Nano-drug for treating osteoarthritis and application thereof

Abstract

The invention provides an inflammation microenvironment responsive nano-drug for regulating and controlling osteoarthritis. Specifically, the invention provides a nano-drug, the drug is obtained by self-assembly of polyethylene glycol-thioketal and bortezomib, and the polyethylene glycol-thioketal is a block copolymer with polyethylene glycol and thioketal as blocks. The nano-drug can be passively targeted to osteoarthritis tissues, under the action of active oxygen, bortezomib is rapidly released, macrophage polarization in an inflammatory microenvironment is regulated and controlled, synovial hyperplasia is reduced, cartilage degeneration is relieved, and a new strategy is provided for treatment of osteoarthritis.

Description

Technical Field

[0001] The present invention relates to the field of nano-drugs, and particularly to a nano-drug for treating osteoarthritis and its uses. Background Art

[0002] Osteoarthritis (OA) is a common chronic degenerative joint disease. Progressive articular cartilage degeneration and chronic synovial inflammation run through the pathological process of OA. Clinically, it is mainly manifested as joint pain, swelling and joint deformity. In the late stage, joint replacement surgery must be performed due to severe joint deformity and limited mobility, which is expensive and brings heavy pressure to patients and social economy. Currently, there is still no effective drug to delay the pathological process of OA. Therefore, searching for and developing drugs to delay the pathological process of OA has important clinical significance and social and economic value.

[0003] In recent years, more and more studies have confirmed that synovial inflammation in the pathological process of OA precedes cartilage degeneration. In particular, the polarization of macrophages in the synovium plays an important role in the process of OA cartilage degeneration. Inhibiting M1 macrophages or promoting the polarization of M1 to M2 is the key point for treating OA.

[0004] Therefore, there is a need in the art for a nano-drug that can effectively treat osteoarthritis. Summary of the Invention

[0005] The object of the present invention is to provide a nano-drug that can be responsive to the inflammatory microenvironment, regulate macrophage polarization in the inflammatory microenvironment, thereby reducing synovial hyperplasia, alleviating cartilage degeneration, and effectively treating osteoarthritis.

[0006] In the first aspect of the present invention, a nano-drug is provided, which is self-assembled from a polyethylene glycol-thioketal copolymer and bortezomib;

[0007] Wherein the polyethylene glycol-thioketal copolymer contains the following polymerization units:

[0008]

[0009] Wherein, m is an integer from 10 to 200, n is an integer from 5 to 500, and p is an integer from 0 to 5.

[0010] In another preferred example, the polyethylene glycol-thioketal copolymer is a block copolymer.

[0011] In another preferred example, the polyethylene glycol-thioketal copolymer uses polyethylene glycol and thioketal-isothiocyanate as two blocks respectively.

[0012] In another preferred example, m is an integer from 50 to 200, preferably an integer from 100 to 150, such as 100, 110, 120, 130, 140, 150.

[0013] In another preferred example, n is an integer from 20 to 200, preferably an integer from 50 to 200.

[0014] In another preferred example, p is an integer from 1 to 5, such as 1, 2, 3, 4, 5.

[0015] In another preferred example, the molecular weight of the polyethylene glycol-thioketal copolymer is about 1×10 3 ~50×10 4 preferably 5×10 3 ~20×10 4 more preferably 1×10 4 ~10×10 4 such as 28400.

[0016] In another preferred example, the structural formula of bortezomib is

[0017]

[0018] In another preferred example, the mass ratio of bortezomib to the polyethylene glycol-thioketal copolymer is 1:2 - 50, preferably 1:5 - 30, such as 1:6, 1:8, 1:10, 1:12, 1:18, 1:20, 1:25.

[0019] In another preferred example, the nano-drug is obtained by co-assembly of bortezomib and the polyethylene glycol-thioketal copolymer at the oil-water interface of a first organic solvent-water system.

[0020] In another preferred example, the first organic solvent is selected from the group consisting of: tetrahydrofuran, dichloromethane, dimethyl sulfoxide, or a combination thereof.

[0021] In another preferred example, the nano-drug is prepared by the following method, which includes the following steps:

[0022] (1) Disperse bortezomib and the polyethylene glycol-thioketal copolymer in the first organic solvent and mix them;

[0023] (2) Mix the mixture obtained in step (1) with water to co-assemble and obtain the nano-drug.

[0024] In another preferred example, the preparation method of the nano-drug is as described in the second aspect of the present invention.

[0025] In another preferred example, the polyethylene glycol-thioketal copolymer is prepared by the following method, which includes the following steps:

[0026] (a) In the presence of a catalyst, acetone and 3-mercaptopropionic acid are mixed to obtain a compound of formula a;

[0027]

[0028] (b) In a second organic solvent, the compound of formula a obtained in step (a) is mixed with a reducing agent to carry out a reduction reaction to obtain a compound of formula b;

[0029]

[0030] (c) In a third organic solvent, the compound of formula b obtained in step (b) is mixed with an isocyanate to carry out a reaction, and then mixed with hydroxy polyethylene glycol to carry out copolymerization to obtain the polyethylene glycol-thioketone copolymer

[0031]

[0032] In another preferred example, the preparation method of the polyethylene glycol-thioketone copolymer is as described in the third aspect of the present invention.

[0033] In another preferred example, the nano-drug comprises one or more characteristics selected from the following group:

[0034] (1) The size of the nano-drug particles is 50 - 500 nm, preferably 100 - 200 nm;

[0035] (2) The drug loading amount of bortezomib in the nano-drug is 5% or more, preferably 7% or more, more preferably 9% or more;

[0036] (3) The polydispersity index of the nano-drug particles is 0.5 or less, preferably 0.2 or less.

[0037] In another preferred example, the nano-drug comprises one or more characteristics selected from the following group:

[0038] (1) Having the ability to respond to reactive oxygen species: dissociating under the stimulation of reactive oxygen species;

[0039] (2) Being able to scavenge reactive oxygen species, preferably scavenging 50% or more, more preferably scavenging 70% or more;

[0040] (3) Inhibiting the secretion of pro-inflammatory factors and reducing the inflammatory response, preferably inhibiting the secretion of TNF-α and IL-1β;

[0041] (4) After 8 weeks, the volume of synovial fluid in the joint cavity is reduced to be basically the same as that of the normal control group;

[0042] (5) Increasing the cartilage area, increasing the thickness of hyaline cartilage, and increasing the flatness of the joint surface, preferably increasing the cartilage area to be basically the same as that of the control group;

[0043] (6) Reduce the synovial score, preferably to below 3.0, more preferably to below 2.0;

[0044] (7) Inhibit M1 macrophages in the pathological process;

[0045] (8) Promote the polarization of M1 macrophages to M2 macrophages;

[0046] (9) Promote the expression of key markers for cartilage matrix synthesis and inhibit the expression of cartilage matrix degradation markers. Preferably, promote the expression of Col 2a1 and inhibit the expression of cartilage matrix degradation markers Col X, NITEGE, and MMP-13;

[0047] (10) The release rate of bortezomib reaches 50% within 5 hours under the action of reactive oxygen species, preferably within 3 hours.

[0048] In a second aspect of the present invention, there is provided a method for preparing the nano-drug described in the first aspect of the present invention, and the method includes the following steps:

[0049] (1) Disperse bortezomib and poly(ethylene glycol)-thioacetal copolymer in a first organic solvent and mix;

[0050] (2) Mix the mixture obtained in step (1) with water and self-assemble to obtain the nano-drug.

[0051] In another preferred example, in step (1), the first organic solvent is selected from the group consisting of: tetrahydrofuran, dichloromethane, dimethyl sulfoxide, or a combination thereof.

[0052] In another preferred example, in step (1), the mass ratio of bortezomib to poly(ethylene glycol)-thioacetal copolymer is 1:2 - 50, preferably 1:5 - 30, such as 1:6, 1:8, 1:10, 1:12, 1:18, 1:20, 1:25.

[0053] In another preferred example, in step (1), the mixing time is 0.5 - 3 h, such as 1 h.

[0054] In another preferred example, step (2) includes: dropwise adding the mixture obtained in step (1) to water and stirring.

[0055] In another preferred example, in step (2), the self-assembly time is 1 - 5 h, such as 3 h.

[0056] In another preferred example, the method is carried out at 10 - 40 °C.

[0057] In another preferred example, step (2) further includes a post-treatment step after mixing.

[0058] In another preferred example, the post-treatment includes dialyzing the obtained mixture, removing the organic solvent, and freeze-drying to obtain the nano-drug.

[0059] In another preferred example, the preparation method of the polyethylene glycol-thioketal copolymer is as described in the third aspect of the present invention.

[0060] In the third aspect of the present invention, a preparation method of a polyethylene glycol-thioketal copolymer is provided. The polyethylene glycol-thioketal copolymer is prepared by the following method, and the method includes the following steps:

[0061] (a) In the presence of a catalyst, acetone and 3-mercaptopropionic acid are mixed to obtain a compound of formula a;

[0062]

[0063] (b) In a second organic solvent, the compound of formula a obtained in step (a) is mixed with a reducing agent, and a reduction reaction is carried out to obtain a compound of formula b;

[0064]

[0065] (c) In a third organic solvent, the compound of formula b obtained in step (b) is mixed with an isocyanate, reacted, and then mixed with hydroxy polyethylene glycol to copolymerize to obtain the polyethylene glycol-thioketal copolymer

[0066]

[0067] In another preferred example, in step (a), the catalyst is an acidic catalyst. Preferably selected from the group consisting of hydrogen chloride, sulfuric acid, p-toluenesulfonic acid, trifluoroacetic acid, nitric acid, or a combination thereof.

[0068] In another preferred example, step (a) is optionally carried out in an organic solvent. The organic solvent is selected from tetrahydrofuran, dichloromethane, dimethyl sulfoxide, or a combination thereof.

[0069] In another preferred example, in step (a), the molar ratio of acetone to 3-mercaptopropionic acid is 1-5:1, preferably 1-2:1.

[0070] In another preferred example, step (a) is carried out under anhydrous conditions.

[0071] In another preferred example, step (a) is carried out at 10-40 °C.

[0072] In another preferred example, the reaction time of step (a) is 10-20 h.

[0073] In another preferred example, step (a) includes: mixing anhydrous acetone and 3-mercaptopropionic acid, introducing dry hydrogen chloride gas, and then stirring vigorously at room temperature.

[0074] In another preferred example, in step (b), the second organic solvent is selected from the group consisting of: tetrahydrofuran, dichloromethane, dimethyl sulfoxide, or a combination thereof.

[0075] In another preferred example, in step (b), the reducing agent is selected from the group consisting of: LiAlH 4 , Pd / H 2 , NaBH 4 , or a combination thereof.

[0076] In another preferred example, in step (b), the molar ratio of the compound of formula a to the reducing agent is 1:5 - 10, preferably 1:6 - 8.

[0077] In another preferred example, step (b) further includes: adding a base to the reduced product to obtain the compound of formula b.

[0078] In another preferred example, the base is selected from the group consisting of: sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, or a combination thereof.

[0079] In another preferred example, in step (b), the reduction reaction is carried out under reflux conditions.

[0080] In another preferred example, in step (b), the reaction time of the reduction reaction is 1 - 5 h, preferably 2 h.

[0081] In another preferred example, step (b) is carried out under anhydrous conditions.

[0082] In another preferred example, step (b) includes: dropwise adding a solution of the reducing agent in the second organic solvent to a solution of the compound of formula a in the second organic solvent, heating under reflux for reaction, adding a base for neutralization, to obtain the compound of formula b.

[0083] In another preferred example, in step (c), the third organic solvent is selected from the group consisting of: tetrahydrofuran, dichloromethane, dimethyl sulfoxide, or a combination thereof.

[0084] In another preferred example, in step (c), the molar ratio of the compound of formula b to the isocyanate is 1:1 - 2, preferably 1:1 - 1.5.

[0085] In another preferred example, in step (c), the compound of formula b and the isocyanate are mixed at 0 - 10 °C.

[0086] In another preferred embodiment, in step (c), the compound of formula b and the isocyanate are subjected to a reflux reaction at 35-55 °C.

[0087] In another preferred embodiment, in step (c), the reaction time of the compound of formula b and the isocyanate is 10-20 h.

[0088] In another preferred embodiment, step (c) is carried out under anhydrous conditions.

[0089] In another preferred embodiment, step (c) includes: under a protective gas atmosphere, the isocyanate dissolved in a third organic solvent is slowly dropped into the solution of the compound of formula b in the third organic solvent, stirred and mixed in an ice-water bath, heated to 35-55 °C and reacted overnight, then hydroxy polyethylene glycol is directly added to the reaction mixture obtained after reaction with the isocyanate for copolymerization reaction, and the polyethylene glycol-thioketal copolymer is obtained after post-treatment.

[0090] In another preferred embodiment, in step (c), the molar ratio of the hydroxy polyethylene glycol to the compound of formula b is 1:5-1000, preferably 1:10-100, for example 1:20.

[0091] In another preferred embodiment, in step (c), the reaction time of the hydroxy polyethylene glycol and the reaction mixture is 18-36 h, preferably 24 h.

[0092] In another preferred embodiment, step (c) further includes: rotary evaporation of the reaction mixture after the reaction is completed, and then precipitation with ice ether, and suction filtration to obtain the polyethylene glycol-thioketal copolymer.

[0093] In the fourth aspect of the present invention, a nano-drug is provided, and the nano-drug is a polyethylene glycol-thioketal copolymer or is self-assembled from a polyethylene glycol-thioketal copolymer;

[0094] The polyethylene glycol-thioketal copolymer contains the following polymerization units:

[0095]

[0096] Wherein, m is an integer of 10-200, n is an integer of 5-500, and p is an integer of 0-5.

[0097] In another preferred embodiment, the polyethylene glycol-thioketal copolymer is as described in the first aspect of the present invention.

[0098] In another preferred embodiment, m, n, and p are each independently as described in the first aspect of the present invention.

[0099] In another preferred embodiment, the nano-drug is self-assembled from a polyethylene glycol-thioketal copolymer at the oil-water interface of a first organic solvent-water.

[0100] In another preferred example, the first organic solvent is selected from the group consisting of: tetrahydrofuran, dichloromethane, dimethyl sulfoxide, or a combination thereof.

[0101] In another preferred example, the nano-drug is prepared by the following method, which includes the following steps:

[0102] 1. The step of preparing the polyethylene glycol-thioketal copolymer, including the following steps:

[0103] (a) In the presence of a catalyst, acetone and 3-mercaptopropionic acid are mixed to obtain a compound of formula a;

[0104]

[0105] (b) In a second organic solvent, the compound of formula a obtained in step (a) is mixed with a reducing agent for a reduction reaction to obtain a compound of formula b;

[0106]

[0107] (c) In a third organic solvent, the compound of formula b obtained in step (b) is mixed with an isocyanate for a reaction, and then mixed with hydroxy polyethylene glycol for copolymerization to obtain the polyethylene glycol-thioketal copolymer

[0108]

[0109] Optionally, 2. The step of self-assembling the polyethylene glycol-thioketal copolymer, including the following steps:

[0110] (b1) The polyethylene glycol-thioketal copolymer is dispersed in a first organic solvent and mixed;

[0111] (b2) The mixture obtained in step (b1) is mixed with water for self-assembly to obtain the nano-drug.

[0112] In another preferred example, the preparation method of the nano-drug described in the fourth aspect of the present invention is as described in the fifth aspect of the present invention.

[0113] In another preferred example, the nano-drug includes one or more characteristics selected from the group consisting of:

[0114] (1) The size of the nano-drug particles is 50 - 500 nm, preferably 100 - 200 nm;

[0115] (2) The polydispersity index of the nano-drug particles is below 0.5, preferably below 0.2.

[0116] In another preferred example, the nano-drug includes one or more characteristics selected from the group consisting of:

[0117] (1) Having the ability to respond to reactive oxygen species: dissociating under the stimulation of reactive oxygen species;

[0118] (2) Being able to scavenge reactive oxygen species, preferably scavenging more than 20%, more preferably scavenging more than 30%;

[0119] (3) Inhibiting the secretion of pro-inflammatory factors and reducing the inflammatory response, preferably inhibiting the secretion of TNF-α and IL-1β;

[0120] (4) Reducing the volume of synovial fluid in the joint cavity;

[0121] (5) Increasing the cartilage area, increasing the thickness of hyaline cartilage, and improving the flatness of the joint surface;

[0122] (6) Reducing the synovial score;

[0123] (7) Inhibiting M1 macrophages in the pathological process;

[0124] (8) Promoting the polarization of M1 macrophages to M2 macrophages;

[0125] (9) Promoting the expression of key markers for cartilage matrix synthesis and inhibiting the expression of cartilage matrix degradation markers, preferably promoting the expression of Col 2a1 and inhibiting the cartilage matrix degradation markers Col X, NITEGE, and MMP-13.

[0126] In the fifth aspect of the present invention, there is provided a method for preparing a nano-drug as described in the fourth aspect of the present invention, and the method includes the following steps:

[0127] 1. The step of preparing a polyethylene glycol-thioketal copolymer, including the following steps:

[0128] (a) Mixing acetone and 3-mercaptopropionic acid in the presence of a catalyst to obtain a compound of formula a;

[0129]

[0130] (b) Mixing the compound of formula a obtained in step (a) with a reducing agent in a second organic solvent and performing a reduction reaction to obtain a compound of formula b;

[0131]

[0132] (c) Mixing the compound of formula b obtained in step (b) with an isocyanate in a third organic solvent, performing a reaction, and then mixing with hydroxy polyethylene glycol to copolymerize to obtain the polyethylene glycol-thioketal copolymer

[0133]

[0134] Optionally, 2. The step of self-assembling the polyethylene glycol-thioketal copolymer includes the following steps:

[0135] (b1) Disperse the polyethylene glycol-thioketal copolymer in a first organic solvent and mix;

[0136] (b2) Mix the mixture obtained in step (b1) with water and self-assemble to obtain the nano-drug.

[0137] In another preferred example, the preparation method of the polyethylene glycol-thioketal copolymer is as described in the third aspect of the present invention.

[0138] In another preferred example, in step (b1), the first organic solvent is selected from the group consisting of: tetrahydrofuran, dichloromethane, dimethyl sulfoxide, or a combination thereof.

[0139] In another preferred example, in step (b1), the mixing time is 0.5 - 3 h, such as 1 h.

[0140] In another preferred example, step (b2) includes: adding the mixture obtained in step (1) dropwise to water and stirring.

[0141] In another preferred example, in step (b2), the self-assembly time is 1 - 5 h, such as 3 h.

[0142] In another preferred example, the method is carried out at 10 - 40 °C.

[0143] In another preferred example, step (b2) further includes a post-treatment step after mixing.

[0144] In another preferred example, the post-treatment includes dialyzing the obtained mixture, removing the organic solvent, and lyophilizing to obtain the nano-drug.

[0145] In the sixth aspect of the present invention, a pharmaceutical composition is provided, which includes the nano-drug described in the first aspect of the present invention and / or the nano-drug described in the third aspect of the present invention, and a pharmaceutically acceptable carrier.

[0146] In another preferred example, the pharmaceutical composition is an injection.

[0147] In another preferred example, the administration method of the pharmaceutical composition is intra-articular injection.

[0148] In another preferred example, in the pharmaceutical composition, the administration concentration of the nano-drug described in the first aspect of the present invention and / or the nano-drug described in the third aspect of the present invention is 0.01 - 1 mg / kg, such as 0.05 mg / kg.

[0149] In the seventh aspect of the present invention, there is provided the use of the nano-drug described in the first aspect of the present invention, the nano-drug described in the fourth aspect of the present invention, or the pharmaceutical composition described in the sixth aspect of the present invention for preparing a drug for preventing and / or treating osteoarthritis.

[0150] In the eighth aspect of the present invention, there is provided the use of the nano-drug described in the fourth aspect of the present invention for preparing a drug delivery system for targeted drug delivery.

[0151] In another preferred example, the drug is a drug for preventing and / or treating osteoarthritis.

[0152] In the ninth aspect of the present invention, there is provided a method for treating osteoarthritis, comprising the step of administering the nano-drug described in the first aspect of the present invention, the nano-drug described in the fourth aspect of the present invention, or the pharmaceutical composition described in the sixth aspect of the present invention to a subject in need thereof.

[0153] In another preferred example, the subject is a mammal, preferably a human.

[0154] 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 specifically described hereinafter (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0155] Figure 1 Shows a schematic diagram of the preparation of PTK in Example 1.

[0156] Figure 2 Shows the preparation of BTZ@PTK nanoparticles and the change in morphology and the drug release curve of BTZ under the stimulation of ROS environment. A: UV absorption spectra of BTZ and BTZ@PTK; B-C: Changes in particle size and polydispersity of BTZ@PTK before and after ROS stimulation; D: Drug release curve of BTZ@PTK nanoparticles after adding H 2 O 2 2O2; E-F: Transmission electron microscope photos of BTZ@PTK nanoparticles before and after ROS stimulation.

[0157] Figure 3 Shows the results of the effect of BTZ@PTK on the activity of RAW264.7 cells. A: Effect on cell activity after 24 hours; A: Effect on cell activity after 48 hours.

[0158] Figure 4Shows the results of the ROS-responsive performance evaluation of BTZ@PTK in in vitro cell experiments. A: DCFH-DA immunofluorescence intensity analysis; B: Quantitative analysis of DCFH-DA immunofluorescence intensity; C: Analysis of the number of ROS-positive cells detected by flow cytometry; D: Quantitative analysis of ROS-positive cells.

[0159] Figure 5 Shows the results of the anti-inflammatory effect of BTZ@PTK in in vitro cell experiments. A: TNF-α level; B: IL-1β level; C: Relative mRNA expression of TNF-α; D: Relative mRNA expression of Il-1β; E: Relative mRNA expression of Il-6.

[0160] Figure 6 Shows the results of ultrasonic detection of joint synovial fluid in in vivo animal experiments (A); Changes in joint synovial fluid volume (B-C).

[0161] Figure 7 Shows the histological staining of knee joint tissues in in vivo animal experiments (A-C), and the results of cartilage and synovium scoring (D-G).

[0162] Figure 8 Shows the detection results of the immunofluorescence expression of M1 pro-inflammatory macrophage markers inhibited by BTZ@PTK in in vivo animal experiments.

[0163] Figure 9 Shows the detection results of immunohistochemical staining of knee joint cartilage matrix generation and metabolic indexes in in vivo animal experiments (A-D), and the quantitative analysis diagrams of the indexes (E-H). Detailed implementation

[0164] Through extensive and in-depth research, the present inventors have first discovered a nano-drug, which is a high drug-loading polymer nanoparticle responsive to high concentrations of reactive oxygen species in the inflammatory microenvironment, and the drug-loaded nanoparticle is obtained by self-assembly of the block copolymer polyethylene glycol-thioketal and bortezomib (BTZ). This drug delivery system can passively target osteoarthritis tissues, rapidly release bortezomib under the action of reactive oxygen species, and regulate macrophage polarization in the inflammatory microenvironment, reduce synovial hyperplasia, and alleviate cartilage degeneration, providing a new strategy for the treatment of osteoarthritis.

[0165] In addition, the present inventors have also unexpectedly found that the block copolymer polyethylene glycol-thioketal itself also has excellent activity in treating osteoarthritis. Therefore, the nano-drug obtained by self-assembly of the block copolymer polyethylene glycol-thioketal can be used both as a drug delivery system and as a drug for treating osteoarthritis itself.

[0166] Based on this, the present invention has been completed.

[0167] Terms

[0168] Unless otherwise defined, 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.

[0169] As used herein, the terms "comprising", "including", and "containing" are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include "consisting of" and "consisting essentially of".

[0170] As used herein, when used in reference to a specifically recited numerical value, the term "about" means that the value can vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0171] As used herein, the term "substantially consistent between A and B" means that the error between A and B is within ±5%.

[0172] Polyethylene glycol-thioketal copolymer

[0173] The present invention provides a polyethylene glycol-thioketal copolymer, which is a block copolymer.

[0174] The polyethylene glycol-thioketal copolymer contains the following polymerization units:

[0175]

[0176] Wherein, m is an integer from 10 to 200, n is an integer from 5 to 500, and p is an integer from 0 to 5.

[0177] The polyethylene glycol-thioketal copolymer is prepared by the following method, which includes the following steps:

[0178] (a) In the presence of a catalyst, acetone and 3-mercaptopropionic acid are mixed to obtain a compound of formula a;

[0179]

[0180] (b) In a second organic solvent, the compound of formula a obtained in step (a) is mixed with a reducing agent to carry out a reduction reaction to obtain a compound of formula b;

[0181]

[0182] (c) In a third organic solvent, the compound of formula b obtained in step (b) is mixed with an isocyanate to carry out a reaction, and then mixed with hydroxy polyethylene glycol to carry out copolymerization to obtain the polyethylene glycol-thioketal copolymer

[0183]

[0184] In the present invention, the polyethylene glycol-thioketal copolymer can self-assemble into nanoparticles by virtue of its amphiphilic property. As a drug delivery system, it can passively target osteoarthritis tissues for effective treatment. In addition, the copolymer itself also has the efficacy of treating osteoarthritis, can significantly inhibit inflammatory factors, scavenge reactive oxygen species (ROS), and reduce the synovitis score.

[0185] The nano-drug of the present invention

[0186] In the present invention, there are two nano-drugs.

[0187] One of the nano-drugs, namely the high drug-loading polymer nanoparticles responsive to the inflammatory microenvironment, refers to a drug delivery system obtained by self-assembly of the block copolymer polyethylene glycol-reactive oxygen species-responsive part thioketal, and then a drug-loaded nanoparticle obtained by self-assembly of the hydrophobic core and bortezomib (BTZ).

[0188] The nano-drug responsive to the inflammatory microenvironment studied in the present invention can promote the polarization of M1 to M2 macrophages, reduce the OARSI score of osteoarthritis mice, alleviate synovial hyperplasia and cartilage damage, thereby alleviating the pathological process of osteoarthritis.

[0189] The nano-drug is obtained by co-assembly of bortezomib and the polyethylene glycol-thioketal copolymer at the oil-water interface of a first organic solvent-water.

[0190] The other nano-drug, namely the nanoparticles obtained by self-assembly of polyethylene glycol-reactive oxygen species-responsive part thioketal, can not only be used as a drug delivery system for delivering drugs, but also can be used as a drug for treating osteoarthritis.

[0191] Drug composition and administration method

[0192] The nano-drug of the present invention, and the drug composition containing the nano-drug of the present invention as the main active ingredient can be used for preventing and / or treating (stabilizing, alleviating or curing) osteoarthritis and the like.

[0193] The drug composition of the present invention contains the nano-drug of the present invention within a safe and effective amount range and a pharmaceutically acceptable excipient or carrier. The "safe and effective amount" herein refers to: the amount of the active ingredient is sufficient to significantly improve the condition without causing serious side effects. Generally, the drug composition contains 1 - 2000 mg of the nano-drug of the present invention per dose, more preferably, contains 10 - 200 mg of the nano-drug of the present invention per dose. Preferably, the "one dose" is one dose of injection or one injection.

[0194] "Pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the nanopharmaceuticals of the present invention and with each other without significantly reducing their drug efficacy.

[0195] There is no particular limitation on the administration method of the nanopharmaceuticals or pharmaceutical compositions of the present invention. Representative administration methods include (but are not limited to): oral administration, parenteral (intravenous, intramuscular, subcutaneous or near the lesion).

[0196] The composition for parenteral injection may contain a physiologically acceptable sterile aqueous or non-aqueous solution, dispersion, suspension or emulsion, and a sterile powder for re-dissolving into a sterile injectable solution or dispersion. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and their suitable mixtures.

[0197] The nanopharmaceuticals of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds (such as drugs for treating osteoarthritis).

[0198] When administered in combination, the pharmaceutical composition further includes one or more (two, three, four, or more) other pharmaceutically acceptable compounds. One or more (two, three, four, or more) of the other pharmaceutically acceptable compounds can be used simultaneously, separately or sequentially with the nanopharmaceuticals of the present invention for preventing and / or treating osteoarthritis.

[0199] When using the pharmaceutical composition, a safe and effective amount of the nanopharmaceuticals of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically recognized effective dosage. For a person weighing 60 kg, the daily dosage is usually 1 to 2000 mg, preferably 20 to 500 mg. Of course, the specific dosage should also consider factors such as the administration route and the patient's health condition, which are all within the scope of the skills of a skilled physician.

[0200] "Safe and effective amount" refers to an amount of the active ingredient sufficient to significantly improve the condition without causing serious side effects. Generally, the pharmaceutical composition contains 1 to 2000 mg of the active ingredient per dose, more preferably, it contains 10 to 200 mg of the active ingredient per dose. Preferably, the "per dose" is an injection dose.

[0201] The main advantages of the present invention include:

[0202] (1) The nanopharmaceuticals of the present invention have active oxygen responsiveness.

[0203] (2) The nano-drug of the present invention can rapidly release bortezomib under the action of reactive oxygen species and regulate macrophage polarization in the inflammatory microenvironment, reduce synovial hyperplasia, and alleviate cartilage degeneration.

[0204] (3) The preparation method of the nano-drug of the present invention is simple and low-cost, and has the prospect of industrial production.

[0205] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0206] Example 1: Preparation of the nano-delivery system

[0207] As Figure 1 shown, prepare the reactive oxygen species-responsive amphiphilic block polymer PTK.

[0208] (1) Preparation method of the reactive oxygen species-responsive thioacetal TK-OH:

[0209] Mix anhydrous acetone (2.94 g, 50 mmol) and 3-mercaptopropionic acid (5.64 g, 25 mmol), introduce dry hydrogen chloride gas, then stir vigorously at room temperature. After reacting overnight, quench in an ice-water bath, filter to obtain a solid and wash it three times with hexane and ice water respectively. Finally, vacuum dry to obtain the intermediate (TK-COOH) as a white powder;

[0210] Completely dissolve TK-COOH (0.5 g, 2 mmol) in anhydrous tetrahydrofuran, and gradually add 8 mL of a tetrahydrofuran solution of LiAlH 4 (0.45 g, 12 mmol). After heating under reflux for 2 hours, add an aqueous sodium hydroxide solution with a mass fraction of 15% dropwise to the reaction solution until no bubbles appear. Then filter to remove the solid, and finally further purify the filtrate by column chromatography to obtain the yellow viscous liquid TK-OH;

[0211] (2) Preparation method of the reactive oxygen species-responsive amphiphilic block polymer PTK:

[0212] TK-OH (200 mg, 0.9 mmol) was completely dissolved in 10 mL of dry dichloromethane. Under a nitrogen atmosphere, isocyanate (168 mg, 1 mmol) dissolved in 8 mL of dry dichloromethane was slowly added dropwise into the reaction flask. After stirring for 1 hour under an ice-water bath atmosphere, the temperature was raised to 40 °C and refluxed overnight. Then, hydroxy polyethylene glycol (with a molecular weight of about 5000, and the added amount was about 280 mg) dissolved in 5 mL of dry dichloromethane was added, and the reaction was continued with stirring for 24 hours. Finally, part of the dichloromethane was removed by rotary evaporation, and then precipitated three times with ice ether. The solid was collected by suction filtration and dried in vacuo to obtain a white solid powder PTK.

[0213] (3) Preparation of bortezomib-loaded polymer nanoparticles (BTZ@PTK):

[0214] An appropriate amount of 10 mg of bortezomib and 100 mg of polymer PTK were fully dissolved in dimethyl sulfoxide. The two were mixed and stirred for 1 hour, and then gradually added dropwise to deionized water. After stirring at room temperature for 3 hours, it was transferred to a dialysis bag and dialyzed for 2 days to remove organic solvents. The dialysis solution was collected and freeze-dried to obtain bortezomib-loaded polymer nanoparticles BTZ@PTK.

[0215] Example 2: Experimental method

[0216] 2.1 Determination of drug loading

[0217] The nanoparticles were dissolved in DMSO to destroy the nanoparticle structure and detected by UV-vis.

[0218] Drug loading = (amount of drug encapsulated in the delivery system / weight of the drug-loaded delivery system) × 100%

[0219] 2.2 Evaluation of reactive oxygen species responsiveness

[0220] 1. An appropriate amount of drug-loaded nanoparticles was weighed and dissolved in different drug release systems, transferred into a dialysis bag, and sampled at specific time points. The drug release of the nanoparticles at different concentrations of H 2 O 2 was determined by UV-vis.

[0221] 2. An appropriate amount of drug-loaded nanoparticles was weighed and dissolved in different reactive oxygen species response systems. After a period of time, the particle morphology was observed by a dynamic light scattering instrument and a transmission electron microscope.

[0222] 2.3 Evaluation of the protective effect of the drug on cells

[0223] Mouse macrophages RAW264.7 were seeded in a 96-well plate, 4×10 3 / Holes. After overnight incubation, PTK, BTZ, and BTZ@PTK were added for culture. CCK-8 solution was added at 24 hours and 48 hours respectively. After incubation for 2 hours, the OD value was read at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and a cell survival rate curve was plotted. The cell survival rate was calculated according to the formula.

[0224] Cell survival rate (%) = (OD value of the experimental group / OD value of the blank control group) × 100%

[0225] 2.4 Evaluation of the ability of the drug to scavenge ROS

[0226] RAW264.7 cells were seeded in 12-well plates at a density of 50,000 cells per well. After overnight incubation, LPS (1 μg / mL) was added to induce the polarization of RAW264.7 into pro-inflammatory M1 macrophages. After 6 hours of stimulation, different drugs were added for intervention for 24 hours. Then, the ROS fluorescent probe DCFH-DA was added for detecting the immunofluorescence intensity and quantitative analysis. Further, flow cytometry was used to analyze the number of intracellular ROS-positive cells and perform quantitative analysis.

[0227] 2.5 Evaluation of the anti-inflammatory effect of the drug on lipopolysaccharide-stimulated RAW264.7 macrophages

[0228] RAW264.7 cells were seeded in 12-well plates at a density of 50,000 cells per well. After overnight incubation, LPS (1 μg / mL) was added to induce the polarization of RAW264.7 into pro-inflammatory M1 macrophages. After 6 hours of stimulation, different drugs were added for intervention for 24 hours. Then, the cell supernatant was collected for ELISA detection to analyze the levels of TNF-α and IL-1β. The cells were collected for qPCR to detect the expression of pro-inflammatory factors TNF-α, IL-1β, and IL-6 and the anti-inflammatory factor Arg-1.

[0229] 2.6 Prevention and treatment effects of the drug on osteoarthritis induced by meniscus instability in mice

[0230] Fifty SPF-grade 8-week-old male C57BL / 6 mice were randomly divided into a Con group, a PBS group, a PTK group, a BTZ group, and a BTZ@PTK group. At 10 weeks of age, a mouse DMM osteoarthritis model was established by surgically inducing instability of the medial meniscus of the right knee joint in the mice. In the Con group, only the tibial ligament of the medial meniscus was exposed without cutting. Each group started intervention 4 weeks after surgery, once a week, by intra-articular injection, for a total of 8 weeks. The Con group was not given any intervention. The BTZ@PTK group was administered into the joint cavity at a dose of 0.05 mg / kg in a volume of 10 μL. The BTZ group was administered into the joint cavity with the same volume and the same dose. The PBS group and the PTK group were injected with the same volume. The changes in the volume of synovial fluid in the knee joint cavity were detected before surgery and at 4, 8, and 12 weeks after DMM. At 12 weeks after surgery, the right knee joint was harvested, paraffin specimens were made, and serial sections of 4 μm were cut for Alcian blue and Orange G (ABOG) staining to observe the tissue injury, synovium, and cartilage changes in the knee joint. Immunofluorescence was used to detect the markers F4 / 80-iNOS and F4 / 80-CD 206 of M1 and M2 macrophages, and immunohistochemistry was used to observe the cartilage matrix synthesis marker Col 2a1 and the degradation markers Col X, NITEGE, and MMP-13.

[0231] 2.6.1 Small animal ultrasound detection of the volume of synovial fluid in the mouse knee joint

[0232] After satisfactory isoflurane inhalation anesthesia of the mice, the mice were placed on a thermostatic heating plate (28 °C), and anesthesia was maintained with a face mask. The knee joint of the mice was routinely depilated with depilatory cream, and the knee joint of the mice was placed under the probe of a Vevo2100 small animal ultrasound. The volume of knee joint fluid was scanned by B-mode and 3D-mode, and 3D reconstruction was completed by software.

[0233] 2.6.2 ABOG staining detection of mouse joint specimens

[0234] After harvesting the mouse knee joint, it was fixed in neutral fixative for 24 hours, soaked and washed in PBS, decalcified for 28 - 30 days, and the decalcifying solution was changed weekly, followed by dehydration and paraffin embedding. Sagittal sections of the knee joint were taken with a thickness of 4 μm. After picking up the sections on adhesive slides in a 42 °C spreading machine and air-drying, they were baked at 60 °C for 2 hours and then placed in an oven at 60 °C overnight. After dewaxing and rehydration, they were treated with 1% hydrochloric acid / ethanol for 30 s, 1% Alcian blue / hematoxylin for 30 min, 1% hydrochloric acid / ethanol for 5 s, immersed in water for 30 s, 1% ammonia water for 15 s, 95% ethanol for 1 min, and Orange G / eosin staining solution (0.5% Orange G + 2% eosin) for 1 min. They were dehydrated through three cylinders of 95% ethanol and two cylinders of 100% ethanol, cleared with xylene and then sealed. Scanning and analysis were performed with a VS120 whole-slide scanner.

[0235] 2.6.3 Immunofluorescence detection of M1 and M2 macrophage phenotypic markers in mouse joint specimens

[0236] Paraffin tissue sections were routinely dewaxed to water, antigen retrieval was performed with 0.1% trypsin antigen retrieval solution for 15 min at 37 °C; washed 3 times with PBS, blocked with 5% BSA for 1 h, corresponding primary antibodies F4 / 80, iNOS and CD 206 were added dropwise, incubated at 4 °C for 16 - 18 hours, washed 3 times with PBS, an appropriate amount of anti-fluorescence quenching mounting medium (containing DAPI) was added dropwise, and scanned and analyzed with a VS120 whole-slide scanner.

[0237] 2.6.4 Immunohistochemical detection of the expression of cartilage matrix synthesis and degradation markers in mouse joint specimens

[0238] Paraffin tissue sections were routinely dewaxed to water, antigen retrieval was performed with 0.1% trypsin antigen retrieval solution for 15 min at 37 °C; washed 3 times with PBS, and operated according to the instructions of the immunohistochemical hypersensitive two-step method PV-9001 / 9002 kit provided by Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.; DAB color development was observed under the microscope, if positive staining appeared, the color development was terminated with tap water; counterstained with hematoxylin for 4 min; differentiated with hydrochloric acid for 5 s, rinsed with water; blued with ammonia water for 2 min, rinsed with water; routinely dehydrated, cleared and mounted; baked the sections in an oven at 60 °C.

[0239] SPSS 26.0 software was used for statistical analysis, and the data were expressed as mean ± standard deviation. Normality and homogeneity of variance tests were performed on the data. If they were met, one-way ANOVA and LSD pairwise comparison were used. If the variance was not homogeneous, Dunnett test was used. P < 0.05 was considered statistically significant.

[0240] Example 3. Synthesis and apparent characteristics of BTZ@PTK

[0241] Both the small molecule BTZ and BTZ@PTK nanoparticles had obvious ultraviolet absorption at 273 nm. Compared with the absorption spectrum of free BTZ in organic solution, the absorption spectrum of BTZ@PTK had a 5 nm red shift ( Figure 2 in A), which was mainly caused by the hydrophobic interaction and π-π interaction of BTZ.

[0242] Based on the ultraviolet absorption, the drug loading of the nanoparticles was obtained as 9.1%.

[0243] As Figure 2 shown in B - C, Figure 2 and E - F in, it was measured by DLS that BTZ@PTK could assemble into nanoparticles with a particle size of 124 nm in PBS, the polydispersity index PDI was 0.15, and under different exogenous ROS stimulations, the particle size became larger, and the polydispersity index also became 0.45, and the nanoparticles dissociated, proving that the assembled body had good ROS response ability.

[0244] As Figure 2 shown in D, from the drug release curve of polymer nanoparticles, it was found that the release of BTZ drug showed an H 2 O 2 concentration-dependent characteristic. After co-incubation in 1 mM H 2 O 2 environment for 24 h, about 60% of the encapsulated drug was released, proving that after the nanoparticles entered the lesion site, they could rapidly release BTZ, providing a possibility for the treatment of osteoarthritis.

[0245] Example 3: Effect of BTZ@PTK on the activity of RAW264.7 cells

[0246] As Figure 3 shown, compared with the PTK and free BTZ groups, at 24 hours and 48 hours, BTZ@PTK had no obvious toxicity to RAW264.7 cells in the concentration range of 0.25 nM to 1 nM, and there was no obvious difference among the groups, suggesting that BTZ@PTK had good cell protection.

[0247] Example 4: Evaluation of the ability of BTZ@PTK to scavenge intracellular ROS

[0248] Compared with the Con group (control group), LPS in the PBS group could significantly increase the immunofluorescence expression of the reactive oxygen species ROS fluorescent probe DCFH-DA, suggesting an increase in the ROS level under LPS stimulation. After treatment with BTZ, PTK, and BTZ@PTK, the immunofluorescence intensity of DCFH-DA was significantly reduced ( Figure 4 in A); compared with the PBS group, the fluorescence quantitative analysis value in the BTZ@PTK group was nearly down-regulated by 70% ( Figure 4 in B); further, flow cytometry was used to detect the number of ROS-positive cells, and the results were consistent with immunofluorescence, and could also inhibit the level of intracellular ROS ( Figure 4 in C); the quantitative analysis of ROS-positive cells showed that it was 1 / 3 of the PBS group ( Figure 4 in D); suggesting that BTZ@PTK had good ROS scavenging ability.

[0249] Example 5: Anti-inflammatory effect of BTZ@PTK on LPS-induced RAW264.7 cells

[0250] ELISA results showed that compared with the Con group, LPS in the PBS group could significantly promote the secretion of TNF-α and IL-1β, while BTZ@PTK significantly down-regulated the levels of TNF-α and IL-1β increased by LPS. Among them, the TNF-α level was about 1 / 5 of the PBS group, and the IL-1β level was 1 / 3 of the PBS group, suggesting that BTZ@PTK could significantly inhibit the secretion of pro-inflammatory factors TNF-α and IL-1β.Figure 5 In (A-B); qPCR results showed that compared with PBS, BTZ@PTK significantly inhibited the levels of Tnf-α, Il-1β, and Il-6 ( Figure 5 in (C-E)). The above results indicate that BTZ@PTK can inhibit the secretion of pro-inflammatory factors and reduce the inflammatory response.

[0251] Example 6: Effect of BTZ@PTK on the volume of knee joint synovial fluid in a knee osteoarthritis (KOA) model mouse

[0252] The size of the synovial fluid volume can reflect whether the synovial tissue proliferates. The larger the volume, the more obvious the proliferation. Ultrasonography results showed that compared with the Con group, the volume of the joint cavity synovial fluid increased significantly after the DMM model surgery, which was approximately 1.6 times that of the normal group, indicating obvious proliferation of the synovial tissue during KOA; as time increased, although the synovial fluid volume decreased slightly, it was still significantly higher than that of normal mice. The synovial fluid volume in the BTZ@PTK group decreased significantly after 4 weeks of intervention and was almost close to the volume of normal mice at 8 weeks, indicating that BTZ@PTK is a drug that can be used to prevent and treat KOA synovial inflammation ( Figure 6 ).

[0253] Example 7: Effect of BTZ@PTK on knee joint tissue damage in a KOA model mouse

[0254] Figure 7 The results in (A-C) showed that the knee joint cartilage surface of the Con group mice was flat and smooth, but the knee joint surface of the KOA mice was uneven, locally rough with cracks, indicating damage to the articular cartilage. The OARSI score was 4.70 ± 0.57, and the cartilage area represented by Alcian blue staining was significantly reduced, indicating severe cartilage loss. In comparison with the PBS group, the joint surface of the BTZ@PTK group was relatively flat and the cartilage area was significantly increased.

[0255] Figure 7 The results in (D-G) showed that compared with the PBS group, the OARSI score in the BTZ@PTK group decreased significantly to 1.50 ± 0.70, the synovitis score also decreased significantly, the cartilage area increased to almost close to the level of normal mice, and the thickness of the articular cartilage also increased significantly.

[0256] The above results suggest that BTZ@PTK has good cartilage protection. In addition, the synovial tissue of KOA mice proliferates and the synovial score increases, while the arrangement of the synovial basal layer and basal cells in the BTZ@PTK group is flat and the score decreases. The above research results suggest that BTZ@PTK can reduce cartilage damage in KOA, reduce synovial inflammation, and effectively delay the pathological process of KOA.

[0257] Example 8: Effects of BTZ@PTK on M1 and M2 type markers of synovial macrophages in the knee joints of KOA model mice

[0258] The results showed that compared with the Con group, the pro-inflammatory M1 type macrophage marker iNOS in the knee joint synovium was significantly increased in the PBS group, and the administration of BTZ@PTK could significantly inhibit the secretion of iNOS ( Figure 8 in A); the secretion of the anti-inflammatory M2 type macrophage marker CD206 in the knee joint synovium of the PBS group was relatively decreased, and the administration of BTZ@PTK could significantly promote the expression of CD206 ( Figure 8 in B). It is suggested that BTZ@PTK can inhibit M1 type macrophages in the pathological process of KOA, promote the polarization of M1 to M2 type, and thus play an anti-inflammatory role.

[0259] Example 9: Effects of BTZ@PTK on knee joint cartilage metabolism indexes in KOA model mice

[0260] As Figure 9 shown in A-D, compared with the Con group, the expression of the key marker Col2a1 for cartilage matrix synthesis in the KOA group was significantly decreased, while the expressions of the cartilage matrix degradation markers Col X, NITEGE, and MMP-13 were significantly increased, suggesting that the joint cartilage metabolism was imbalanced, the degradation increased, and the synthesis decreased, resulting in cartilage degeneration; after the treatment with BTZ@PTK, the imbalance in the expression of cartilage metabolism-related markers in the KOA group could be reversed.

[0261] Among them, BTZ@PTK could increase the loss of Col 2a1 expression in the KOA model, and could increase the positive expression area of Col 2a1 by nearly 50% ( Figure 9 in E), while inhibiting the expressions of Col X, MMP-13, and NITEGE, and the positive cell numbers were 30%, 30%, and 50% of those in the KOA group respectively ( Figure 9 in F-H). The above results suggest that BTZ@PTK has the function of maintaining cartilage homeostasis.

[0262] All the documents mentioned in the present invention are incorporated herein by reference as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

1. A nano-drug, which is obtained by self-assembly of a polyethylene glycol-thioketal copolymer and bortezomib; wherein the polyethylene glycol-thioketal copolymer contains the following polymerization units: Wherein, m is an integer from 10 to 200, n is an integer from 5 to 500, and p is an integer from 0 to 5.

2. The nano-drug according to claim 1, characterized in that the mass ratio of bortezomib to the polyethylene glycol-thioketal copolymer is 1:2 - 50, preferably 1:5 - 30.

3. The nano-drug according to claim 1, characterized in that the nano-drug is obtained by self-assembly of bortezomib and the polyethylene glycol-thioketal copolymer at the oil-water interface of a first organic solvent-water.

4. The nano-drug according to claim 1, characterized in that the polyethylene glycol-thioketal copolymer is prepared by the following method, and the method includes the following steps: (a) In the presence of a catalyst, acetone and 3-mercaptopropionic acid are mixed to obtain a compound of formula a; (b) In a second organic solvent, the compound of formula a obtained in step (a) is mixed with a reducing agent to carry out a reduction reaction to obtain a compound of formula b; (c) In a third organic solvent, the compound of formula b obtained in step (b) is mixed with an isocyanate, reacted, and then mixed with hydroxy polyethylene glycol to copolymerize to obtain the polyethylene glycol-thioketone copolymer 5. The preparation method of the nano-drug according to claim 1, characterized in that the method includes the following steps: (1) Disperse bortezomib and the polyethylene glycol-thioketal copolymer in a first organic solvent and mix; (2) Mix the mixture obtained in step (1) with water to self-assemble to obtain the nano-drug.

6. A nano-drug, characterized in that the nano-drug is a polyethylene glycol-thioketal copolymer or is obtained by self-assembly of a polyethylene glycol-thioketal copolymer; the polyethylene glycol-thioketal copolymer contains the following polymerization units: Wherein, m is an integer from 10 to 200, n is an integer from 5 to 500, and p is an integer from 0 to 5.

7. The preparation method of the nano-drug according to claim 6, characterized in that the method includes the following steps: i. The step of preparing a polyethylene glycol-thioketal copolymer, including the following steps: (a) In the presence of a catalyst, acetone and 3-mercaptopropionic acid are mixed to obtain a compound of formula a; (b) In a second organic solvent, the compound of formula a obtained in step (a) is mixed with a reducing agent to carry out a reduction reaction to obtain a compound of formula b; (c) In a third organic solvent, the compound of formula b obtained in step (b) is mixed with an isocyanate, reacted, and then mixed with hydroxy polyethylene glycol to copolymerize to obtain the polyethylene glycol-thioketal copolymer and, Optionally, ii. The step of self-assembling the polyethylene glycol-thioketal copolymer, including the following steps: (b1) Disperse the polyethylene glycol-thioketal copolymer in a first organic solvent and mix; (b2) Mix the mixture obtained in step (b1) with water to self-assemble to obtain the nano-drug.

8. A pharmaceutical composition, characterized in that the pharmaceutical composition includes the nano-drug according to claim 1 and / or the nano-drug according to claim 6, and a pharmaceutically acceptable carrier.

9. The use of the nano-drug according to claim 1, the nano-drug according to claim 6, or the pharmaceutical composition according to claim 8, characterized in that for preparing a drug for preventing and / or treating osteoarthritis.

10. The use of the nano-drug according to claim 6, characterized in that For preparing a drug delivery system for targeted drug delivery.

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