralize the positive charge of r9 and prevent non-specific cellular uptake;

[0125] d) C6 (6-aminohexanoic acid): spacer group;

[0126] e)GPLGVRG (7 amino acids): a specific cleavage sequence of matrix metalloproteinase 13 (an endopeptidase, abbreviated as MMP13);

[0127] f)r9 (9 D-arginine residues): A positively charged cell-penetrating peptide that can quickly and efficiently cross the cell membrane;

[0128] g) Cys: C-terminal cysteine ​​provides a thiol group for coupling with maleimide groups on the micelle surface.

[0129] In this embodiment, IOX4 was chosen as the model drug representing the disease-modifying drug because:

[0130] First, IOX4 is a selective inhibitor of hypoxia-inducible factor-1α (HIF-1α) prolyl hydroxylase-2 (PHD2), and HIF-1α is crucial for maintaining cartilage homeostasis by coordinating the metabolic adaptation of chondrocytes to their hypoxic microenvironment through upregulation of glycolytic enzymes and glucose transporters.

[0131] Secondly, HIF-1α promotes the expression of key cartilage matrix components such as type II collagen and proteoglycans, while inhibiting hypertrophic differentiation. IOX4 requires a sustained high intracellular concentration to inhibit enzyme activity and maintain HIF-1α levels. Therefore, IOX4 is an ideal model drug for disease modification.

[0132] It can be seen that the intelligent drug delivery system provided in this embodiment has a multi-level mechanism of action, including:

[0133] Cartilage retention mechanism: Since CM-Ms can bind to type II collagen on the surface of cartilage matrix and chondrocytes via the WYRGRL sequence, they can achieve initial anchoring and prevent being washed away by synovial fluid.

[0134] Disease cell recognition mechanism: Studies have found that diseased chondrocytes selectively overexpress MMP13 (overexpressed in articular cartilage of OA patients, but almost undetectable in normal adult tissues), and MMP13 selectively accumulates around diseased chondrocytes in OA patients. Therefore, the intelligent drug delivery system provided in this embodiment can recognize diseased cells through MMP13.

[0135] Activation of endocytosis mechanism; after MMP13 cleaves the GPLGVRG sequence, the negatively charged polyglutamate fragment (WYRGRL-PEG3-e9-C6-GPLG) is detached, exposing the positively charged CPP fragment ((VRG)-r9-C6-Cys), and the surface potential changes from near neutral to positive, driving cellular uptake;

[0136] The intelligent drug delivery system provided in this embodiment has a hydrodynamic diameter precisely controlled at 16±5nm. This size is smaller than the pore size of the cartilage matrix (60nm), which can ensure effective penetration, and can also achieve cartilage retention through the type II collagen adhesion of CMP and the endocytosis triggered by MMP13, thus achieving an ideal balance between penetration and retention.

[0137] It should be noted that:

[0138] The above preparation example uses the optimal solution in the technical solution provided in this embodiment, including the optimal ratio and the optimal process control parameters. However, in fact, experiments have shown that using all the feeding ratios and all the process control (range) parameters provided in this embodiment can yield products with the same or basically the same properties and characteristics as those obtained in the above preparation example. This embodiment is only for the purpose of simplifying its description, so it has not been described in detail. It is only a representative description using the above preparation example as an example. Therefore, it can be understood that the above preparation example cannot be used as a limiting case of the present invention.

[0139] From the above preparation examples, it can be seen that:

[0140] The preparation method provided in this embodiment is relatively simple, the production process is stable, and it can ensure the quality of the product and achieve the corresponding technical effects.

[0141] To further aid in understanding the technical solution provided in this embodiment, as well as the specific operation process and the effects that can be obtained, the preparation method and intelligent drug delivery system provided in this embodiment will be further explained below through specific effect examples.

[0142] Of course, those skilled in the art should understand that the examples described below are illustrative and not restrictive, and should not be used to limit the scope of protection claimed by the present invention.

[0143] To provide a clear and effective comparison, the following preparations are made: first, a control peptide is prepared according to the CMP preparation method described in Example 1 above, and fluorescently labeled micelles are prepared according to the drug-loaded micelle preparation method. Then, the relevant technical effects are compared through the following effect examples:

[0144] The following control peptides were prepared according to the CMP preparation method described in Example 1 above:

[0145] 1) Type II collagen adhesion peptide, abbreviated as CP, is a peptide with the sequence WYRGRL-PEG3-e9-C6-(gplgvrg)-r9-C6-Cys, which is the peptide in CMP in which (GPLGVRG) is replaced by D-amino acid (gplgvrg), and the (gplgvrg) sequence cannot be cleaved by MMP13;

[0146] 2) MMP13 is a cell-penetrating peptide, abbreviated as MP, whose sequence is e9-C6-(GPLGVRG)-r9-C6-Cys), meaning that CMP lacks the peptide sequence for adhering to type II collagen;

[0147] Fluorescently labeled micelles were prepared according to the preparation method of drug-loaded micelles in Example 1 above: that is, according to the preparation method of Example 1 above, when preparing drug-loaded micelles, DiR or coumarin 6 (C6) dye (accounting for 0.08% of the lipid weight) was added to the lipid mixture in chloroform, and then the preparation method described in Example 1 was continued to be operated to obtain a fluorescently labeled intelligent drug delivery system. Example 1: Quality control and characterization methods and test results of CMP, CP, and MP.

[0148] 1.1) Identification and purity of peptides

[0149] Characterization methods: The purity and structure of the synthesized CMP, CP and MP were identified by high performance liquid chromatography-mass spectrometry. The analytical conditions of HPLC were as follows: C18 column, mobile phase A was an aqueous solution containing 0.1% TFA, mobile phase B was an acetonitrile solution containing 0.1% TFA, and gradient elution was used.

[0150] Through testing, we can obtain: such as Figure 1 The image shown is the HPLC chromatogram of the viral glycoprotein mimic peptide CMP (molecular weight: 4587.1) provided in Example 1 of the present invention. Figure 2 The HPLC chromatogram of type II collagen adhesion peptide CP (molecular weight: 4587.1) provided in Example 1 of the present invention is shown below. Figure 3 The HPLC chromatogram of MMP13-activated cell-penetrating peptide, i.e., MP (molecular weight: 3552.0), provided for Example 1 of the present invention, and Figure 4 MS spectrum of CMP (molecular weight: 4587.1), a viral glycoprotein mimic peptide provided in Example 1 of the present invention. Figure 5 MS spectrum of type II collagen adhesion peptide CP (molecular weight: 4587.1) provided in Example 1 of the present invention. Figure 6 MS spectrum of MMP13-activated cell-penetrating peptide, i.e. MP (molecular weight: 3552.0), provided as an example of the efficacy of the present invention.

[0151] The test results show that:

[0152] The retention time of the obtained CMP peptide was 11.88 minutes, and the purity was 99.2%.

[0153] Mass spectrometry analysis showed multiple multicharged ion peaks, including [M+5]5+ m / z 918.29, [M+6]6+ m / z 765.41, [M+7]7+ m / z 656.21, and [M+8]8+ m / z 574.43, which are consistent with the theoretical molecular weight (4587.1 Da).

[0154] 1.2) Determination of peptide coupling efficiency

[0155] Assay method: The determination is indirectly performed by measuring the unreacted maleimide groups. Specifically, 3 molar amounts of cysteine ​​are added to the micelle suspension and reacted for 30 minutes to block unreacted maleimide. Excess cysteine ​​reacts with Ellman's reagent, releasing an equal amount of free 5-thio-2-nitrobenzoic acid anion (TNB). - ), quantitative detection of TNB by HPLC - Calculate the coupling efficiency.

[0156] The test results show that the coupling efficiency of CMP in Example 1 is 88.89%.

[0157] 1.3) Particle size and potential determination

[0158] Measurement methods: Measurements were performed using dynamic light scattering (DLS), transmission electron microscopy (TEM), and zeta potential.

[0159] Through testing, we can obtain, for example:

[0160] Figure 7 The diagram shown is a dynamic light scattering (DLS) particle size distribution of the micelle formulation provided in Example 1 of the present invention;

[0161] Figure 8 The image shown is a transmission electron microscope (TEM) image of CM-Ms provided in Example 1 of the present invention, wherein the scale bar is 100 nm; and

[0162] Figure 9 The diagram shown is a Zeta potential distribution of CM-Ms processed by Ms, CM-Ms and MMP13 provided in Example 1 of the present invention. The data is shown as average value ± SD (n=3).

[0163] from Figure 7 As can be seen, the particle size of Ms is 11.67 nm, the particle size of Ms@IOX4 is 13.58 nm, and the particle size of CM-Ms@IOX4 is 16.22 nm.

[0164] from Figure 8 As can be seen from the transmission electron microscope (TEM), CM-Ms@IOX4 is a uniform sphere.

[0165] from Figure 9 As can be seen, the Zeta potential of Ms@IOX4 is -2.85±0.35 mV, the Zeta potential of CM-Ms@IOX4 is 0.214±0.28 mV, and the Zeta potential of CM-Ms@IOX4 after MMP13 treatment is +7.43±0.5 mV.

[0166] 1.4) X-ray photoelectron spectroscopy (XPS) analysis

[0167] The test revealed that sulfur (0.17%) was detected on the surface of CM-Ms, while no sulfur was detected on the surface of Ms, confirming that CMP coupling was successful.

[0168] 1.5) Stability Evaluation

[0169] The storage stability of CM-Ms@IOX4 at 37℃ or 4℃ was evaluated by monitoring the change in particle size over time, and the results were as follows: Figure 10 The figure shown is the particle size change curve of CM-Ms@IOX4 provided in Example 1 of the present invention during the storage stability evaluation at 37°C or 4°C. It can be seen that after 7 days of storage at 4°C and 37°C, the particle size change of CM-Ms@IOX4 is less than 5%, indicating that it has excellent storage stability.

[0170] 1.6) Drug Encapsulation and Release

[0171] Through testing, we can obtain results such as Figure 11 The figures shown are the cumulative release curves of 10X4 in different formulations provided in Example 1 of the present invention, where the data are expressed as mean ± SD (n=3).

[0172] from Figure 11 As can be seen, the drug loading (DLC) of CM-Ms@IOX4 is 5.86±0.41%, the encapsulation efficiency (EE) is 97.6±0.73%, and the cumulative release after 96 hours is about 65%, exhibiting sustained-release characteristics. Example 2: In vitro experimental verification

[0173] 2.1) Establishment of a diseased chondrocyte model

[0174] Primary chondrocytes were extracted from the distal femur and proximal tibial epiphysis of 1- to 5-day-old mice, digested with 2 mg / mL type II collagenase for 12 hours, the isolated cells were collected, and treated with IL-1β (10 ng / mL) for 48 hours to induce a diseased phenotype, and the following results could be obtained Figure 12 as shown in the graph of the expression level of MMP13 mRNA in chondrocytes after different time intervals of IL-1β stimulation provided in Effect Example 2 of the present invention, and Figure 13 as shown in the graph of the expression level of MMP13 protein in normal or IL-1β-induced diseased chondrocytes provided in Effect Example 2 of the present invention.

[0175] It can be seen from Figure 12 that the expression of MMP13 mRNA continued to increase; and it can be seen from Figure 13 that the protein level increased from 0.37 ng / mL to 14.60 ng / mL, a 39.5-fold increase.

[0176] 2.2) Quantitative analysis of cell uptake

[0177] Through detection, the following results could be obtained: the flow cytometry histogram provided in Effect Example 2 of the present invention as shown in Figure 14 , the graph of the average fluorescence intensity of coumarin-6 (C6) calculated based on Figure 15 provided in Effect Example 2 of the present invention, and Figure 14 the graph of the cell uptake of CM-Ms@C6 in the presence of an MMP13 inhibitor provided in Effect Example 2 of the present invention as shown in Figure 16 , where DB04760 is a selective MMP-13 inhibitor, GM6001 is a broad-spectrum MMP inhibitor, the blue is DAPI dye, the green is C6 dye, and the scale bar is 50 µm.

[0178] It can be seen from Figure 14 and Figure 15 that flow cytometry shows that the uptake ability order of each formulation is: Ms < C-Ms < M-Ms < CM-Ms; the uptake amount of CM-Ms is 3.12 times higher than that of Ms, 1.93 times higher than that of C-Ms, and 1.16 times higher than that of M-Ms;

[0179] It can be seen from Figure 16 that pretreatment with the specific MMP13 inhibitor DB04760 (10 μM) or the broad-spectrum MMP inhibitor GM6001 (10 μM) significantly inhibits the uptake of CM-Ms, thus demonstrating the importance of MMP13 activation.

[0180] 2.3) Perfusate experiment on human cartilage explants

[0181] Cartilage explants with a diameter of 4 mm and a thickness of 1 mm were harvested from the femoral head surface of patients undergoing hip replacement surgery. A microfluidic device was constructed, and the explants were fixed in a groove with their surface flush with the device surface. After incubation for 2 hours, the explants were continuously flushed at a flow rate of 200 μL / min for 24 hours to obtain the desired results. Figure 17 The image shown is a representative fluorescence image of a cross section of a human cartilage explant provided in Example 2 of the present invention after incubation with a C6-containing preparation for 2 hours and rinsing for 24 hours. In the image, red represents MMP13, green represents C6, blue represents DAPI, and the scale bar is 100 µm.

[0182] from Figure 17 As can be seen above, CM-Ms maintain high fluorescence intensity in the lesion area, while the fluorescence intensity in the normal area is significantly reduced, proving selective retention.

[0183] 2.4) Verification of MMP13-mediated CMP fragmentation

[0184] Incubation with 400 μg / mL CMP and 200 ng / mL MMP13 for 8 hours yielded a degradation product peak (retention time 12.75 min) detected by HPLC, which can be used to obtain... Figure 18 The figure shown is an HPLC detection result of CMP solution treated with recombinant human MMP13 (rhMMP13) for 8 hours according to Example 2 of the present invention. The black circles represent peaks of the CMP peptide and its degradation products, respectively; and... Figure 19 The example shown is provided by Example 2 of the effect of the present invention. Figure 18 Mass spectrum of the degradation peak (WYRGRL-PEG3-(d-Glu)9-Acp-GPLG, molecular weight: 2652.96), Figure 20 The figure shown is a graph showing the change in CMP degradation rate at different MMP13 concentrations provided in Example 2 of the present invention.

[0185] from Figure 18 , Figure 19 and Figure 20 As can be seen, the degradation product is WYRGRL-PEG3-(d-Glu)9-Acp-GPLG (molecular weight 2652.96), confirming that it cleaved at the expected location, and the CMP degradation rate was dose-dependent on the MMP13 concentration. Example 3: Small animal model experiment

[0186] 3.1) Establishment of the OA Model

[0187] Eight-week-old male C57 mice were anesthetized with sodium pentobarbital intraperitoneally and OA was induced by right anterior cruciate ligament transection (ACLT). The sham surgery group underwent the same procedure but without ligament transection. The heterogeneous cartilage degeneration model was performed using medial meniscus instability (DMM) surgery.

[0188] Dosing regimen (fluorescently labeled smart drug delivery system):

[0189] Experimental group: The dose was 1 μg IOX4 / mouse, administered via intra-articular injection, once a week or once a month, for a treatment period of 8 weeks;

[0190] Control group: PBS, free IOX4, Ms@IOX4, C-Ms@IOX4, M-Ms@IOX4.

[0191] After testing, the following can be obtained: Figure 21 The image shown is a representative IVIS image at different time points after a single intra-articular injection of the DiR-labeled preparation into the OA knee joint provided in Example 3 of the present invention. Figure 22 Example 3 of the present invention provides a basis for... Figure 21 Semi-quantitative analysis of calculated fluorescence radiation efficiency of the knee joint in OA mice, where n=5; and Figure 23 Example 3 of the present invention provides a basis for... Figure 22 Calculate the area under the curve (AUC), where n=5;

[0192] from Figure 21 As can be seen from the in vivo pharmacokinetic studies, CM-Ms@DiR remained in the joint for more than 38 days, and its AUC (area under the curve) was 2.45 times higher than that of unmodified micelles. Furthermore, in vivo clearance studies showed that the liver was its main clearance organ.

[0193] from Figure 22 and Figure 23 The results show that the liver / joint fluorescence intensity ratio indicates that at 24 hours, CM-Ms is 3.19 times lower than free DiR, at 48 hours it is 6.36 times lower, and at 72 hours it is 1.82 times lower.

[0194] 3.2) Intracartilaginous distribution:

[0195] After testing, the following can be obtained: Figure 24 The image shown is a representative fluorescence image of mice after intra-articular injection of CM-Ms, as provided in Example 3 of the present invention. Figure 25 The bar chart shown is a comparison of the fluorescence radiative efficiency ratio of the liver and joints in the CM-Ms groups isolated with DiR and DiR labels at specified time points, provided in Example 3 of the present invention. Data are expressed as mean ± SD (n=3), **P < 0.01, ***P < 0.001, and nonparametric two-tailed ANOVA was used in the labeled groups, followed by Tukey's post-hoc test; and as shown in Example 3 of the present invention. Figure 26The image shown is a representative Safranin O / Fix Green stained image of the knee joint of an OA mouse model of medial meniscus instability (DMM) provided in Example 3 of the present invention. The green box represents the normal area, the blue box represents the diseased area, and the scale bar is 200 µm. Figure 27 The image shown is an example of the effect of a single injection of the C6-labeled formulation provided in Example 3 of the present invention. Figure 26 Fluorescence imaging of selected areas, where green represents C6, red represents MMP13, and blue represents DAPI, with a scale bar of 50 µm; Figure 28 A bar chart showing the average fluorescence intensity analysis of C6 in diseased and normal regions provided in Example 3 of the present invention; Figure 29 Example 3 of the present invention provides a basis for... Figure 27 The corresponding ratios are shown in the bar chart. Data are expressed as mean ± SD. ***P < 0.001, ns indicates no significant difference between groups.

[0196] from Figure 24 and Figure 25 It can be seen that after 48 hours, the fluorescence intensity of CM-Ms in the diseased chondrocytes was 4.38 times higher than that of Ms, and the ratio of diseased area to normal area was 3.80; while after 96 hours, the fluorescence intensity of CM-Ms in the diseased chondrocytes was 15.06 times higher than that of Ms, and the ratio of diseased area to normal area was 9.26.

[0197] from Figures 26-29 As can be seen, there are no significant differences between the labeled groups.

[0198] 3.3) Pain assessment (Von Frey test)

[0199] Experiments can yield results such as... Figure 30 The results of the Von Frey test (n=5) during the treatment of the mouse osteoarthritis model with CM-Ms@IOX4 in Example 3 of the present invention are shown in the figure. **P < 0.01, and ns indicates no significant difference between groups.

[0200] The results showed that CM-Ms@IOX4 significantly improved the 50% foot withdrawal threshold (PWT), reaching a level comparable to the sham surgery group, indicating that it effectively relieved joint pain.

[0201] 3.4) Histological evaluation

[0202] The results of the tests show that:

[0203] Figure 31 Representative images of the proximal tibial articular cartilage stained with Safranin O / Fix Green at weeks 4 and 8 of treatment, provided as an example of the efficacy of the present invention, wherein the scale bar is 100 µm; Figure 32 Example 3 of the present invention provides a basis for... Figure 31Bar chart analyzing OARSI scores for knee cartilage in week 4; Figure 33 Example 3 of the present invention provides a basis for... Figure 31 Bar chart analyzing the OARSI score of knee cartilage in week 8.

[0204] Figures 31-33 The results showed that CM-Ms@IOX4 treatment significantly improved cartilage structure and reduced the degree of degeneration.

[0205] 3.5) Synovial membrane evaluation

[0206] After testing, the following can be obtained: Figure 33 The image shown is a representative H&E staining image of the synovial membrane of a mouse joint after 8 weeks of treatment, provided in Example 3 of the present invention. The scale bar is 400 µm.

[0207] Figure 33 The results showed that CM-Ms@IOX4 can effectively inhibit synovial thickening and inflammatory cell infiltration.

[0208] 3.6) MicroCT Analysis

[0209] After testing, the following can be obtained: Figure 35 The representative three-dimensional sagittal imaging image of the medial inferior tibial cartilage at the 8th week of treatment provided for Example 3 of the efficacy of the present invention, wherein the scale bar is 200 µm; Figure 36 A bar chart of bone volume fraction analysis (BV / TV, %) based on three-dimensional reconstruction results provided for Example 30 of the present invention; Figure 37 A bar chart analyzing the beam thickness (Tb.Th, mm) based on the three-dimensional reconstruction results is provided for Example 3 of the present invention. Figure 38 Two-dimensional micro-CT coronal images of the medial inferior tibial cartilage at week 8 of treatment, provided as an example of the efficacy of the present invention, wherein white arrows indicate the location of osteophyte formation; Figure 39 Example 3 of the present invention provides a basis for... Figure 38 A bar chart of the number of osteophytes was generated. All data in the bar charts are expressed as mean ± standard deviation (n=5). *P < 0.05 and **P < 0.01 indicate significant differences, and ns indicates no statistical significance. The statistical analysis was performed using nonparametric two-tailed ANOVA and Tukey post-hoc test.

[0210] from Figures 35-39 The results show that the bone volume fraction (BV / TV) in the CM-Ms@IOX4 group was 57.02%, which was significantly lower than that in other treatment groups; the trabecular bone thickness (Tb.Th) in the CM-Ms@IOX4 group was the thinnest; and the osteophyte formation in the CM-Ms@IOX4 group was significantly reduced.

[0211] 3.7) Molecular mechanism verification

[0212] The results show that:

[0213] like Figure 40 The images shown represent HIF-1α immunofluorescence staining images of the tibial articular cartilage of the mouse osteoarthritis model provided in Example 3 of the present invention after treatment with CM-Ms@IOX4 for 4 weeks and 8 weeks.

[0214] like Figure 41 The bar chart shown is a histogram of the average fluorescence intensity of HIF-1α provided in Example 3 of the present invention.

[0215] like Figure 42 The image shown is a representative immunohistochemical (IHC) image of type II collagen (COL2) in the tibial articular cartilage after treatment with CM-Ms@IOX4 in the mouse osteoarthritis model provided in Example 3 of the present invention.

[0216] like Figure 43 The figure shown is a bar chart of the percentage of COL2 positive area provided in Example 3 of the present invention;

[0217] like Figure 44 The image shown is an immunohistochemical image of MMP13 in the tibial articular cartilage of a mouse osteoarthritis model provided in Example 3 of the present invention after treatment with CM-Ms@IOX4.

[0218] like Figure 45 The figure shown is a bar chart of the percentage of MMP13 positive area provided in Example 3 of the present invention;

[0219] like Figure 46 The image shown is an immunohistochemical image of tibial articular cartilage aggregate glycan (ACAN) in a mouse osteoarthritis model provided in Example 3 of the present invention after treatment with CM-Ms@IOX4.

[0220] like Figure 47 The figure shown is a bar chart of the percentage of ACAN positive area provided in Example 3 of the present invention;

[0221] like Figure 48 The image shown is an immunohistochemical image of ADAMTS-5 in the tibial articular cartilage of a mouse osteoarthritis model provided in Example 3 of the present invention after treatment with CM-Ms@IOX4.

[0222] like Figure 49The bar chart shown is a percentage of positive area of ​​ADAMTS-5 in the tibial articular cartilage after treatment with CM-Ms@IOX4 in the mouse osteoarthritis model provided in Example 3 of the present invention. All data in the bar charts are expressed as mean ± SD standard deviation (n = 5). *P < 0.05, **P < 0.01 indicate significant difference, and ns indicates no statistically significant difference. Statistical analysis was performed using nonparametric two-tailed ANOVA, and Tukey's post-hoc test was conducted.

[0223] like Figure 50 The bar chart shown is a graph illustrating the regulatory effect of CM-Ms@IOX4 on downstream genes of HIF-1α provided in Example 3 of the present invention.

[0224] from Figure 40 and Figure 41 As can be seen, after 8 weeks of treatment with CM-Ms@IOX4 in a mouse osteoarthritis model, the HIF-1α level in the CM-Ms@IOX4 group was 10.98 times, 4.53 times, and 3.08 times higher than that in the PBS, IOX4, and Ms@IOX4 groups, respectively.

[0225] from Figure 42 and Figure 43 As can be seen, after 8 weeks of treatment with CM-Ms@IOX4 in the mouse osteoarthritis model, the COL2 immunohistochemical area of ​​the CM-Ms@IOX4 group was the largest.

[0226] from Figure 44 and Figure 45 As can be seen, after 8 weeks of treatment with CM-Ms@IOX4 in the mouse osteoarthritis model, the MMP13 positive area in the CM-Ms@IOX4 group was the smallest.

[0227] from Figure 46 and Figure 47 As can be seen, after 8 weeks of treatment with CM-Ms@IOX4 in a mouse osteoarthritis model, the expression of aggregated proteoglycan (ACAN) was highest in the CM-Ms@IOX4 group.

[0228] from Figure 48 and Figure 49 As can be seen, after 8 weeks of treatment with CM-Ms@IOX4, the expression of ADAMTS-5 (ACAN lyase) in the mouse osteoarthritis model was lowest in the CM-Ms@IOX4 group.

[0229] And from Figure 50 The results show that after 8 weeks of treatment with CM-Ms@IOX4, the expression of Col2α1, Acan, and Sox9 mRNA in the mouse osteoarthritis model was significantly upregulated by RT-qPCR.

[0230] 3.8) HIF-1α knockdown verification

[0231] By constructing the AAV-shHif1a virus and performing HIF-1α knockdown verification, the following results can be obtained: Figure 51 The image shown is a diagram of the results of Safranin O / Fix Green staining of the tibial articular cartilage in the negative control group NC and the Hif1a silent AAV group at week 8 after sham surgery or anterior cruciate ligament transection (ACLT) provided in Example 3 of the present invention. The scale bar is 200 µm. Figure 52 The example shown is based on the effect of the present invention, Example 3. Figure 50 The OARSI score analysis of knee cartilage is shown in the bar chart. Data are expressed as mean ± standard deviation (n=5). P < 0.05, P < 0.01, P < 0.001, and ns indicate no significant difference.

[0232] from Figure 51 and Figure 52 As can be seen, at week 8 after sham surgery or anterior cruciate ligament transection (ACLT), in the negative control group (NC) and the Hif1a-silenced AAV group, the knockdown efficiency of the constructed AAV-shHif1a virus after CM-Ms@IOX4 treatment was 88.68%. This shows that HIF-1α knockdown had little effect on the OARSI score in the sham surgery control group, but significantly promoted cartilage degeneration in the ACLT group, thus confirming that HIF-1α plays a key role in maintaining OA cartilage homeostasis. Example 4: Validation using a large animal model

[0233] Establishment of the sheep OA model:

[0234] Adult female sheep weighing 75±10 kg were selected and fasted for 12 hours before the operation. They were sedated by intramuscular injection of teretamine hydrochloride and zoprazepam hydrochloride (6.0 mg / kg). After tracheal intubation, anesthesia was maintained with 1-5% isoflurane. An open medial partial anterior meniscectomy and anterior cruciate ligament transection of the right knee joint were performed. The left knee was used as a sham control.

[0235] Dosage regimen:

[0236] The experimental group received 4 mL CM-Ms@IOX4 (0.1 mg / mL), while the control group received normal saline, sodium hyaluronate (HA), and free IOX4. The first treatment was one week after the operation. The dosing frequency was once every 10 days. The treatment cycle was 3 months. The evaluation time points were 1, 2, and 3 months after treatment.

[0237] 4.1) Functional Evaluation

[0238] Through experimentation, the following can be obtained: Figure 53This is a collection of representative photographs of sheep walking gait after treatment, provided as an example of the effectiveness of this invention, in Example 4.

[0239] from Figure 53 The results show that: in terms of standing posture, sheep in the CM-Ms@IOX4 group could stand on both hind hooves, while sheep in other groups could not stand on their right hind hoof due to pain; in terms of gait analysis, the CM-Ms@IOX4 group had a gait close to normal, while sheep in other groups had a limp on their right hind leg and could hardly bear weight.

[0240] 4.2) MRI evaluation

[0241] After testing, the following can be obtained: Figure 54 The images shown are X-ray images of the articular cartilage of a sheep osteoarthritis model provided in Example 4 of the present invention, wherein the yellow dashed circle indicates the bone marrow lesion area; and Figure 55 Example 4 of the present invention provides a basis based on Figure 54 The MOCART score analysis is shown in the bar chart. Data are expressed as mean ± standard deviation (n=3 / group). P < 0.05, **P < 0.001, and ns indicate no significant difference.

[0242] from Figure 54 and Figure 55 As can be seen from the baseline MRI, the anterior cruciate ligament and medial meniscus are normal. In the CM-Ms@IOX4 group, the articular cartilage layer is intact and the subchondral bone is normal; while in the control group, there is subchondral bone edema and varying degrees of cartilage destruction. The MOCART score of the CM-Ms@IOX4 group is significantly higher than that of other groups as treatment progresses.

[0243] 4.3) Gross observation (ICRS score)

[0244] After testing, the following can be obtained: Figure 56 The set of representative photographs of osteoarthritis cartilage after treatment with saline, sodium hyaluronate injection, IOX4, and CM-Ms@IOX4, provided as an example of the effectiveness of this invention, and Figure 57 Example 4 of the present invention provides a basis based on Figure 56 Macroscopic scoring bar chart of femoral condyle and tibial plateau joint surface injuries;

[0245] Figure 56 and Figure 57 The results showed that the femoral condyle and tibial plateau cartilage of the CM-Ms@IOX4 group remained nearly intact, and the joint score was significantly lower than that of the control group.

[0246] 4.4) Histopathology (OARSI classification)

[0247] After testing, the following can be obtained: Figure 58 The image shown is a representative set of Safranin O / Fix Green stained femoral condyle tissue after 12 weeks of treatment, as provided in Example 4 of the present invention, and as shown in... Figure 59 The example shown is based on the effect of the present invention, Example 4. Figure 58 The OARSI score bar chart is shown, with the upper scale bar at 500 µm and the lower scale bar at 2 cm.

[0248] from Figure 58 and Figure 59 As can be seen, the femoral condyle OARSI grade of the CM-Ms@IOX4 group was significantly lower than that of the saline, HA and IOX4 groups; the tibial plateau showed that the cartilage thickness of the CM-Ms@IOX4 group was maintained, and the OARSI grade was significantly reduced.

[0249] 4.5) H&E staining (HC / CC ratio)

[0250] After testing, the following can be obtained: Figure 60 The image shown is a representative set of H&E staining images of femoral condyle tissue after 12 weeks of treatment, provided in Example 4 of the present invention. Figure 61 The bar chart shown is a comparison of the thickness ratio of hyaline cartilage to calcified cartilage provided in Example 4 of the present invention. The upper scale bar is 500 µm and the lower scale bar is 2 cm. All data are expressed as mean ± standard deviation (n=3). ***P < 0.001, ns indicates no significant difference.

[0251] from Figure 60 and Figure 61 As can be seen, the thickness of the hyaline cartilage layer and the HC / CC ratio in the CM-Ms@IOX4 group were comparable to those in the sham surgery group, but significantly better than those in other treatment groups. Example 5: Safety Evaluation Data

[0252] 5.1) Cytotoxic CCK-8 assay

[0253] After testing, we can obtain the following:

[0254] like Figure 62 The bar chart shown is an evaluation bar graph of the cytotoxicity of CM-Ms against primary chondrocyte CCK-8 after 24 hours of incubation, provided in Example 5 of the present invention. Figure 63 The bar chart shown is an evaluation of the cytotoxicity of 10X4 on primary chondrocytes CCK-8 after 24 hours of incubation, provided in Example 5 of the present invention.

[0255] Depend on Figure 62 and Figure 63 It can be seen that in the CCK-8 assay, the cell viability of CM-Ms at a concentration of 5.0 mg / mL was >95%, and the cell viability of IOX4 at a concentration of 100 μg / mL was >95%.

[0256] 5.2) Synovial irritation

[0257] After testing, the following can be obtained: Figure 64 The image shown is a representative set of H&E staining images of the knee joints of sham-operated mice on days 0, 3, and 10 after intra-articular injection of different formulations, provided in Example 5 of the present invention. The scale bar is 200 µm.

[0258] Depend on Figure 64 It can be seen that on days 0, 3, and 10 after intra-articular injection of PBS, IOX4, Ms@IOX4, C-Ms@IOX4, M-Ms@IOX4, and CM-Ms@IOX4, there were no obvious morphological changes in the synovium H&E staining.

[0259] 5.3) Organ toxicity assessment

[0260] After testing, the following can be obtained: Figure 65 The image shown is a representative H&E staining set of the heart, liver, spleen, lungs, and kidneys of OA mice after 8 weeks of treatment, as provided in Example 5 of the present invention. The scale bar is 200 µm. Figure 66 The image shown is a representative H&E staining set of the heart, liver, spleen, lungs and kidneys of OA sheep after 8 weeks of treatment, provided in Example 5 of the present invention. The scale bar is 200 µm.

[0261] from Figure 65 and Figure 66 As can be seen, after long-term treatment, the H&E staining of the heart, liver, spleen, lungs and kidneys of OA mice and sheep showed a healthy appearance with no pathological changes.

[0262] 5.4) Hematological examination

[0263] After testing, it can be obtained Figure 67 The results shown are the hematological index test results provided in Example 5 of the present invention. The orange dashed line represents the normal range of each index, and the data are expressed as mean ± standard deviation (n=3).

[0264] from Figure 67 As can be seen from the results, hematological examinations showed that the white blood cell (WBC), red blood cell (RBC), platelet (PLT), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) of the OA mice and sheep in the experiment were all within the normal range.

[0265] 5.5) Immunogenicity test: No obvious immune response or allergic reaction was observed after testing.

[0266] Based on the above description of the effects, the intelligent drug delivery system provided in this embodiment has the following characteristics.

[0267] 1) Technological innovation and advantages:

[0268] a) For the first time, the dual functions of viral glycoproteins (non-specific adhesion and specific activation) are integrated into a single peptide sequence, achieving the unity of cartilage retention and selective delivery to diseased cells, with the innovative advantage of biomimetic design;

[0269] b) Because biomimetic particles smaller than 60nm can penetrate the cartilage matrix, and biomimetic particles with a diameter of 16nm are within the ideal range and can effectively penetrate cartilage, they have a size advantage.

[0270] c) The intelligent drug delivery system provided in this embodiment utilizes MMP13 as an endogenous biomarker for diseased chondrocytes, realizing "on-demand activation" intelligent delivery, improving the precision of treatment, and having the advantage of intelligent response;

[0271] d) The intelligent drug delivery system provided in this embodiment can simultaneously achieve structural protection (delaying degeneration of cartilage and subchondral bone) and symptom improvement (relieving pain and improving function), which meets the definition criteria of DMOADs and has the advantage of dual therapeutic effects;

[0272] e) The intelligent drug delivery system provided in this embodiment is produced using FDA-approved DSPE-PEG material and is prepared using a mature thin-film hydration method. The designed peptides can be mass-produced through solid-phase synthesis and have good industrialization prospects. Therefore, it has the advantage of clinical translatability.

[0273] f) The intelligent drug delivery system provided in this embodiment can be extended to other DMOAD candidate drugs that need to target intracellular sites of action, such as Wnt inhibitors, gene drugs, and metabolic regulators. Therefore, it has the potential advantage of platform technology.

[0274] g) Experiments have confirmed that the intelligent drug delivery system provided in this embodiment can maintain good efficacy even with monthly administration. Therefore, it can reduce the frequency of injections, reduce the risk of infection, and improve patient compliance, thus having the advantage of convenient drug administration.

[0275] 2) Potential application areas:

[0276] Based on the above advantages, it can be seen that, in addition to its use in treating osteoarthritis, the intelligent drug delivery system provided in this embodiment may also have the following potential applications:

[0277] Delivery of agents that promote bone injury repair, intra-articular gene therapy vectors, drug delivery for other degenerative joint diseases, and delivery of growth factors through cartilage tissue engineering. Example 2

[0278] This embodiment provides a pharmaceutical composition comprising the intelligent drug delivery system described in Embodiment 1.

[0279] The pharmaceutical composition provided in this embodiment is a ready-to-use pharmaceutical composition, wherein the active ingredient is CMP-IOX4-Micelles prepared in Example 1, with a concentration of 1 mg / mL based on IOX4; the pharmaceutical carrier is PBS buffer with a pH of 7.4, which contains 5% (w / v) mannitol as an excipient to maintain the stability of the micelles and to play a protective role during the lyophilization process. The above mixture is sterile filtered, then dispensed into vials, and lyophilized to obtain a lyophilized powder for injection.

[0280] Instructions for use: For clinical use, reconstitute with 2 mL of sterile water for injection. Gently shake to restore a homogeneous micelle solution for intra-articular injection. Example 3

[0281] This embodiment provides an application of the viral glycoprotein mimic peptide (CMP) obtained in Example 1 in the preparation of a targeted delivery system.

[0282] In this embodiment, the viral glycoprotein mimic peptide (with the sequence: WYRGRL-PEG3-e9-C6-(GPLGVRG)-r9-C6-Cys) is used as a key functional element.

[0283] Application: Viral glycoprotein mimic peptides (CMPs) can be used as targeting ligands to prepare any drug delivery system designed to treat or diagnose degenerative joint diseases such as osteoarthritis and cartilage damage.

[0284] Specific implementation methods include: clicking chemical coupling of the CMP with maleimide groups on the surface of other types of nanocarriers (such as liposomes, polymer nanoparticles, exosomes, etc.) via its C-terminal cysteine ​​residue.

[0285] Functions and effects: This modified delivery system can mimic the affinity of viruses for cartilage matrix, achieving non-specific adhesion and enrichment of diseased articular cartilage. At the same time, its enzyme response sequence can ensure that the loaded drug or diagnostic agent is specifically activated and released in the diseased microenvironment. Therefore, the application of this CMP can significantly improve the targeting efficiency and therapeutic / diagnostic specificity of various delivery systems for degenerative joint diseases. Example 4

[0286] This embodiment provides an application of the intelligent drug delivery system obtained in Example 1 as a fluorescence imaging reagent for the diagnosis of degenerative joint diseases.

[0287] Preparation of imaging reagents: Following the preparation method described in Example 1, in the step of forming the lipid film, in addition to the disease-modifying drug (which may be omitted or added as irrelevant drugs in diagnostic applications) and lipid material, an additional 0.08% of the total lipid weight of the near-infrared fluorescent dye DiR is added. Subsequent steps, including hydration, coupling with CMP peptide and purification, are the same as in Example 1, and finally a fluorescently labeled intelligent drug delivery system (CMP-DiR-Micelles) is obtained.

[0288] Application as a diagnostic reagent: The prepared CMP-DiR-Micelles are diluted with physiological saline to an appropriate concentration and used as a contrast agent for intra-articular imaging examinations.

[0289] Instructions for use: Inject the imaging reagent into the joint of a subject suspected of having osteoarthritis via intra-articular injection.

[0290] Imaging and diagnosis: 24-48 hours after injection, the joint area is scanned using a near-infrared fluorescence imaging system.

[0291] Diagnostic principle: As shown in Examples 3 and 5, this reagent specifically accumulates in the cartilage region of degenerative diseases. By detecting the intensity and distribution of the fluorescence signal, doctors can non-invasively and visually assess the extent and severity of cartilage damage, enabling early diagnosis and monitoring of degenerative joint diseases. This reagent is also suitable for real-time navigation in minimally invasive arthroscopic surgery, accurately locating the lesion area.

[0292] In conclusion, it can be seen that:

[0293] This invention provides an intelligent drug delivery system for treating osteoarthritis. Its core is a drug-loaded micelle covalently coupled with a viral glycoprotein mimic peptide (CMP). The system's hydrodynamic diameter is precisely controlled at 16±5 nm, enabling effective penetration of dense cartilage matrix (pore size approximately 60 nm). The key innovation lies in the CMP sequence (WYRGRL-PEG3-e9-C6-(GPLGVRG)-r9-C6-Cys), which, through the WYRGRL sequence, achieves non-specific adhesion to the cartilage matrix, ensuring long-term retention. It also contains the MMP13-specific cleavage sequence GPLGVRG, ensuring activation only in the microenvironment of diseased chondrocytes that overexpress MMP13, exposing positively charged cell-penetrating peptides and driving efficient intracellular drug delivery.

[0294] Compared with existing technologies, the significant features and advancements of this invention are mainly reflected in the following aspects: Firstly, it achieves a breakthrough from "cartilage targeting" to "precise targeting of diseased cells," that is, selectively acting on the diseased area through the MMP13 response mechanism, solving the problem that existing targeting ligands cannot distinguish between diseased and healthy cartilage cells. Secondly, through precise size design and biomimetic peptide sequences, it systematically solves the core contradiction between penetrating cartilage and resisting synovial fluid clearance, achieving an ideal balance between penetration and retention. Furthermore, this system exhibits genuine disease-modifying therapeutic effects, simultaneously achieving the dual efficacy of delaying cartilage degeneration and improving functional symptoms in animal models. The monthly dosing regimen is significantly superior to existing regimens requiring frequent injections, greatly improving patient compliance and reducing the risk of infection. Moreover, the delivery system adopts a modular design, capable of carrying multiple disease-modifying drugs such as IOX4 and Wnt inhibitors, possessing strong platform technology potential. Its fully synthetic and biocompatible materials also ensure good industrialization feasibility.

[0295] In the description process of the above instruction manual:

[0296] The terms “this embodiment,” “this embodiment of the invention,” “this case,” “this comparative example,” “as shown,” “further,” etc., are used to indicate that the specific features, structures, materials, or characteristics described in the embodiment or case or comparative example are included in at least one embodiment or case or comparative example of the present invention.

[0297] In this specification, the illustrative expressions of the above terms are not necessarily directed at the same embodiments, cases, or comparative examples. Moreover, the specific features, structures, materials, or characteristics described may be combined or combined in any suitable manner in one or more embodiments, cases, or comparative examples. Furthermore, without causing contradiction, those skilled in the art may combine or combine the different embodiments, cases, or comparative examples described in this specification, as well as the features in the different embodiments, cases, or comparative examples.

[0298] Finally, it should be noted that the above embodiments and comparative examples are only used to illustrate the technical solutions and technical effects of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, examples, and comparative examples, those skilled in the art should understand that modifications or supplements can still be made to the technical solutions or technical effects described in the foregoing embodiments, examples, and comparative examples, or equivalent substitutions can be made to some or all of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions described in the embodiments of the present invention. Non-essential improvements, adjustments, or substitutions made by those skilled in the art based on the content of this specification are all within the scope of protection claimed by the present invention.

Claims

1. A smart drug delivery system for treating osteoarthritis, characterized in that: The system is a drug-loaded micelle with a hydrodynamic diameter of 16±5 nm and a viral glycoprotein mimic peptide covalently coupled to its surface. The viral glycoprotein mimic peptide has the sequence WYRGRL-PEG3-e9-C6-(GPLGVRG)-r9-C6-Cys, which can achieve non-specific adhesion to the cartilage matrix and specific activation in the disease cell microenvironment.

2. The intelligent drug delivery system as described in claim 1, characterized in that: The drug-loaded micelles comprise a disease-modifying drug for treating osteoarthritis, DSPE-PEG3400-maleimide, and DSPE-PEG2000, wherein the molar ratio of the disease-modifying drug to the DSPE-PEG3400-maleimide and the DSPE-PEG2000 is 5:1:

9.

3. The intelligent drug delivery system as described in claim 2, characterized in that: The disease-modifying drug is any one of the following: hypoxia-inducible factor-1α prolyl hydroxylase-2 inhibitor IOX4, Wnt pathway inhibitor, gene therapy drug, or metabolic regulator.

4. The intelligent drug delivery system as described in claim 1, characterized in that: The viral glycoprotein mimic peptide is linked to the maleimide group on the surface of the drug-loaded micelle via a click chemistry reaction, with the thiol group of its C-terminal cysteine ​​residue being linked to the maleimide group on the surface of the drug-loaded micelle.

5. A method for preparing the intelligent drug delivery system as described in any one of claims 1 to 4, characterized in that, Includes the following steps: First, the disease-modifying drug, DSPE-PEG3400-maleimide, and DSPE-PEG2000 are dissolved in an organic solvent in a certain proportion, and then the organic solvent is removed to form a lipid film. The lipid film was hydrated with a buffer solution, and then the lipid film was dispersed and filtered to obtain drug-loaded micelles. The viral glycoprotein mimic peptide is dissolved, and then the dissolved viral glycoprotein mimic peptide is mixed with the drug-loaded micelles. The mixture is reacted for 10 to 12 hours under conditions of pH 7.2 to 8.0, reaction temperature of 25°C and inert gas protection to obtain the reaction product. The reaction product is purified to obtain the intelligent drug delivery system.

6. The preparation method according to claim 5, characterized in that: The organic solvent is chloroform, the buffer is PBS buffer with a pH of 7.4, the inert gas is nitrogen, the solvent for dissolving the viral glycoprotein mimic peptide is 6M guanidine hydrochloride, and the dissolved viral glycoprotein mimic peptide is matched with the molar ratio of the thiol group of its C-terminal cysteine ​​to the maleimide on the surface of the drug-loaded micelles at 4:

1. The purification is performed by Sepharose CL-4B gel chromatography.

7. The use of the intelligent drug delivery system as described in any one of claims 1 to 4 in the preparation of drugs for treating osteoarthritis or promoting cartilage damage repair, wherein the drugs are administered via intra-articular injection at a frequency of once a month or less.

8. A pharmaceutical composition, characterized in that: It includes an intelligent drug delivery system as described in any one of claims 1 to 4, and a pharmaceutically acceptable carrier or excipient.

9. The application of a viral glycoprotein mimic peptide in the preparation of a targeted drug delivery system for degenerative joint diseases, characterized in that: The sequence of the viral glycoprotein mimic peptide is WYRGRL-PEG3-e9-C6-(GPLGVRG)-r9-C6-Cys.

10. A reagent for the diagnosis or imaging of degenerative joint diseases, characterized in that, The intelligent drug delivery system comprising fluorescent labeling as described in any one of claims 1 to 4, wherein the intelligent drug delivery system comprising fluorescent labeling is prepared by the preparation method as described in claim 5, and in the preparation step of forming the lipid film, 0.08% by weight of the fluorescent dye DiR or coumarin 6 is added.

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