Micron self-assembly material with joint retention, lubrication and anti-inflammatory effects as well as preparation method and application of micron self-assembly material

Abstract

The invention provides a micron self-assembly material with joint retention, lubrication and anti-inflammatory functions as well as a preparation method and application of the micron self-assembly material. The micron self-assembly material is formed by self-assembly of avidin modified mesoporous polydopamine nanoparticles and biotinylated XII type collagen targeted lubricating peptide modified mesoporous polydopamine nanoparticles, and the mesoporous polydopamine nanoparticles and the biotinylated XII type collagen targeted lubricating peptide are combined to form the micron self-assembly material. In the avidin modified mesoporous polydopamine nanoparticles, avidin and the mesoporous polydopamine nanoparticles are connected through a covalent bond; in the biotinylated XII type collagen targeted lubricating peptide modified mesoporous polydopamine nanoparticle, the N end of the XII type collagen targeted lubricating peptide is connected with biotin through a covalent bond, and the C end of the XII type collagen targeted lubricating peptide is connected with the mesoporous polydopamine nanoparticle through an amino acid and a covalent bond. The micron self-assembly material can be specifically combined with a cartilage focus area, the retention time in joints is prolonged, good lubrication and excellent anti-inflammatory activity are shown, and the pathological progress of osteoarthritis can be effectively inhibited.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a micron self-assembly material with joint retention, lubrication and anti-inflammatory effects, and a preparation method and application thereof. Background Art

[0002] Osteoarthritis (OA) is a chronic disabling disease with progressive degeneration of articular cartilage as its core pathological feature. Its typical pathological manifestations include articular cartilage degeneration, subchondral bone sclerosis, osteophyte formation, synovial inflammation and eventual loss of joint function. According to statistics, about 250 million people are affected by osteoarthritis worldwide. At present, the treatment of OA mainly includes surgical treatment (such as microfracture, cartilage transplantation, joint replacement, etc.) and drug treatment (such as oral non-steroidal anti-inflammatory drugs, analgesics, or intra-articular injection of hyaluronic acid, glucocorticoids, etc.). However, due to the lack of blood vessels and lymphatic return in cartilage tissue, whether it is administered orally or injected into the articular cavity, the drug will be quickly cleared by the blood vessels and lymphatic system in the synovial tissue, making it difficult to exert its biological effect at the lesion site. For example, the half-life of non-steroidal anti-inflammatory drugs in synovial fluid is only 1-4 hours. Oral medications usually require increasing dosages to maintain efficacy, but this can lead to significant systemic side effects; while intra-articular injections face problems such as rapid degradation and insufficient retention time, requiring multiple injections, which not only increases the patient's physical and mental burden, but also increases the risk of infection. Currently, there is still a lack of clinical treatments that can effectively prevent the progression of osteoarthritis.

[0003] CN113967199A discloses a resveratrol-polylactic acid long-acting nano-microsphere, which has good stability and can significantly alleviate symptoms such as cartilage damage and synovial inflammation after being injected into the knee joint cavity.

[0004] CN118903058A discloses a drug-loaded core-shell microsphere for sequentially regulating cell autophagy and chondrogenesis, which comprises a shell layer and a core layer from the outside to the inside, wherein the shell layer comprises a polymer matrix with photo-crosslinking properties and first drug-loaded nanoparticles loaded with drugs for regulating chondrocyte autophagy activity, and the core layer comprises a water-soluble polymer matrix, targeted cartilage matrix molecular modifications and second drug-loaded nanoparticles loaded with drugs for promoting chondrogenesis, thereby improving the retention time and bioavailability of the drugs in the joint cavity.

[0005] Although a series of nanomaterials or nano drug delivery carriers have been developed and applied to the treatment of arthritis, the cartilage matrix is ​​composed of a type II collagen network entangled with highly negatively charged proteoglycans, and as the depth of the cartilage increases, the density of proteoglycans increases significantly and presents a brush-like structure, resulting in a serious steric hindrance effect, making it difficult for nano drugs to effectively penetrate into the cartilage matrix. In addition, nano drug carriers free in the joint cavity are also easily cleared by synovial blood vessels, limiting their therapeutic effect.

[0006] In response to the problems of short residence time and limited efficacy of traditional oral drugs, intra-articular injection drugs and nanomaterials in the joints, the present invention has developed a micron self-assembled material with joint residence, lubrication and anti-inflammatory effects, aiming to solve the above problems and provide a new, efficient and long-term strategy for the treatment of osteoarthritis. Summary of the invention

[0007] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a micron self-assembly material with joint retention, lubrication and anti-inflammatory effects, as well as a preparation method and application thereof. The micron self-assembly material can specifically bind to the cartilage lesion area, significantly prolong the retention time in the joint, and has excellent lubrication performance and anti-inflammatory activity, effectively solving the technical problems of the existing drug preparations being easy to metabolize and decompose in the joint, difficult to retain in the joint cavity, and unable to effectively prevent the pathological process of osteoarthritis.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a micron self-assembled material with joint retention, lubrication and anti-inflammatory effects, wherein the micron self-assembled material is formed by self-assembly of avidin-modified mesoporous polydopamine nanoparticles and biotinylated type XII collagen targeted lubricating peptide-modified mesoporous polydopamine nanoparticles.

[0010] In the avidin-modified mesoporous polydopamine nanoparticles, the avidin and the mesoporous polydopamine nanoparticles are connected via covalent bonds.

[0011] In the biotinylated type XII collagen targeted lubricating peptide modified mesoporous polydopamine nanoparticles, the N-terminus of the type XII collagen targeted lubricating peptide is connected to biotin via a covalent bond, and the C-terminus is covalently connected to the mesoporous polydopamine nanoparticles via amino acids.

[0012] The present invention constructs a micron-scale self-assembly material by modifying mesoporous polydopamine nanoparticles with avidin and biotinylated type XII collagen targeting lubricating peptides, respectively, using the "avidin-biotin" specific binding mechanism. Since the diameter of the sinusoids in the synovial blood vessels is 10-50μm and the endothelial gap is 100-500nm, usually only particles with a size of less than 500nm are allowed to pass through, and micron-scale particles are difficult to pass through the synovial blood vessels. Therefore, the micron self-assembly material can effectively reside in the joint cavity, avoiding the problem of being quickly cleared by the synovial tissue. In addition, during the development of OA, the cartilage matrix undergoes progressive degradation, resulting in a large amount of glycosaminoglycans in the middle and deep layers exposed; at the same time, chondrocytes undergo dedifferentiation and hypertrophy, causing changes in the expression of type XII collagen (Col.XII). Based on the above-mentioned OA pathological characteristics, the present invention utilizes the positive charge effect of avidin and the specific binding ability of type XII collagen targeted lubricating peptide to OA pathological products, thereby achieving the close binding of the self-assembled material to the cartilage lesion area and the joint lubrication function; at the same time, with the help of the anti-inflammatory effect of polydopamine, the OA pathological process is synergistically prevented, thereby achieving the goal of long-term intervention in OA progression with a single injection.

[0013] The self-assembly of the present invention can be performed in vivo and / or in vitro.

[0014] Preferably, the amino acid sequence of the type XII collagen targeting lubricating peptide includes SEQ ID NO: 1, and the specific sequence of SEQ ID NO: 1 is: DLQYWYPIWDTH.

[0015] The type XII collagen targeted lubricating peptide with the amino acid sequence of DLQYWYPIWDTH can accurately identify type XII collagen, quickly focus on the injured site, and has excellent joint lubrication effect, reducing wear on the joints.

[0016] Preferably, in the biotinylated type XII collagen targeting lubricating peptide modified mesoporous polydopamine nanoparticles, the N-terminus of the type XII collagen targeting lubricating peptide is covalently linked to biotin, and the C-terminus is covalently linked to the mesoporous polydopamine nanoparticles via the thiol group on cysteine.

[0017] Preferably, the avidin comprises a cationic protein having an isoelectric point of 10-15.

[0018] Among them, the specific point values ​​10-15 can be selected as 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, etc.

[0019] In the present invention, the cationic protein with an isoelectric point of 10-15 can not only rapidly self-assemble with biotin, but also efficiently bind to the cartilage matrix (highly negatively charged glycosaminoglycan entanglement), so that the micron self-assembly material has a more stable binding effect on the surface of damaged cartilage.

[0020] Preferably, the mass ratio of the avidin-modified mesoporous polydopamine nanoparticles to the biotinylated type XII collagen targeted lubricating peptide-modified mesoporous polydopamine nanoparticles is (1-10):(1-10).

[0021] Among them, the specific point values ​​1-10 can be selected as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.

[0022] Preferably, the particle size of the mesoporous polydopamine nanoparticles is 100-300 nm, and the pore size is 10-50 nm.

[0023] Among them, the specific point values ​​in 100-300nm can be selected from 100nm, 120nm, 140nm, 160nm, 180nm, 200nm, 220nm, 240nm, 260nm, 280nm, 300nm, etc., and the specific point values ​​in 10-50nm can be selected from 10nm, 20nm, 30nm, 40nm, 50nm, etc.

[0024] In the present invention, the mesoporous polydopamine nanoparticles with a particle size of 100-300 nm have good biocompatibility, are not easily quickly recognized and cleared by the immune system, and can remain in the body for a relatively long time.

[0025] In addition, by controlling the pore size to 10-50nm, the micron self-assembled material can be further used as a drug delivery platform to load anti-inflammatory drugs that can be used in combination to further prevent the pathological progression of osteoarthritis.

[0026] Preferably, the mesoporous polydopamine nanoparticles are prepared by a preparation method comprising the following steps:

[0027] (1) Dopamine hydrochloride and a template agent are mixed and dissolved in a solvent, a pore-enlarging agent is added, and a polymerization reaction occurs in an alkaline environment.

[0028] (2) The solution after the reaction is stirred and centrifuged to obtain a precipitate.

[0029] (3) The precipitate is repeatedly washed with an organic solvent to remove the template, thereby obtaining the mesoporous polydopamine nanoparticles.

[0030] Preferably, in step (1), the template agent comprises poloxamer, the pore-enlarging agent comprises mesitylene, and the solvent comprises an aqueous solution of ethanol.

[0031] Preferably, the stirring speed in step (2) is 300-500 rpm.

[0032] Among them, the specific point values ​​in 300-500rpm can be selected as 300rpm, 350rpm, 400rpm, 450rpm, 500rpm, etc.

[0033] Preferably, the organic solvent in step (3) comprises ethanol and / or acetone.

[0034] In the present invention, dopamine forms mesoporous polydopamine nanoparticles by intramolecular ring self-polymerization under the conditions of base catalysis, template agent and 300-500rpm shear force, and the mesoporous polydopamine nanoparticles have the molecular structure of o-quinone, catechol and pyrrole. The mesoporous polydopamine nanoparticles prepared by the method have uniform particle size, clear pore structure and orderly arrangement.

[0035] Preferably, the avidin-modified mesoporous polydopamine nanoparticles are prepared by a preparation method comprising the following steps:

[0036] The mesoporous polydopamine nanoparticles are mixed with thiol-modified avidin, and Michael addition reaction is carried out. The pH value of the reaction is adjusted to 7-9, and the precipitate is collected by centrifugation to obtain the avidin-modified mesoporous polydopamine nanoparticles.

[0037] Among them, the specific point values ​​​​from 7 to 9 can be selected as 7, 7.5, 8, 8.5, 9, etc.

[0038] Preferably, the Michael addition reaction is carried out for 10-40 hours at a temperature of 10-50°C.

[0039] Among them, the specific point values ​​in 10-40h can be selected as 10h, 20h, 30h, 40h, etc., and the specific point values ​​in 10-50℃ can be selected as 10℃, 20℃, 30℃, 40℃, 50℃, etc.

[0040] Preferably, the biotinylated type XII collagen targeted lubricating peptide modified mesoporous polydopamine nanoparticles are prepared by a preparation method comprising the following steps:

[0041] The biotin-modified type XII collagen targeted lubricating peptide and mesoporous polydopamine nanoparticles are mixed and reacted, the pH value of the reaction is adjusted to 7-9, and the precipitate is collected by centrifugation to obtain biotinylated type XII collagen targeted lubricating peptide-modified mesoporous polydopamine nanoparticles.

[0042] Among them, the specific point values ​​​​from 7 to 9 can be selected as 7, 7.5, 8, 8.5, 9, etc.

[0043] Preferably, the Michael addition reaction is carried out for 10-40 hours at a temperature of 10-50°C.

[0044] Among them, the specific point values ​​in 10-40h can be selected as 10h, 20h, 30h, 40h, etc., and the specific point values ​​in 10-50℃ can be selected as 10℃, 20℃, 30℃, 40℃, 50℃, etc.

[0045] In a second aspect, the present invention provides a method for preparing the micron self-assembled material having joint retention, lubrication, and anti-inflammatory effects as described in the first aspect, the preparation method comprising:

[0046] Avidin-modified mesoporous polydopamine nanoparticles and biotinylated type XII collagen targeted lubricating peptide-modified mesoporous polydopamine nanoparticles were mixed in PBS and self-assembled to form micron self-assembled materials.

[0047] Preferably, the self-assembly time is 0.1-30h and the temperature is 4-50°C.

[0048] Among them, the specific point values ​​in 0.1-30h can be selected as 0.1h, 0.2h, 1h, 4h, 7h, 10h, 13h, 17h, 20h, 23h, 27h, 30h, etc., and the specific point values ​​in 4-50℃ can be selected as 4℃, 10℃, 20℃, 30℃, 40℃, 50℃, etc.

[0049] In a third aspect, the present invention provides a use of the micron self-assembled material as described in the first aspect in preparing a drug for treating arthritis.

[0050] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] The present invention relates to a micron-scale self-assembly material, which is constructed by modifying mesoporous polydopamine nanoparticles with avidin and biotinylated type XII collagen targeting lubricating peptides, respectively, and utilizing the "avidin-biotin" specific binding mechanism. The size of the micron self-assembly material is larger than the endothelial gap of the synovial blood vessels, and can effectively avoid being quickly cleared by the synovial blood vessels, thereby significantly prolonging its retention time in the joint cavity. At the same time, the present invention utilizes the positive charge effect of avidin and the specific binding ability of the Col.XII targeting lubricating peptide to OA pathological products, thereby achieving a close combination of the self-assembly material with the OA cartilage lesion area and a joint lubrication function. In addition, through the anti-inflammatory effect of polydopamine, the OA pathological process is synergistically inhibited, achieving the goal of long-term intervention in the progression of OA with a single injection. The present invention effectively solves the problems of the existing drug preparations being easy to metabolize and clear in the joints, difficult to retain in the joint cavity, and unable to prevent the pathological process of OA. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 These are the transmission electron microscope and energy spectrum elemental analysis diagrams of different materials in Verification Example 1.

[0054] Figure 2 This is a scanning electron microscope image of the assembly process of the micron self-assembled material in Verification Example 2.

[0055] Figure 3 This is a fluorescence imaging analysis diagram of the combination of different experimental groups and pig cartilage in Test Example 1.

[0056] Figure 4 This is a quantitative analysis of the fluorescence intensity of different experimental groups combined with pig cartilage in Test Example 1.

[0057] Figure 5 This is an in vivo fluorescence imaging analysis of the retention of different materials in the joints of arthritic mice in Test Example 2.

[0058] Figure 6 This is a diagram showing the distribution of different materials in various organs of arthritic mice in Test Example 2.

[0059] Figure 7 This is an in vivo imaging analysis of the oxidative stress probe in mice of different experimental groups in Test Example 3.

[0060] Figure 8 This is a quantitative analysis chart of the expression of oxidative stress probes in mice in different experimental groups in Test Example 3.

[0061] Fig. 9 This is a micro-CT analysis result of osteophytes in mice of different experimental groups in Test Example 3.

[0062] Fig.10 It is a statistical graph of osteophyte width of mice in different experimental groups in Test Example 3. DETAILED DESCRIPTION

[0063] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0064] Preparation Example 1-1

[0065] Synthesis of mesoporous polydopamine nanoparticles:

[0066] (1) 0.3 g dopamine hydrochloride monomer (DA, Sigma, H8502) and 0.5 g poloxamer 407 (F127, Sigma, P2443) were dissolved in 50 mL of 50% ethanol aqueous solution and stirred at 500 rpm to dissolve.

[0067] (2) After dissolution, 520 μL of mesitylene pore expander (TMB, Sigma, M7200) was added dropwise at 500 rpm, ultrasonicated for 30 min, and then 1 mL of ammonia (NH4OH) was added dropwise at 500 rpm. 4 OH, 28-30%, Sigma, 221228), stirred at 500 rpm for 30 min and then at 300 rpm for 3 h to carry out polymerization reaction, and after the reaction, centrifuged at 12000 rpm for 30 min to collect the material.

[0068] (3) preparing a mixed solution of ethanol and acetone (volume ratio 2:1), using the mixed solution and the material collected in step (2) to wash to remove the template agent poloxamer 407, centrifuging at 12000 rpm for 30 min to collect the material, and dispersing the collected material in a mixed solution of ethanol and acetone (volume ratio 2:1) and ultrasonicating for 30 min, and repeating this process 3 times.

[0069] (4) The material obtained in step (3) was washed three times with anhydrous ethanol, and the material was collected each time by centrifugation at 12000 rpm for 30 min. The material collected each time by centrifugation was dispersed in anhydrous ethanol and ultrasonicated for 30 min.

[0070] (5) The material obtained in step (4) was dried in a vacuum drying oven at 60° C. for 3 h, and then sealed and stored at 4° C. to obtain mesoporous polydopamine nanoparticles.

[0071] Preparation Example 1-2

[0072] Synthesis of Fluorescently Labeled Mesoporous Polydopamine Nanoparticles

[0073] To facilitate fluorescence imaging observation, the mesoporous polydopamine nanoparticles prepared in Preparation Example 1 were modified with thiol-anthocyanin (SH-Cy5) as follows:

[0074] Weigh 10 mg of mesoporous polydopamine nanoparticles and 10 mg of SH-Cy5 (Ponsure, PS2-SC5-2K), dissolve in 20 mL and 4 mL of PBS, respectively, to prepare a concentration of 0.5 mg / mL mesoporous polydopamine nanoparticles and 2.5 mg / mL SH-Cy5. Then take 5 mL of 0.5 mg / mL mesoporous polydopamine nanoparticles and 1 mL of 2.5 mg / mL SH-Cy5 and stir at 500 rpm at room temperature in the dark for 24 hours.

[0075] During the reaction, the pH was adjusted to 8.0 with 3 mol / L NaOH. After the reaction, the supernatant was washed with PBS until it was colorless. The precipitated material was collected by centrifugation at 12000 rpm for 30 min each time to obtain Cy5-labeled mesoporous polydopamine nanoparticles.

[0076] Preparation Example 2-1

[0077] Synthesis of avidin-modified mesoporous polydopamine nanoparticles

[0078] Weigh 2.5 mg of the mesoporous polydopamine nanoparticles obtained in Preparation Example 1-1 and disperse them in 5 mL of simulated body fluid (PBS, pH = 8.0), add 500 μL of 2 mg / mL thiol-modified avidin (Protein Mods, 320124AVT8), stir and react at room temperature and in the dark at 500 rpm for 24 hours, during which the pH is adjusted to 8.0 with 3 mol / L NaOH. After the reaction, centrifuge at 12000 rpm for 30 min to collect the precipitate, wash 3 times with PBS, and obtain avidin-modified mesoporous polydopamine nanoparticles.

[0079] Preparation Example 2-2

[0080] Synthesis of Fluorescently Labeled Avidin-Modified Mesoporous Polydopamine Nanoparticles

[0081] The preparation method of fluorescently labeled avidin-modified mesoporous polydopamine nanoparticles refers to Preparation Example 2-1, and the only difference between it and Preparation Example 2-1 is that 1 mL 2.5 mg / mL SH-Cy5 is added for fluorescence modification at the same time as the thiol-modified avidin.

[0082] Preparation Example 3-1

[0083] Synthesis of biotinylated Col.XII-targeted lubricating peptide-modified mesoporous polydopamine nanoparticles

[0084] (1) Synthesis of biotinylated Col.XII targeting lubricating peptide: modify Col.XII targeting lubricating peptide, modify its N-terminus with biotin, and modify its C-terminus with cysteine ​​to obtain biotinylated Col.XII targeting lubricating peptide, weigh 2.5 mg of biotinylated Col.XII targeting lubricating peptide and dissolve it in 1 mL of DMSO;

[0085] (2) Weigh 2.5 mg of the mesoporous polydopamine nanoparticles obtained in Preparation Example 1-1 and disperse them in 5 mL of PBS solution (pH = 8.0);

[0086] (3) The solutions prepared in step (1) and step (2) were mixed, stirred at 500 rpm for 24 h at room temperature in the dark, during which the pH value was adjusted to 8.0 with 3 mol / L NaOH. After the reaction, the precipitate was collected by centrifugation at 12000 rpm for 30 min, and washed three times with PBS to obtain biotinylated Col.XII targeted lubricating peptide-modified mesoporous polydopamine nanoparticles.

[0087] Preparation Example 3-2

[0088] Synthesis of Fluorescently Labeled Biotinylated Col.XII Targeted Lubricating Peptide Modified Mesoporous Polydopamine Nanoparticles

[0089] The preparation method of fluorescently labeled biotinylated Col.XII targeted lubricating peptide modified mesoporous polydopamine nanoparticles refers to Preparation Example 3-1, and the only difference between it and Preparation Example 3-1 is that in step (3), 1 mL 2.5 mg / mL SH-Cy5 is added for fluorescence modification at the same time as the biotinylated Col.XII targeted lubricating peptide is added.

[0090] Preparation Example 4-1

[0091] Synthetic micronized self-assembled materials

[0092] The avidin-modified mesoporous polydopamine nanoparticles prepared in Preparation Example 2-1 and the biotinylated Col.XII targeted lubricating peptide-modified mesoporous polydopamine nanoparticles prepared in Preparation Example 3-1 were mixed in PBS at a mass ratio of 2:1 and self-assembled for 30 minutes to obtain a micron self-assembled material with joint retention, lubrication and anti-inflammatory effects.

[0093] Preparation Example 4-2

[0094] Synthesis of fluorescently labeled micronized self-assembled materials

[0095] The preparation method of the micron self-assembled material refers to Preparation Example 4-1, and the only difference between it and Preparation Example 4-1 is that the nanoparticles of Preparation Examples 2-1 and 3-1 are replaced by the nanoparticles of Preparation Examples 2-2 and 3-2 respectively.

[0096] Verification Example 1

[0097] The mesoporous polydopamine nanoparticles obtained in Preparation Example 1-1, the avidin-modified mesoporous polydopamine nanoparticles obtained in Preparation Example 2-1, and the biotinylated Col.XII targeted lubricating peptide-modified mesoporous polydopamine nanoparticles obtained in Preparation Example 3-1 were subjected to transmission electron microscopy and energy spectrum elemental analysis. The results are as follows: Figure 1 As shown in the figure, transmission electron microscopy results show that the modification of avidin and biotinylated Col.XII targeted lubricating peptide did not change the size, morphology and pore structure of mesoporous polydopamine nanoparticles. All three nanoparticles showed a spherical morphology with a size of 100-200nm, and the mesoporous structure with a size of about 14nm was clearly visible. The energy spectrum results showed that the newly added S element distribution was visible on the surface of the mesoporous polydopamine nanoparticles modified with avidin and targeted lubricating peptide, proving that it exists on the surface of the mesoporous polydopamine nanoparticles.

[0098] Verification Example 2

[0099] This verification example verifies the assembly process of Preparation Example 4-1. The avidin-modified mesoporous polydopamine nanoparticles obtained in Preparation Examples 2-1 and 3-1 were mixed with the biotinylated Col.XII targeted lubricating peptide-modified mesoporous polydopamine nanoparticles in a PBS buffer at a mass ratio of 2:1. The mixture was dried, gold-sprayed, and observed under a scanning electron microscope 5 minutes, 30 minutes, and 60 minutes after mixing. The results are shown in the figure. Figure 2 The results show that as time goes by, the nanoparticles gradually form a state of interconnection through the high affinity between "avidin-biotin" and finally self-assemble into micron-sized materials.

[0100] Test Example 1

[0101] Testing the material's binding to the natural cartilage matrix

[0102] (1) Preparation of pig cartilage samples: Fresh pig bones were purchased and the whole-layer cartilage was separated and cut into uniform round pieces using a 6-mm diameter circular punch. The pieces were divided into a healthy group and an injured group. The healthy group consisted of normal pig cartilage washed with ultrapure water only, and the injured group consisted of injured pig cartilage treated with 2.5% trypsin at 37°C for 15 min.

[0103] (2) Experimental methods

[0104] The Cy5-labeled micron self-assembled material obtained in Preparation Example 4-2 was dripped onto the surface of healthy and damaged pig cartilage at a concentration of 10 μL of 10 mg / mL, incubated in the dark for 2, 4, 8, and 12 hours, and washed 5 times with PBS. The material binding conditions on the cartilage surface were observed by fluorescence imaging and quantitative analysis of fluorescence intensity using a small animal in vivo optical three-dimensional imaging system (PerkinElmer, IVIS spectrum);

[0105] The Cy5-labeled mesoporous polydopamine nanoparticles obtained in Preparation Example 1-2 with the same concentration were used as the control of this experiment.

[0106] (3) Test results

[0107] Fluorescence imaging analysis results of pig cartilage Figure 3 As shown in the figure, whether it is healthy porcine cartilage or damaged porcine cartilage, mesoporous polydopamine nanoparticles and micron self-assembled materials show a binding enhancement effect over time. Compared with mesoporous polydopamine nanoparticles, micron self-assembled materials modified with positively charged avidin and biotinylated Col.XII targeted lubricating peptide have more significant cartilage binding ability after dynamic co-incubation with cartilage for 24 hours. Especially for damaged porcine cartilage, due to the large amount of collagen and negatively charged glycosaminoglycans in the cartilage matrix exposed by enzymatic digestion, the material has a more stable binding effect on the surface of damaged cartilage.

[0108] The fluorescence intensity of the damaged pig cartilage surface was quantitatively analyzed. Figure 4 As shown. The analysis showed that although the cartilage binding effect of mesoporous polydopamine nanoparticles increased over time within 12 hours, there was no significant difference in the fluorescence intensity between the time points, and as time went on, the average fluorescence intensity decreased, indicating that the binding stability of the mesoporous polydopamine nanoparticles was insufficient, and some particles would fall off and become free in the buffer. In contrast, the micron self-assembled material showed a significantly enhanced cartilage binding effect over time, and the fluorescence intensity after 24 hours increased by 2.60 times and 2.32 times compared with 2 hours and 4 hours, respectively. In addition, the fluorescence intensity of the micron self-assembled material was significantly higher than that of the mesoporous polydopamine nanoparticles, the former being 2.39 times that of the latter, further demonstrating its excellent binding stability.

[0109] Test Example 2

[0110] Testing the residence time of micronized self-assembled materials in joints

[0111] (1) Construction of arthritis model:

[0112] The anterior cruciate ligament transection method was used to establish the arthritis animal model. All experimental protocols were evaluated and approved by the Institutional Animal Care and Use Committee of Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences (YSB-20220316-TW-A0526). The experimental procedures are as follows:

[0113] Ten-month-old male C57BL / 6 mice (purchased from Guangdong Medical Experimental Animal Center) were selected as modeling animals. After inducing anesthesia with 3% isoflurane, the mice were fixed in a supine position and wore an anesthesia mask for continuous anesthesia. The anesthesia concentration was maintained at about 2%. The concentration was appropriately adjusted according to the anesthesia state of the animals during the operation. The mice were fixed to the operating table, the right knee surgical area was prepared and disinfected with iodine, and the knee was bent 90° to make a sagittal incision of about 1.0 cm above the medial side of the knee joint. Subsequently, the right knee was straightened and the patella was removed to expose the joint capsule. Fine scissors were used to open the joint capsule under a stereo microscope (ZEISS, STEMIDV4 SPOT), expose and cut the anterior cruciate ligament, and cause joint mechanical instability. Based on this model, progressive joint damage was induced by transection of the anterior cruciate ligament for 4 weeks to simulate the formation of osteoarthritis.

[0114] (2) Joint injection of materials

[0115] The Cy5-labeled mesoporous polydopamine nanoparticles obtained in Preparation Example 1-2 and the Cy5-labeled micron self-assembled materials obtained in Preparation Example 4-2 were injected into the joint cavity at a volume of 10 μL 10 mg / mL, respectively. The micron self-assembled materials were prepared and used immediately, and the retention of the materials in the joints of arthritic mice was evaluated at designated time points (2 hours, 4 hours, 12 hours, 24 hours, 2 days, 4 days, 6 days, 8 days, 10 days, 12 days, and 14 days) using a small animal in vivo optical three-dimensional imaging system, as well as the distribution of the materials in various organs (heart, liver, spleen, lungs, and kidneys) 14 days after joint injection.

[0116] (3) Test results

[0117] In vivo fluorescence imaging analysis of material retention in the joints of arthritic mice Figure 5 As shown. Both mesoporous polydopamine nanoparticles and micron self-assembled materials showed strong fluorescence signals within 12 hours after joint injection. However, the fluorescence intensity of the mesoporous polydopamine nanoparticle group was significantly lower than that of the micron self-assembled material group within 12 hours, and its fluorescence signal began to decay significantly after 1 day, and showed a significant downward trend with time. Specifically, 2 days after injection, the fluorescence intensity of 60% of the mouse joints dropped sharply; 6 days after injection, 60% of the mouse joints could no longer detect fluorescence signals. The above results show that although polydopamine has universal adhesion properties, due to the metabolic clearance of synovial blood vessels, mesoporous polydopamine nanoparticles are difficult to reside in the joint cavity for a long time. In contrast, the micron self-assembled material can be stably retained in the joint cavity, and a strong fluorescence signal can still be detected 8 days after injection. Fluorescence expression can still be observed in the joint area of ​​some mice 14 days after injection.

[0118] To further verify whether the weakening of the fluorescence signal was caused by the metabolism or attenuation of the fluorescent molecules, we euthanized the animals 14 days later and removed the knee joints for ex vivo tissue imaging analysis. Figure 6 The joint areas of animal samples in all avidin-biotinylated lubricating polypeptide assembly layer groups showed obvious fluorescent signals, and no abnormal aggregation of materials in other major organs (such as heart, liver, spleen, lung, and kidney) was observed.

[0119] Test Example 3

[0120] Analysis of oxidative stress in vivo during the early stage of arthritis treatment

[0121] (1) Experimental groups and intervention methods:

[0122] ① PBS group: arthritis mice of test example 2 were injected with 10 μL PBS;

[0123] ② Dexamethasone group: The arthritis mice of Test Example 2 were injected with 10 μL of 2 mg / mL clinical drug dexamethasone;

[0124] ③ Micron self-assembled material group: The arthritic mice of Test Example 2 were injected with 10 μL of 10 mg / mL micron self-assembled material prepared in Preparation Example 4-1.

[0125] (2) Analysis of oxidative stress in the body during the early stages of arthritis treatment:

[0126] On the 3rd and 7th days after the intervention, 10 μL of 8 mg / mL active oxygen probe L-012 sodium salt (C 13 H 8 C1N 4 O 2 ·Na) were injected into the joint cavity, and the bioluminescence of reactive oxygen probes in the joint was detected using a small animal in vivo optical three-dimensional imaging system, and the bioluminescence intensity of the reactive oxygen probes in each group was statistically analyzed.

[0127] The in vivo imaging results and statistical analysis of reactive oxygen species probes are shown in Figure 7 and Figure 8 As shown. The results showed that after local injection of dexamethasone into the joint cavity, the expression level of oxidative stress probe was not significantly different from that of the PBS group 7 days after injection, indicating that the relief effect of dexamethasone on arthritis inflammation failed to be sustained and significantly improved. In contrast, after in situ intervention with micron self-assembled materials, the expression level of oxidative stress, an indicator of arthritis pathology, was significantly and continuously reduced on the 3rd and 7th days, indicating that it has a sustained improvement effect on arthritis inflammation. This result shows that micron self-assembled materials are significantly superior to traditional drug treatments in reducing arthritis inflammation.

[0128] (3) Micro-CT analysis of osteophytes:

[0129] On the 14th and 28th days after the intervention, the model joints were removed and fixed with 4% paraformaldehyde. Micro-computed tomography (micro-CT) was used to reconstruct the pericartilaginous osteophytes in three dimensions. The micro-CT test parameters were as follows: resolution 10.4 μm, current 114 μA, voltage 55 kV, and integration time 500 ms. Based on the micro-CT two-dimensional images, the osteophyte width of the largest osteophyte section was measured, and the data of each group were statistically analyzed.

[0130] Three-dimensional reconstruction and two-dimensional CT images such as Fig. 9 The statistical results are shown in Fig.10As shown. The results showed that a large amount of irregular bone hyperplasia appeared around the joints in both the PBS group and the dexamethasone group, and osteophyte formation was significant. Over time, osteophyte hyperplasia, an important pathological indicator of arthritis, continued to worsen. Statistical analysis showed that the therapeutic effect of the dexamethasone group was limited and there was no significant difference compared with the PBS group; 28 days after injection, the average maximum osteophyte widths of the two groups were 474.20μm and 541.92μm, respectively.

[0131] In contrast, in situ injection of micron self-assembly materials significantly slowed down the development of osteophytes. During the period from 14 to 28 days, no new osteophytes were observed, the joint edge morphology was clear, and there was less bone hyperplasia. Long-term therapeutic effects can be achieved by only one joint injection of avidin-biotinylated lubricating polypeptide self-assembly material. After 28 days of injection, the width of the osteophytes was reduced by 2.48 times and 2.70 times compared with the PBS group and the dexamethasone group, respectively. The above results show that the micron self-assembly material involved in the present invention has a significant effect in preventing the pathological progression of osteoarthritis.

[0132] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0133] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0134] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A micron self-assembled material with joint retention, lubrication and anti-inflammatory effects, characterized in that: The micron self-assembly material is formed by self-assembly of avidin-modified mesoporous polydopamine nanoparticles and biotinylated type XII collagen targeted lubricating peptide-modified mesoporous polydopamine nanoparticles; In the avidin-modified mesoporous polydopamine nanoparticles, the avidin and the mesoporous polydopamine nanoparticles are connected by a covalent bond; In the biotinylated type XII collagen targeted lubricating peptide modified mesoporous polydopamine nanoparticles, the N-terminus of the type XII collagen targeted lubricating peptide is connected to biotin via a covalent bond, and the C-terminus is covalently connected to the mesoporous polydopamine nanoparticles via amino acids.

2. The micron self-assembly material according to claim 1, characterized in that: The amino acid sequence of the type XII collagen targeting lubricating peptide includes SEQ ID NO: 1; Preferably, in the biotinylated type XII collagen targeting lubricating peptide modified mesoporous polydopamine nanoparticles, the N-terminus of the type XII collagen targeting lubricating peptide is covalently linked to biotin, and the C-terminus is covalently linked to the mesoporous polydopamine nanoparticles via the thiol group on cysteine.

3. The micron self-assembled material according to claim 1 or 2, characterized in that: The avidin includes a cationic protein with an isoelectric point of 10-15.

4. The micron self-assembled material according to any one of claims 1 to 3, characterized in that: The mass ratio of the avidin-modified mesoporous polydopamine nanoparticles to the biotinylated type XII collagen targeted lubricating peptide-modified mesoporous polydopamine nanoparticles is (1-10):(1-10).

5. The micron self-assembled material according to any one of claims 1 to 4, characterized in that: The particle size of the mesoporous polydopamine nanoparticles is 100-300 nm, and the pore size is 10-50 nm.

6. The micron self-assembled material according to any one of claims 1 to 5, characterized in that: The mesoporous polydopamine nanoparticles are prepared by a preparation method comprising the following steps: (1) mixing and dissolving dopamine hydrochloride and a template agent in a solvent, adding a pore-enlarging agent, and causing a polymerization reaction in an alkaline environment; (2) stirring and centrifuging the solution after the reaction to obtain a precipitate; (3) repeatedly washing the precipitate with an organic solvent to remove the template to obtain the mesoporous polydopamine nanoparticles; Preferably, in step (1), the template agent comprises poloxamer, the pore-enlarging agent comprises mesitylene, and the solvent comprises an aqueous solution of ethanol; Preferably, the stirring speed in step (2) is 300-500 rpm; Preferably, the organic solvent in step (3) comprises ethanol and / or acetone.

7. The micron self-assembled material according to any one of claims 1 to 6, characterized in that: The avidin-modified mesoporous polydopamine nanoparticles are prepared by a preparation method comprising the following steps: The mesoporous polydopamine nanoparticles are mixed with thiol-modified avidin, and Michael addition reaction is performed, the pH value of the reaction is adjusted to 7-9, and the precipitate is collected by centrifugation to obtain avidin-modified mesoporous polydopamine nanoparticles; Preferably, the Michael addition reaction is carried out for 10-40 hours at a temperature of 10-50°C.

8. The micron self-assembled material according to any one of claims 1 to 7, characterized in that: The biotinylated type XII collagen targeted lubricating peptide modified mesoporous polydopamine nanoparticles are prepared by a preparation method comprising the following steps: The biotin-modified type XII collagen targeted lubricating peptide is mixed with mesoporous polydopamine nanoparticles to perform Michael addition reaction, the pH value of the reaction is adjusted to 7-9, and the precipitate is collected by centrifugation to obtain biotinylated type XII collagen targeted lubricating peptide-modified mesoporous polydopamine nanoparticles; Preferably, the Michael addition reaction is carried out for 10-40 hours at a temperature of 10-50°C.

9. A method for preparing a micron self-assembled material having joint retention, lubrication, and anti-inflammatory effects as claimed in any one of claims 1 to 8, characterized in that: The preparation method comprises: Avidin-modified mesoporous polydopamine nanoparticles and biotinylated type XII collagen-targeted lubricating peptide-modified mesoporous polydopamine nanoparticles were mixed in PBS and self-assembled to form micron self-assembled materials; Preferably, the self-assembly time is 0.1-30h and the temperature is 4-50°C.

10. Use of the micron self-assembled material with joint residence, lubrication, and anti-inflammatory effects as claimed in any one of claims 1 to 8 in the preparation of a drug for treating arthritis.

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