Application of platelets modified by matrix metalloproteinase 13 in prevention and treatment of arthritis

Abstract

The invention relates to application of platelets modified by matrix metalloproteinase 13 in prevention and treatment of arthritis. Specifically, the invention provides a platelet complex, which is characterized by comprising platelet cells, a chemical linker element and a specific protein, wherein the platelet cells are linked to the specific protein through the chemical linker element, and the specific protein comprises an antibody targeting a cartilage injury specific expression molecule. The platelet complex (platelet at MMP13) disclosed by the invention is combined with MMP13 released by active targeting OA cartilage injury lesion sites, and cartilage repair and regeneration can be promoted, so that the effect in drugs for preventing and / or treating OA can be achieved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to an application of platelets modified with matrix metalloproteinase 13 in preventing and treating arthritis. Background Art

[0002] Osteoarthritis (OA) is the most common chronic joint disease, characterized by degenerative damage to articular cartilage, leading to progressive loss of joint structure and function. The prevalence of OA has increased significantly in my country with the aging population. According to global epidemiological studies, my country has the highest number of OA patients in the world, with an estimated 400 million people affected by the disease by 2025. A 2023 survey of chronic diseases among over 220,000 Chinese individuals aged 60 and older revealed that OA is one of the most common chronic diseases in this age group, with a prevalence rate of 43.7%. These data not only highlight the severity of OA in China but also reflect its significant socioeconomic burden. The pathogenesis of OA is extremely complex, with a multi-dimensional interplay of genetics, metabolic pathways, biomechanical loads, and environmental factors. The combined effects of these factors on joint structure lead to progressive wear and structural destruction of the cartilage layer. The clinical manifestations of OA are diverse, including but not limited to chronic pain, localized swelling, joint stiffness, limited range of motion, and joint deformity. As the disease progresses, the reduction in joint range of motion has a significant impact on the patient's daily activities, and in the late stage of the disease, joint nodules or knee flexion deformities may occur.

[0003] Platelet-rich plasma (PRP) is used in orthopedic diseases because platelets in it secrete multiple growth factors to promote tissue repair after activation. However, the lack of targeting of PRP and the large differences in storage methods and joint cavity treatment plans have affected the effectiveness of OA treatment.

[0004] Therefore, there is an urgent need to develop a new type of drug and preparation method for effectively preventing and treating arthritis in this field, which will provide new insights into the clinical application of preventing and controlling OA degenerative lesions and has important scientific significance and clinical value. Summary of the Invention

[0005] In view of the above problems, the first object of the present invention is to provide a preparation method for modifying autologous platelets using biocoupling and separation gel centrifugation methods.

[0006] The second purpose of the present invention is to provide modified autologous platelets (Platelet@MMP13) to promote the effectiveness verification of OA treatment.

[0007] The third purpose of the present invention is to provide new technology and new strategy for clinical treatment of OA with Platelet@MMP13.

[0008] In the first aspect of the present invention, a platelet complex is provided, which comprises: platelet cells, a chemical linker element and a specific protein; wherein the platelet cells are connected to the specific protein via the chemical linker element; and the specific protein comprises an antibody targeting a molecule specifically expressed in cartilage damage.

[0009] In another preferred embodiment, the platelet cells are autologous platelet cells.

[0010] In another preferred embodiment, the platelet cells are mammalian platelet cells.

[0011] In another preferred embodiment, the mammal is a human, a rodent or a non-human primate, preferably including a human, a mouse, a rat, a rabbit or a monkey.

[0012] In another preferred embodiment, the chemical linker element comprises: a maleimide linker, a PEG linker, PASylation or HESylation.

[0013] In another preferred embodiment, the chemical linker element is a thiol-maleimide group.

[0014] In another preferred embodiment, the specific protein includes at least one of an antibody, a ligand, and a receptor targeting a molecule specifically expressed in cartilage damage.

[0015] In another preferred embodiment, the cartilage damage-specifically expressed molecule includes metalloproteinase 13 (MMP13).

[0016] In another preferred embodiment, the specific protein includes at least one of an antibody, a ligand, and a receptor targeting MMP13.

[0017] In another preferred embodiment, the specific protein is an MMP13 antibody.

[0018] In a second aspect of the present invention, a method for preparing the platelet complex according to the first aspect of the present invention is provided, comprising the following steps:

[0019] S1: performing thiol modification on the platelets to obtain activated platelets;

[0020] S2: performing maleimide modification on the specific protein to obtain an activated protein;

[0021] S3: mixing and incubating the activated platelets and the activated protein to couple the activated platelets with the activated protein, thereby obtaining the platelet complex.

[0022] In another preferred embodiment, the specific protein includes at least one of an antibody, a ligand, and a receptor targeting a molecule specifically expressed in cartilage damage.

[0023] In another preferred embodiment, the cartilage damage-specifically expressed molecule includes metalloproteinase 13 (MMP13).

[0024] In another preferred embodiment, the specific protein includes at least one of an antibody, a ligand, and a receptor targeting MMP13.

[0025] In another preferred embodiment, the specific protein is an MMP13 antibody.

[0026] In another preferred embodiment, the platelets are thiol-modified using the Trants reagent in S1, and the number of the platelets is 0.8×10^9~1.2×10^9, preferably 1×10^9; the amount of the Trants reagent is 2800-3200 μg, preferably 3000 μg.

[0027] In another preferred embodiment, in step S2, the reagent used for maleimidation modification of the specific protein is Sulfo-SMCC; the specific protein is MMP13 antibody, and the amount of the MMP13 antibody is 0.5 μg / ml to 5 μg / ml, preferably 5 μg / ml of MMP13 antibody; the amount of the Sulfo-SMCC is 2800-3200 μg, preferably 3000 μg.

[0028] In a third aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0029] (a1) a first active ingredient for preventing and / or treating arthritis-related diseases, the first active ingredient comprising: the platelet complex according to the first aspect of the present invention;

[0030] (a2) a second active ingredient for preventing and / or treating arthritis-related diseases, wherein the second active ingredient includes: other drugs for preventing and / or treating arthritis-related diseases; and

[0031] (b) a pharmaceutically acceptable carrier.

[0032] In another preferred embodiment, in the pharmaceutical composition, the component (a1) accounts for 1-99 wt %, preferably 10-90 wt %, and more preferably 30-70 wt % of the total weight of the pharmaceutical composition.

[0033] In another preferred embodiment, in the pharmaceutical composition, the component (a2) accounts for 1-99 wt %, preferably 10-90 wt %, and more preferably 30-70 wt % of the total weight of the pharmaceutical composition.

[0034] In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is 1:100 to 100:1, preferably 1:10 to 10:1.

[0035] In another preferred embodiment, the pharmaceutical composition further comprises an additional component that inhibits arthritis.

[0036] In another preferred embodiment, the pharmaceutical composition further comprises an additional component that inhibits cartilage degeneration.

[0037] In another preferred embodiment, the other drugs for preventing and / or treating arthritis-related diseases are selected from the following group: non-steroidal anti-inflammatory drugs, analgesics (non-anti-inflammatory), chondroprotective agents, intra-articular injection drugs, Chinese patent medicines and external patches, or a combination thereof.

[0038] In another preferred embodiment, the nonsteroidal anti-inflammatory drug comprises celecoxib.

[0039] In another preferred embodiment, the bone protective agent comprises chondroitin sulfate.

[0040] In another preferred embodiment, the intra-articular injection drug includes sodium hyaluronate and glucocorticoid.

[0041] In another preferred embodiment, the arthritis-related disease is selected from the group consisting of aging-related arthritis, lipid metabolism arthritis, traumatic arthritis, or a combination thereof.

[0042] In another preferred embodiment, the pharmaceutical composition may contain a single compound or a mixture of multiple compounds.

[0043] In another preferred embodiment, the pharmaceutical composition is used to prepare a drug or preparation for treating or preventing arthritis-related diseases.

[0044] In another preferred embodiment, the pharmaceutical dosage form is an oral or parenteral dosage form.

[0045] In another preferred embodiment, the oral dosage form is a tablet, powder, granule or capsule, or an emulsion or syrup.

[0046] In another preferred embodiment, the non-oral dosage form is an injection or injection.

[0047] In another preferred embodiment, the total content of the active ingredient (a1) and the active ingredient (a2) is 1 to 99 wt %, more preferably 5 to 90 wt %, of the total weight of the composition.

[0048] In a fourth aspect of the present invention, a medicine kit is provided, comprising:

[0049] (i) a first container, and the active ingredient (a1) in the first container is the platelet complex according to the first aspect of the present invention, or a drug containing the active ingredient (a1); and

[0050] (ii) a second container, and the active ingredient (a2) in the second container; other drugs for preventing and / or treating arthritis-related diseases, or drugs containing the active ingredient (a2).

[0051] In another preferred embodiment, the first container and the second container are the same or different containers.

[0052] In another preferred embodiment, the medicine in the first container is a single-ingredient preparation containing the platelet complex according to the first aspect of the present invention.

[0053] In another preferred embodiment, the medicine in the second container is a single-ingredient preparation containing other medicines for preventing and / or treating arthritis-related diseases.

[0054] In another preferred embodiment, the dosage form of the drug is an oral dosage form or an injection dosage form.

[0055] In another preferred embodiment, the kit further comprises instructions for administering the active ingredient (a1) and the active ingredient (a2) in combination to (i) prevent and / or treat arthritis-related diseases; and / or (ii) inhibit arthritis.

[0056] In another preferred embodiment, the dosage forms of the preparation containing the active ingredient (a1) such as the platelet complex described in the first aspect of the present invention or the preparation containing other drugs for preventing and / or treating arthritis-related diseases include capsules, tablets, suppositories, or intravenous injections.

[0057] In another preferred embodiment, the platelet complex preparation containing active ingredient (a1) as described in the first aspect of the present invention has a concentration of 0.0001-100 mg / kg body weight, preferably 0.1-50 mg / kg body weight, more preferably 1-20 mg / kg body weight.

[0058] In a fifth aspect of the present invention, a method for inhibiting arthritis is provided, comprising the steps of:

[0059] In the presence of the platelet complex described in the first aspect of the present invention, bone and joint related cells are cultured to inhibit arthritis.

[0060] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0061] In another preferred embodiment, the method is therapeutic.

[0062] In another preferred embodiment, the effective concentration of the platelet complex is 0.0001-100 mg / kg body weight, preferably 1-50 mg / kg body weight, and more preferably 5-20 mg / kg body weight.

[0063] In the sixth aspect of the present invention, provided is the use of the platelet complex as described in the first aspect of the present invention, or the platelet complex prepared by the method described in the second aspect of the present invention, or the pharmaceutical composition as described in the third aspect of the present invention, or the drug kit as described in the fourth aspect of the present invention in the preparation of products targeting cartilage damage.

[0064] In another preferred embodiment, the product is selected from the following group: the bone defect repair product is a spinal injury repair product, a skull injury repair product, a maxillofacial bone defect repair product, a limb bone defect repair product or a joint bone defect repair product; preferably, the spinal injury repair product is selected from cervical vertebrae, thoracic vertebrae or lumbar vertebrae injury repair products, or the limb bone defect repair product is selected from femur, fibula or humerus injury repair products, or the joint bone defect repair product is selected from hip joint or knee joint bone injury repair products.

[0065] In the seventh aspect of the present invention, provided is the use of the platelet complex as described in the first aspect of the present invention, or the platelet complex prepared by the method described in the second aspect of the present invention, or the pharmaceutical composition as described in the third aspect of the present invention, or the drug kit as described in the fourth aspect of the present invention in the preparation of drugs for preventing and / or treating arthritis.

[0066] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one.

[0067] Compared with the prior art, the present invention has the following technical effects:

[0068] (1) The present invention discloses for the first time a preparation method for modifying autologous platelets using bioconjugation and separation gel centrifugation methods, and successfully prepares Platelet@MMP13.

[0069] (2) The present invention discloses for the first time the use of Platelet@MMP13 in the preparation of drugs for the prevention and / or treatment of arthritis. By actively targeting the MMP13 released from the OA cartilage damage lesion and combining with each other, the effect of preventing and / or treating OA drugs can be achieved by promoting cartilage repair and regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 The diagram shows the modification principle of Platelet@MMP13 in an embodiment of the present invention. A shows the modification of thiol groups on platelets (platelets) using Traut's reagent to enable the subsequent attachment of anti-MMP13 antibodies to the platelets; B shows the activation of the anti-MMP13 antibodies using Sulfo-SMCC to enable their reaction with thiol groups on platelets; and C shows the bioconjugation linking of thiol groups on platelets to maleimide groups on the anti-MMP13 antibodies, enabling the loading of MMP13 antibodies onto the platelet membrane. (Hereinafter referred to as Platelet@MMP13).

[0071] Figure 2 Shown are flow cytometry analysis results of Platelet@MMP13 from an example of the present invention. Figure A is a flow cytometry scatter plot of control platelets and Platelet@MMP13; Figure B is a quantitative statistical graph of Platelet@MMP13-positive cells (n = 3).

[0072] Figure 3 Shown are confocal laser images of platelets carrying different concentration gradients of anti-MMP13 antibodies [Alexa Fluor® 594] (0, 0.05, 0.5 μg / ml) in an embodiment of the present invention, including platelets (gray) and MMP13 (red); (n=3); the scale bars on the left and right sides of Figure A are 5 μm and 0.5 μm respectively; Figure B is the statistical results of Figure A.

[0073] Figure 4Figures show the results of an example of the present invention demonstrating that intra-articular injection of modified autologous platelets (Platelet@MMP13) improves cartilage degeneration in OA mice. Figure A shows the experimental flow chart for intra-articular injection of Platelet@MMP13. Six weeks after OA mice (n=10) were established, each group received intra-articular injections of normal saline, conventional platelets, and Platelet@MMP13, once weekly for four times. Following treatment, knee joint samples were collected for knee hyperalgesia, histology, and immunofluorescence analysis. Figure B shows mechanical allodynia testing performed on the right hind paw of each group of mice. Figure C shows thermal hyperalgesia testing performed on the right hind paw of each group of mice. Figure D shows OARSI scores for articular cartilage in each group of mice. Figure E shows Safranin-Fast Green (SO) staining analysis of each group of mice. Scale bar, 200 μm. Figure F shows immunofluorescence analysis of the cartilage-related proteins Col2a1 and Acan in each group of mice. Scale bar, 100 μm. DETAILED DESCRIPTION

[0074] To make the purpose, technical solutions, beneficial effects, and significant improvements of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, all the embodiments described are only some embodiments of the present invention, not all embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0075] the term

[0076] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0077] As used herein, the term “including” or “comprising” encompasses “comprising,” “consisting mainly of,” “consisting essentially of,” and “consisting of;” “consisting mainly of,” “consisting essentially of,” and “consisting of” are subordinate concepts of “containing,” “having,” or “including.”

[0078] As used herein, the terms "matrix metalloproteinase 13-modified platelets", "MMP13-modified platelets", "Platelet@MMP13", "platelet complex", "modified autologous platelets", and "autologous PRP carrying cartilage damage marker MMP13 antibodies" can be used interchangeably and all refer to the platelet complex described in the first aspect of the present invention.

[0079] Platelet-rich plasma (PRP)

[0080] Platelet-rich plasma (PRP) is used in orthopedic diseases because activated platelets secrete multiple growth factors to promote tissue repair. However, PRP therapy currently faces bottlenecks: whether platelets can target cartilage damage after injection, whether they can efficiently secrete growth factors, and how long their effects last are unknown. Studies have shown that matrix metalloproteinase 13 (MMP13) expression is upregulated at sites of osteoarthritis (OA) cartilage damage. This study successfully prepared modified autologous platelets (Platelet@MMP13) through a Traut and Sulfo-SMCC bioconjugation reaction and demonstrated superior efficacy compared to conventional PRP in OA mice.

[0081] Matrix Metalloproteinase-13 (MMP-13)

[0082] Metalloproteinase 13, also known as collagenase 3, is a key member of the matrix metalloproteinase (MMP) family. It is primarily involved in the degradation and remodeling of the extracellular matrix (ECM), playing a key role in cartilage, bone, and certain pathological processes. In arthritis (OA) and rheumatoid arthritis (RA), MMP13 is overactivated by inflammatory factors (such as IL-1β and TNF-α), leading to cartilage destruction, abnormal bone remodeling, and loss of joint function.

[0083] MMP13 antibody

[0084] The role of MMP13 antibodies in the treatment of arthritis is primarily based on their specific inhibition of MMP13 activity, thereby slowing the degradation of joint tissue, protecting cartilage and bone structures, inhibiting the destruction of cartilage matrix, and delaying the progression of arthritis.

[0085] Experimental methods:

[0086] This paper mainly explores the precise modification and verification of autologous PRP loaded with cartilage damage marker MMP13

[0087] Articular cartilage lacks blood vessels, nerves, and lymph nodes, making it difficult to self-repair after cartilage damage. Currently, PRP is widely used in the treatment of OA. However, due to the lack of targeting of PRP, storage methods and joint cavity treatment options vary greatly, which has affected the effectiveness of OA treatment. Therefore, the present invention uses peripheral blood purified platelets from C57BL / 6 mice and a DMM-induced OA mouse model in C57BL / 6 mice as research subjects. Laser confocal immunofluorescence, flow cytometry, and other morphological and molecular biological methods are used to systematically analyze and identify the precise modification of the cartilage damage marker MMP13 by autologous PRP loading. The specific research content and indicators are as follows:

[0088] (1) Peripheral blood (5 ml) was collected from 5 male C57BL / 6 mice at 12 weeks of age. PRP was centrifuged using a separation gel to obtain purified platelets. The platelets were reacted with amino groups on the platelets using Traut's reagent. Subsequently, the platelets were cross-linked with the cartilage damage marker MMP13 antibody using Sulfo-SMCC. Finally, the two products were co-incubated to obtain autologous PRP loaded with the cartilage damage marker MMP13. In other words, the membrane proteins on the PRP platelets were linked to the MMP13 antibody.

[0089] (2) Flow cytometry analysis was performed to analyze the expression of platelet CD41 and MMP13 in autologous PRP loaded with the cartilage damage marker MMP13, confirming that Traut's reagent and Sulfo-SMCC reagent successfully and precisely modified autologous PRP loaded with the cartilage damage marker MMP13.

[0090] (3) Laser confocal immunofluorescence analysis was used to identify the presence of membrane proteins on PRP-loaded platelets linked to MMP13 antibodies, confirming the successful and precise modification of autologous PRP loaded with the cartilage damage marker MMP13.

[0091] (4) A DMM cartilage injury model was established using C57BL / 6 mice. Six weeks after modeling, the mice were injected with normal saline, conventional PRP, and modified autologous PRP (Platelet@MMP13) into the joint cavity. The treatment course was once a week for a total of 4 times. After treatment, the pain index, tissue macroscopic score, and cartilage-related proteins (Col2a1, Acan, etc.) of OA mice were detected, which proved that modified platelets improved the degenerative lesions of OA.

[0092] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. The experimental materials and reagents involved in the present invention can be obtained from commercial sources unless otherwise specified.

[0093] Example 1:

[0094] In this example, peripheral blood (5 ml) was collected from five 12-week-old male C57BL / 6 mice. PRP was centrifuged using separation gel to produce purified platelets. Traut's reagent was reacted with amino groups on the platelets. Subsequently, Sulfo-SMCC was used to cross-link the platelets with an antibody against MMP13, a marker of cartilage damage. Finally, the two products were co-incubated to obtain autologous PRP carrying the antibody against MMP13, a marker of cartilage damage.

[0095] Immunofluorescence and flow cytometry analysis were used to identify the expression of platelet CD41 and MMP13 in autologous PRP carrying the cartilage damage marker MMP13, confirming that Traut's reagent and Sulfo-SMCC reagent successfully and precisely modified the autologous PRP carrying the cartilage damage marker MMP13, specifically modifying the platelets in the PRP to produce the cartilage damage marker MMP13, namely Platelet@MMP13. The specific modification method is shown in the figure below. Figure 1 shown.

[0096] First, platelets in PRP were thiolated using Traut's reagent to introduce active thiol groups. Second, anti-MMP13 antibodies [Alexa Fluor® 594] (NOVUS, NBP2-72740) or anti-MMP13 antibodies (Abcam, ab39012) were chemically activated using Sulfo-SMCC (sulfate-N-succinimidyl-4-methylcyclohexanecarboxylate) (MCE, HY-D0975-100mg) to enable them to react with thiol groups. Finally, efficient conjugation of thiol groups on the platelet surface to maleimide groups on the anti-MMP13 antibody was achieved through bioconjugation technology.

[0097] The specific experimental methods are as follows:

[0098] (1) Peripheral blood (5 ml) was collected from 5 C57BL / 6 mice. PRP was prepared by separation gel centrifugation using sterile blood collection tubes (containing anticoagulant) to obtain purified platelets.

[0099] (2) Dissolve Traut's reagent (3 mg / mL) in HBSS containing 4 mM EDTA (pH = 8) and add platelets to thiolate the proteins on the platelet surface. Incubate at room temperature for 2 hours and wash with HBSS to remove unreacted Traut's reagent (the ratio of the platelets to the Trants reagent is 1×10^9 platelets: 3000 μg Trants reagent).

[0100] Sulfo-SMCC (3 mg / mL, pH 7) was reacted with anti-MMP13 antibody (Alexa Fluor® 594) at varying concentrations (0, 0.5, and 5 μg / ml) at 4°C for 1 hour. Unbound MMP13 was removed by washing. The ratio of the specific protein to the Sulfo-SMCC reagent was 5 μg of MMP13 antibody: 3000 μg of Sulfo-SMCC reagent.

[0101] Thiolated platelets were incubated with antibodies treated with Sulfo-SMCC at room temperature for 1 hour to obtain autologous PRP carrying MMP13 (Platelet@MMP13).

[0102] (3) Using immunofluorescence experiments, the purified platelets were fixed in confocal microplates and fixed with 4% paraformaldehyde. After washing with PBS, the plates were incubated with Traut's reagent (3 mg / mL) for 2 hours, Sulfo-SMCC (3 mg / mL, pH=7) and different concentration gradients (0, 0.5, 5 μg / ml) of Anti-MMP13 antibody [Alexa Fluor® 594] (1:200 dilution) at 4°C for 1 hour, washed with PBS three times for 5 minutes each time, and observed and photographed using a laser confocal fluorescence microscope to analyze the colocalization of platelets and MMP13.

[0103] (4) Using flow cytometry, the concentration of Platelet@MMP13 was adjusted to 1×10^6 / mL, and rabbit anti-594 (Abcam, ab150084) fluorescent secondary antibody (1:500 dilution) and anti-CD41 antibody [Alexa Fluor® 488] (Biolegend, 133908) (1:200 dilution) were added and incubated at room temperature for 1 hour. After washing with PBS, the cells were resuspended in PBS and the expression levels of CD41 and MMP13 were detected by flow cytometry.

[0104] Experimental results:

[0105] Through bioconjugation and separation gel centrifugation technology, efficient conjugation of thiol groups on the platelet surface with maleimide groups on the Anti-MMP13 antibody was achieved. Flow cytometry results showed that platelets were successfully modified to carry Anti-MMP13 antibodies, with a modification efficiency of up to 93% ( Figure 2 AB). The results of laser confocal microscopy showed that different concentration gradients (0, 0.5, 5 μg / ml) of Anti-MMP13 antibodies co-localized on the platelet surface ( Figure 3AB), and the modification effect of 5 μg / ml Anti-MMP13 antibody reached 55.52%, which was better than the modification effect of 0.5 μg / ml (21.51%), confirming the specificity and high efficiency modification of 5 μg / ml Anti-MMP13 antibody.

[0106] In summary, Traut reagent and Sulfo-SMCC reagent successfully achieved specific and efficient modification of platelets in PRP, obtaining Platelet@MMP13, providing a new strategy for the treatment of OA cartilage damage.

[0107] Example 2

[0108] like Figure 4 As shown in the experimental flow chart A, this example uses C57BL / 6 mice to establish a DMM cartilage injury model. Six weeks after modeling, the mice receive intra-articular injections of normal saline, conventional PRP, and Platelet@MMP13 PRP, respectively. The treatment course is once a week for a total of four times. After treatment, the pain index, tissue macroscopic score, and cartilage-related proteins (Col2a1, Acan, etc.) of the OA mice are measured, demonstrating that modified platelets improve the degenerative lesions of OA.

[0109] The specific experimental methods are as follows:

[0110] (1) Twelve-week-old C57BL / 6 mice were selected and randomly divided into a control group (sham operation group), a model group (DMM operation group), a PRP treatment group (DMM operation + conventional PRP treatment), and a Platelet@MMP13 treatment group (DMM operation + modified autologous platelet treatment), with 10 mice in each group.

[0111] (2) The OA cartilage injury model was established by cutting the medial meniscus-tibial ligament of the right knee joint of mice through DMM surgery. The sham surgery group only incised the skin without damaging the meniscus, and antibiotics were given after surgery to prevent infection.

[0112] (3) Six weeks after modeling, treatment intervention began: the control group and the model group were injected with normal saline (20 μL / time) into the joint cavity, the PRP treatment group was injected with conventional PRP (20 μL / time), and the Platelet@MMP13 PRP treatment group was injected with modified autologous platelets (20 μL / time), once a week for a total of 4 times.

[0113] (4) After treatment, the pain indicators of mice in each group (including mechanical pain threshold and thermal pain threshold) were tested. The mice were killed and the right knee joints were separated. The OARSI scoring system was used to perform macroscopic scoring of the articular cartilage to evaluate the integrity, thickness and osteophyte formation of the cartilage surface.

[0114] (5) The joint tissue was decalcified and embedded to prepare knee joint tissue sections. The expression levels of cartilage-related proteins (Col2a1 and Acan) were detected by immunofluorescence staining.

[0115] Experimental results:

[0116] The mechanical pain threshold and thermal pain threshold of the Platelet@MMP13 PRP-treated group were significantly higher than those of the model group and the unmodified PRP-treated group ( Figure 4 B-4C). According to the OARSI score, the cartilage surface integrity, thickness and osteophyte formation of the Platelet@MMP13 PRP treatment group were significantly better than those of the model group and the unmodified PRP treatment group ( Figure 4 D-4E). Immunofluorescence staining results showed that the expression levels of cartilage-related proteins (Col2a1 and Acan) were higher in the model group and PRP treatment group ( Figure 4 F) In summary, Platelet@MMP13 treatment significantly improved pain symptoms, cartilage tissue damage, and cartilage-related protein expression in OA mice, demonstrating that modified platelets can effectively alleviate OA degenerative lesions and provide a new strategy for OA treatment.

[0117] The above describes the preferred embodiments of the present invention. It should be understood that the present invention is not limited to the above-mentioned specific embodiments, and the devices and structures that are not described in detail should be understood to be implemented in a common manner in the art; any technician familiar with the art can use the above-mentioned disclosed methods and technical contents to make many possible changes and modifications to the technical solutions of the present invention without departing from the scope of the technical solutions of the present invention, or modify them into equivalent embodiments of equivalent changes, which does not affect the essential content of the present invention. Therefore, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention that do not depart from the content of the technical solutions of the present invention are still within the scope of protection of the technical solutions of the present invention.

Claims

1. A platelet complex, characterized in that The complex comprises: platelet cells, a chemical linker element and a specific protein; wherein the platelet cells are connected to the specific protein via the chemical linker element; and the specific protein comprises an antibody targeting a molecule specifically expressed in cartilage damage.

2. The platelet complex according to claim 1, characterized in that The chemical linker elements include: thiol-maleimide group, maleimide linker, PEG linker, PASylation or HESylation.

3. The platelet complex according to claim 1, characterized in that The specific protein is MMP13 antibody.

4. A method for preparing the platelet complex according to claim 1, characterized in that: The following steps are involved: S1: performing thiol modification on the platelets to obtain activated platelets; S2: performing maleimide modification on the specific protein to obtain an activated protein; S3: mixing and incubating the activated platelets and the activated protein to couple the activated platelets with the activated protein, thereby obtaining the platelet complex.

5. The method according to claim 4, characterized in that The specific protein is an MMP13 antibody; In S1, the platelets are thiol-modified using a Trants reagent, the number of the platelets is 0.8×10^9 to 1.2×10^9, preferably 1×10^9; the amount of the Trants reagent used is 2800-3200 μg, preferably 3000 μg; In step S2, the reagent used for maleimidation modification of the specific protein is Sulfo-SMCC; the specific protein is MMP13 antibody, and the amount of the MMP13 antibody is 0.5 μg / ml to 5 μg / ml, preferably 5 μg / ml of MMP13 antibody; the amount of the Sulfo-SMCC is 2800-3200 μg, preferably 3000 μg.

6. A pharmaceutical composition, characterized in that include: (a1) a first active ingredient for preventing and / or treating arthritis-related diseases, the first active ingredient comprising: the platelet complex according to claim 1; (a2) a second active ingredient for preventing and / or treating arthritis-related diseases, wherein the second active ingredient includes: other drugs for preventing and / or treating arthritis-related diseases; and (b) a pharmaceutically acceptable carrier; The other drugs for preventing and / or treating arthritis-related diseases are selected from the following group: non-steroidal anti-inflammatory drugs, analgesics (non-anti-inflammatory), chondroprotective agents, intra-articular injection drugs, Chinese patent medicines and external patches, or a combination thereof.

7. A medicine box, characterized in that: include: (i) a first container, and the active ingredient (a1) the platelet complex according to claim 1, or a medicament containing the active ingredient (a1) in the first container; and (ii) a second container, and the active ingredient (a2) in the second container; other medicaments for preventing and / or treating arthritis-related diseases, or medicaments containing the active ingredient (a2); The kit further comprises instructions for administering the active ingredient (a1) and the active ingredient (a2) in combination to (i) prevent and / or treat arthritis-related diseases; and / or (ii) inhibit arthritis.

8. Use of the platelet complex according to claim 1, or the platelet complex prepared by the method according to claim 4, or the pharmaceutical composition according to claim 6, or the drug kit according to claim 7 in preparing a product targeting cartilage damage.

9. The use according to claim 8, characterized in that The product is selected from the following group: the bone defect repair product is a spinal injury repair product, a skull injury repair product, a maxillofacial bone defect repair product, a limb bone defect repair product or a joint bone defect repair product; preferably, the spinal injury repair product is selected from cervical vertebrae, thoracic vertebrae or lumbar vertebrae injury repair products, or the limb bone defect repair product is selected from femur, fibula or humerus injury repair products, or the joint bone defect repair product is selected from hip joint or knee joint bone injury repair products.

10. Use of the platelet complex according to claim 1, or the platelet complex prepared by the method according to claim 4, or the pharmaceutical composition according to claim 6, or the medicine kit according to claim 7 in preparing a drug for preventing and / or treating arthritis.

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