Asymmetric phosphorus-containing dendrimer carrying aza-bisphosphonate terminal, compound as well as preparation and application of asymmetric phosphorus-containing dendrimer and compound

Abstract

The invention relates to an asymmetric phosphorus-containing dendrimer carrying an aza-bisphosphonate terminal, a compound and preparation and application of the asymmetric phosphorus-containing dendrimer, the asymmetric phosphorus-containing dendrimer has the characteristics of uniform molecular weight, simple preparation method, high reaction process controllability, easiness in operation and separation and the like, the intracellular delivery efficiency of bromelain can be enhanced, and the yield of bromelain is increased. The inflammatory factor storm is controlled by synergistically regulating the macrophage phenotype, the cartilage cell protection function is played, and good development prospects and application values are achieved in treatment of osteoarthritis patients.

Description

Technical Field

[0001] The invention belongs to the field of functional nanomaterials, and particularly relates to an asymmetric phosphorus-containing dendrimer carrying an azabisphosphonate terminal, a complex, and preparation and application thereof. Background Art

[0002] Osteoarthritis (OA) is a chronic degenerative disease characterized by chronic inflammation and cartilage degeneration, which leads to cartilage lesions and subchondral bone defects in patients at the end stage (Adv. Sci. 2022, 9, 2105727). The drug treatments recommended by clinical guidelines mainly include analgesics and anti-inflammatory drugs (such as paracetamol and nonsteroidal anti-inflammatory drugs). These treatments have limited effects on slowing the progression of OA and have certain toxic side effects on the patient's gastrointestinal tract; total lateral joint replacement, as a common clinical surgical treatment, may be accompanied by the risk of various postoperative complications and will lead to heavy personal economic burden and significantly increased medical expenses (ACSNano 2023, 17, 12842-12861). Therefore, there is an urgent need to develop new, safe and efficient treatments for osteoarthritis.

[0003] Under OA pathological conditions, M1 synovial macrophages produce a large number of proinflammatory cytokines and reactive oxygen species (ROS), increase chondrocyte apoptosis and secretion of matrix metalloproteinases (MMPs), thereby inducing degradation of the extracellular matrix and accelerating cartilage degeneration (Nat. Biomed. Eng. 2021, 5, 1069–1083). Therefore, controlling the phenotype of synovial macrophages is an important strategy for treating OA.

[0004] Bromelain (Bro) is a natural product derived from pineapple rhizomes. It has excellent anti-inflammatory and immunomodulatory activities and has been shown to be useful in the treatment of inflammatory diseases such as osteoarthritis, rheumatoid arthritis and asthma (Food Funct. 2023, 14, 8101-8128). Bro can exert a strong anti-inflammatory effect by inhibiting the nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) signaling pathways in macrophages. Importantly, Bro can also maintain the integrity of articular cartilage by inhibiting the activity of MMPs (Curr. Issues Mol. Biol. 2021, 43, 93-106). However, Bro has the characteristics of large molecular weight and poor membrane permeability. The cell's biological membrane barrier hinders the efficiency of the molecule entering the cell, thereby limiting the bioavailability of Bro. Therefore, improving the intracellular delivery efficiency of Bro is an important measure to enhance the bioactivity of Bro for the treatment of OA.

[0005] Existing literature reports that phosphorus-containing dendrimers or dendrimers have been developed as nano-delivery platforms for a variety of therapeutic agents, including gene drugs, chemotherapeutic drugs, protein drugs, etc. Among them, phosphorus-containing dendrimers modified with sodium phosphite can stably compound proteins of different molecular weights and efficiently deliver them into cells without affecting the secondary structure and biological activity of the protein (ACSNano 2024, 18, 2195–2209). Importantly, this type of dendrimer also has excellent autoimmune activity, such as promoting the proliferation of natural killer cells and polarizing macrophages (Adv. Mater. 2023, 35, 2208277). However, the effect of the structural symmetry of this type of phosphorus-containing dendrimer on biological activity and its protein delivery performance has not been verified. Therefore, exploring the structure-activity relationship of phosphorus-containing dendrimers is crucial to the development of drug delivery technology based on phosphorus-containing dendrimers.

[0006] A search of domestic and foreign literature and patents has not yet found any reports on the synthesis of asymmetric phosphorus-containing dendrimers carrying azabisphosphonate ends and their use in the treatment of osteoarthritis after complexing with bromelain. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide an asymmetric phosphorus-containing dendrimer carrying an azabisphosphonate terminal, a complex, and its preparation and application. The present invention enhances the bioavailability of Bro by using asymmetric phosphorus-containing dendrimers, and combines phosphorus-containing dendrimers to induce macrophages to polarize to the M2 phenotype, inhibit the inflammatory factor storm, and thus resist chondrocyte apoptosis to achieve efficient treatment of osteoarthritis.

[0008] The present invention provides an asymmetric phosphorus-containing dendrimer, the structural formula of the asymmetric phosphorus-containing dendrimer is:

[0009]

[0010] The molecular formula of the asymmetric phosphorus-containing dendrimer is C 45 H 49 N 4 Na 7 O 27 P 10 , molecular weight is 1548.55g / mol.

[0011] The present invention provides a method for preparing an asymmetric phosphorus-containing dendrimer, comprising:

[0012] (1) Formaldehyde, compound 1 and solvent are mixed, reacted in an ice bath, and then trimethoxyphosphine solution is added, stirred at room temperature for reaction, purified, and dried to obtain dimethyl phosphite-modified tyramine, which is recorded as compound 2; wherein compound 1 is tyramine;

[0013] (2) p-hydroxybenzaldehyde, compound 3, potassium carbonate and solvent are mixed, reacted at room temperature, and purified to obtain 5 p-hydroxybenzaldehyde-modified cyclotriphosphazenes, which are denoted as compound 4; compound 3 is hexachlorocyclotriphosphazene;

[0014] (3) mixing tyramine modified with dimethyl phosphite, 5 cyclotriphosphazenes modified with p-hydroxybenzaldehyde, cesium carbonate, and a solvent, reacting at room temperature, and purifying to obtain compound 5;

[0015] (4) Mixing sodium borohydride, compound 5, and a solvent under ice bath conditions, stirring for reaction, and purifying to obtain compound 6;

[0016] (5) Mix thionyl chloride and compound 6 under ice bath conditions, stir to react, purify, and dry to obtain compound 7; (6) Mix compound 7 and trimethoxyphosphorus solution, heat to reflux for reaction, purify, and dry to obtain compound 8;

[0017] (7) Compound 8, a solvent, and trimethylsilyl bromide are mixed, stirred for reaction, the organic solvent is removed by rotary evaporation, and the mixture is dried in vacuo. The mixture is then suspended in an aqueous solution, a sodium hydroxide solution is added dropwise thereto, the mixture is stirred for reaction in an ice bath, and the mixture is freeze-dried to obtain an asymmetric phosphorus-containing dendrimer.

[0018] Preferably, in step (1), the molar ratio of tyramine to trimethoxyphosphine is 1:1 to 1:4; and the solvent is anhydrous tetrahydrofuran.

[0019] Preferably, the ice bath reaction time in step (1) is 30-60 min; the room temperature stirring reaction time in step (1) is 12-24 h.

[0020] Preferably, the purification process conditions in step (1) are as follows: adding an equal volume of saturated brine to the reaction mixture, then extracting with 4 to 5 times the volume of ethyl acetate solution, the extraction times are 2 to 3 times, then adding excess anhydrous magnesium sulfate to the organic phase, stirring and drying, collecting the solution by filtration, drying the organic solvent by spin drying, and purifying by column chromatography with acetone as the solvent.

[0021] Furthermore, the step (1) comprises adding formaldehyde solution dropwise to anhydrous tetrahydrofuran in which tyramine (compound 1) is dissolved, reacting in an ice bath, then adding trimethoxyphosphine solution dropwise, reacting by stirring at room temperature, purifying, and vacuum drying to obtain dimethyl phosphite-modified tyramine, recorded as compound 2;

[0022] The water content of the formaldehyde solution is 37%; the concentration of tyramine after being dissolved in anhydrous tetrahydrofuran is 0.012-1.2 mmol / mL.

[0023] Preferably, in step (2), the molar ratio of hexachlorocyclotriphosphazene, p-hydroxybenzaldehyde and potassium carbonate is 1:3-7:8-12; the potassium carbonate is anhydrous potassium carbonate; and the solvent includes anhydrous tetrahydrofuran;

[0024] Preferably, the room temperature reaction in step (2) is a stirring reaction at room temperature for 12-24 hours.

[0025] The purification process in step (2) is as follows: the precipitate in the solution after the reaction is removed by filtration, and then purification is performed by column chromatography using ethyl acetate and n-hexane (v / v=3-5 / 5-10) as solvents.

[0026] Furthermore, the step (2) comprises: dissolving p-hydroxybenzaldehyde in anhydrous tetrahydrofuran, and adding dropwise to an anhydrous tetrahydrofuran solution containing hexachlorocyclotriphosphazene (compound 3) and anhydrous potassium carbonate, stirring the mixture at room temperature to react, and obtaining 5 p-hydroxybenzaldehyde-modified cyclotriphosphazenes after purification, which are recorded as compound 4.

[0027] Preferably, in step (3), the molar ratio of the five p-hydroxybenzaldehyde-modified cyclotriphosphazenes, the dimethyl phosphite-modified tyramine, and the cesium carbonate is 1:1-2:2-4; the cesium carbonate is anhydrous cesium carbonate; and the solvent comprises anhydrous tetrahydrofuran;

[0028] Preferably, the room temperature reaction in step (3) is a stirring reaction at room temperature for 12-24 hours;

[0029] The purification process in step (3) is as follows: the precipitate in the solution after the reaction is removed by filtration, and then purification is performed by column chromatography using methanol and dichloromethane (v / v=1 / 20-30) as solvents.

[0030] Furthermore, the step (3) comprises: dissolving compound 2 in anhydrous tetrahydrofuran, and gradually adding the compound 2 dropwise to an anhydrous tetrahydrofuran solution containing compound 4 and anhydrous cesium carbonate, stirring the reaction at room temperature, and obtaining chemical compound 5 after purification.

[0031] Preferably, in step (4), the molar ratio of compound 5 to sodium borohydride is 1:5-10; the solvent comprises an anhydrous tetrahydrofuran / methanol mixture; wherein the volume ratio of anhydrous tetrahydrofuran to methanol is 3-6:1;

[0032] Preferably, the reaction is stirred for 12-24 hours in step (4).

[0033] The purification process conditions in step (4) are as follows: extracting the reaction solution with distilled water and ethyl acetate / tetrahydrofuran (v / v=1 / 2-5) solution for 2-4 times, then adding anhydrous magnesium sulfate to the organic phase, stirring and drying, collecting the solution by filtration, and rotary evaporating the organic solvent.

[0034] Furthermore, the step (4) comprises: adding sodium borohydride solution dropwise into an anhydrous tetrahydrofuran / methanol mixed solution containing compound 5 under ice bath conditions, stirring for reaction, purifying, and vacuum drying to obtain compound 6.

[0035] Preferably, in step (5), the ratio of thionyl chloride to compound 6 is 6-10 mL: 0.5-1 mmol; and the reaction is stirred for 12-24 h in step (5);

[0036] The purification conditions of step (5) are as follows: add 8 to 12 mL of anhydrous tetrahydrofuran to the reaction solution to dissolve it, then add 8 to 12 times the volume of a pentane / ether (v / v=1-4 / 1) mixed solution, stir for 30 to 60 minutes, and filter and collect the precipitate.

[0037] Furthermore, the step (5) comprises: adding the thionyl chloride solution to the compound 6 under ice bath conditions, stirring for reaction, purifying, and vacuum drying to obtain the compound 7.

[0038] Preferably, in step (6), the concentration of compound 7 in the trimethoxyphosphine solution is 10-20 μmol / mL; and the heating reflux reaction is carried out at 100-150° C. for 5-7 days.

[0039] The purification process conditions in step (6) are as follows: after the reaction is completed, trimethoxyphosphine is removed by vacuum drying, dichloromethane is added to dissolve it, and then it is added dropwise to a pentane solution (the volume ratio of dichloromethane to pentane is 1:8-12), stirred for reaction for 10 to 30 minutes, and the precipitate is collected by filtration.

[0040] The drying in step (6) is vacuum drying.

[0041] Furthermore, the step (6) comprises dissolving compound 7 in a trimethoxyphosphine solution, heating to reflux reaction, purifying, and vacuum drying to obtain compound 8.

[0042] Preferably, in step (7), the molar ratio of compound 8 to trimethylsilyl bromide is 1:15-20; the molar ratio of sodium hydroxide to compound 8 is 5-10:1; and the solvent comprises acetonitrile;

[0043] Preferably, the stirring reaction in step (7) is for 12-24 hours; and the stirring reaction in an ice bath is for 30-60 minutes.

[0044] Furthermore, the step (7) comprises: under ice bath conditions, compound 8 is dissolved in an acetonitrile solution, and trimethylsilyl bromide is added dropwise thereto, the reaction is stirred, the organic solvent is removed by rotary evaporation, and vacuum drying is performed, the obtained product is suspended in an aqueous solution, sodium hydroxide solution is added dropwise thereto, the reaction is stirred in an ice bath, and after freeze-drying, an asymmetric 0th generation phosphorus-containing dendrimer (G0.PD-ABP) carrying an azabisphosphonate (ABP) end is obtained, which is recorded as compound 9.

[0045] The concentration of the sodium hydroxide solution is 0.1-0.2 mol / L.

[0046] The invention provides an asymmetric phosphorus-containing dendrimer complex, which comprises a complex formed by the asymmetric phosphorus-containing dendrimer and a protease.

[0047] The invention provides a method for preparing an asymmetric phosphorus-containing dendrimer complex, comprising: mixing the asymmetric phosphorus-containing dendrimer, protease and a solvent, stirring at room temperature, and centrifuging to obtain the asymmetric phosphorus-containing dendrimer complex.

[0048] The solvent is ultrapure water.

[0049] Further, the asymmetric phosphorus-containing dendrimer is added dropwise into the protease solution, stirred at room temperature, and the precipitate is collected by centrifugation.

[0050] Preferably, the mass ratio of the asymmetric phosphorus-containing dendrimer to the protease is 10-14:1; the protease is bromelain Bro;

[0051] Preferably, the stirring time is 2-6 hours; and the centrifugation is performed at 8000-15000 rpm for 10-30 minutes.

[0052] The present invention provides an application of the asymmetric phosphorus-containing dendrimer complex or the asymmetric phosphorus-containing dendrimer complex prepared by the method in preparing a drug for treating osteoarthritis.

[0053] (1) LPS-activated RAW264.7 cells were plated in a 96-well plate and incubated at 37°C and 5% CO 2 After culturing for 24 h under the same environment, the cytotoxicity of Bro, G0.PD / Bro, and G0.PD-ABP / Bro was evaluated by CCK-8 method.

[0054] (2) LPS-activated RAW264.7 cells were seeded in 12-well plates and incubated at 37°C and 5% CO 2 After culturing in the environment for 24 hours, Bro, G0.PD / Bro, and G0.PD-ABP / Bro were added to the cells and incubated for 4 hours, respectively. The cells were collected and washed three times with PBS, and the phagocytosis of different materials by the cells was detected by flow cytometry and laser confocal microscopy.

[0055] (3) RAW264.7 cells were seeded in a 12-well plate and cultured at 37°C and 5% CO2 for 24 hours. After LPS stimulation for 24 hours, the culture medium containing Bro, G0.PD / Bro, and G0.PD-ABP / Bro was replaced and incubated with the cells for 24 hours. The cells were then collected, stained with CD206 and CD86 fluorescent antibodies, washed with PBS, and the effects of different materials on macrophage polarization were detected by flow cytometry.

[0056] (4) Chondrocytes were seeded in a 12-well plate and cultured at 5% CO2 and 37°C for 24 hours. Subsequently, the supernatant of LPS-activated macrophages treated with Bro, G0.PD / Bro, and G0.PD-ABP / Bro for 24 hours was replaced with the chondrocytes for 24 hours. The cells were collected and incubated with AnnexinV-FITC (5 μL) and PI (5 μL) in the dark for 10 minutes, and the apoptosis of chondrocytes after treatment with different conditioned media was detected by flow cytometry.

[0057] (5) Bro, G0.PD / Bro, and G0.PD-ABP / Bro were injected into the lesion site of osteoarthritis mice through the joint cavity, and the mice in the normal group were treated with PBS. The treatment cycle was 24 days. From the date of treatment, the weight of the mice was recorded every 4 days, and the serum of the mice in each experimental group was obtained after the treatment to detect the expression of inflammatory factors by enzyme-linked immunosorbent assay (ELISA). (6) After the treatment, the mice in different treatment groups were euthanized and the knee joint tissues of the mice were collected. After soaking in tissue fixative for 24 hours, the cartilage degradation and bone changes of the knee joints of the mice were analyzed by safranin-O-fast green staining and small animal micro-computed tomography (Micro-CT).

[0058] The present invention uses Zeta potential and dynamic light scattering analysis (DLS), transmission electron microscopy (TEM), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and other means to characterize the physical and chemical properties of the prepared nanocomplex. Then, the cytotoxicity of G0.PD-ABP / Bro and related control materials is evaluated by CCK-8 method; the phagocytosis of the materials by cells is detected by flow cytometry and laser confocal microscopy; the effect of the nanomaterials on macrophage typing is evaluated by flow cytometry; the effect of the nanomaterials on chondrocyte apoptosis is evaluated by flow cytometry; finally, an in vivo osteoarthritis animal model is established, and the therapeutic effect of the nanodrug on osteoarthritis is evaluated by enzyme-linked immunosorbent assay (ELISA), safranin-O-fast green staining and Micro-CT, and the biosafety of the nanocomplex is evaluated by monitoring the weight changes of mice during treatment.

[0059] The present invention designs and synthesizes an asymmetric phosphorus-containing dendrimer carrying an azabisphosphonate terminal and complexes it with bromelain to construct a composite nanomedicine. On the one hand, the nanomedicine can promote the intracellular delivery of bromelain, and in combination with the phosphorus-containing dendrimer, inhibit the inflammatory response by regulating the relevant inflammatory signaling pathways in the cell, and induce macrophages to polarize to the M2 phenotype; on the other hand, the immune effect generated by macrophage polarization is used to inhibit chondrocyte apoptosis, thereby achieving OA inflammation regression and cartilage protection.

[0060] The asymmetric phosphorus-containing dendrimer of the present invention has the characteristics of uniform molecular weight, simple preparation method, high controllability of reaction process, easy operation and separation, etc. It can enhance the intracellular delivery efficiency of bromelain, control the inflammatory factor storm by synergistically regulating the phenotype of macrophages, and exert the protective function of chondrocytes, and has good development prospects and application value in the treatment of osteoarthritis patients.

[0061] Beneficial Effects

[0062] (1) The process of the present invention is simple, easy to operate and separate, low in cost, and the raw materials are commercially available, thus having good development prospects;

[0063] (2) The asymmetric phosphorus-containing dendrimer prepared by the present invention has a uniform molecular weight and can be physically complexed with Bro to prepare a stable nanocomposite, thereby enhancing the intracellular delivery efficiency of Bro and improving the bioavailability of Bro;

[0064] (3) The nanocomplex prepared by the present invention can combine with the immunomodulatory activity of the phosphorus-containing dendrimers themselves to inhibit the inflammatory response by regulating the relevant inflammatory signaling pathways in the cells, thereby inducing macrophage polarization to the M2 phenotype; on the other hand, the immune effect produced by macrophage polarization is used to inhibit chondrocyte apoptosis, thereby achieving OA inflammation resolution and cartilage protection, which has potential clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 (A) and (B) are synthetic routes of asymmetric phosphorus-containing dendrimers carrying azabisphosphonate termini prepared by the present invention;

[0066] Figure 2 The hydrogen nuclear magnetic resonance spectrum (A) and phosphorus spectrum (B) of compound 2 prepared in Example 1;

[0067] Figure 3 The hydrogen nuclear magnetic resonance spectrum (A) and phosphorus spectrum (B) of compound 4 prepared in Example 1;

[0068] Figure 4 The hydrogen nuclear magnetic resonance spectrum (A) and phosphorus spectrum (B) of compound 5 prepared in Example 1;

[0069] Figure 5 The hydrogen nuclear magnetic resonance spectrum (A) and phosphorus spectrum (B) of compound 6 prepared in Example 1;

[0070] Figure 6 The hydrogen nuclear magnetic resonance spectrum (A) and phosphorus spectrum (B) of compound 7 prepared in Example 1;

[0071] Figure 7 The hydrogen nuclear magnetic resonance spectrum (A) and phosphorus spectrum (B) of compound 8 prepared in Example 1;

[0072] Figure 8 The hydrogen nuclear magnetic resonance spectrum (A) and phosphorus spectrum (B) of compound 9 prepared in Example 1;

[0073] Fig. 9 The hydration kinetic size distribution diagram (A) and surface potential diagram (B) of Bro, G0.PD / Bro, and G0.PD-ABP / Bro;

[0074] Fig.10SDS-PAGE images of Bro, G0.PD / Bro, and G0.PD-ABP / Bro;

[0075] Fig.11 TEM images of (A) G0.PD / Bro and (B) G0.PD-ABP / Bro;

[0076] Fig.12 Cell viability graph of Bro, G0.PD / Bro, G0.PD-ABP / Bro and LPS-activated RAW264.7 cells co-incubated for 24 hours;

[0077] Fig.13 Flow cytometry quantitative results (A) and laser confocal microscopy images (B) of intracellular fluorescence intensity after Bro, G0.PD / Bro, G0.PD-ABP / Bro were co-incubated with LPS-activated RAW264.7 cells for 24 hours;

[0078] Fig.14 Representative flow cytometry images of intracellular CD86 and CD206 expression levels after Bro, G0.PD / Bro, G0.PD-ABP / Bro and LPS-activated RAW264.7 cells were co-incubated for 24 h (A), M1 macrophage percentage (B), and M2 macrophage percentage (C);

[0079] Fig.15 Flow cytometry (A) and quantitative results (B) of chondrocyte apoptosis and necrosis after 24 hours of culture of chondrocytes with different conditioned medium (supernatant collected after 24 hours of co-incubation of Bro, G0.PD / Bro, G0.PD-ABP / Bro prepared by the present invention with LPS-activated RAW264.7);

[0080] Fig.16 The weight change curve of mice in each experimental group during the treatment period in Example 9;

[0081] Fig.17 The expression levels of pro-inflammatory cytokines (TNF-α and IL-1β) and anti-inflammatory cytokine IL-10 in the serum of mice in each experimental group in Example 9;

[0082] Fig.18 The results of the safranin-O-fast green staining of the inflamed knee joint tissues of the mice in each experimental group in Example 10;

[0083] Fig.19 Micro-CT imaging of the inflamed knee joints of mice in each experimental group in Example 11 (A) and quantitative results of bone volume fraction (B), bone density (C) and trabecular thickness (D). DETAILED DESCRIPTION

[0084] 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 are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0085] Unless otherwise specified, all chemical reagents were used directly without further purification. Phosphite-modified symmetrical phosphorus-containing dendrimers (G0.PD) were obtained from the group of Professor JP Majoral, Laboratory of Coordination Chemistry, National Center for Scientific Research, France. Bro was purchased from SigmaAldrich. RAW264.7 cells (mouse macrophage cell line) were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. DMEM medium, fetal bovine serum, penicillin-streptomycin, and trypsin were purchased from Hangzhou Gino Biomedical Technology Co., Ltd. BCA kit was purchased from Shanghai Biyuntian Biotechnology Co., Ltd. Cell apoptosis kit was purchased from Jiangsu Keygen Biotechnology Co., Ltd. BALB / c mice were purchased from Shanghai Regen Biotechnology Co., Ltd. Antibodies such as Anti-CD86-PE and Anti-CD206-FITC were purchased from Thermo Fisher Scientific. Water with a resistivity higher than 18.2 MΩ.cm used in all experiments was purified by a laboratory water purification system (PURIST UV Ultrapure, Shanghai Lefeng Biotechnology Co., Ltd., Shanghai).

[0086] Example 1

[0087] (1) Under ice bath conditions, 8 mL of formaldehyde solution (37% water content) was added dropwise to 50 mL of anhydrous tetrahydrofuran containing 51.2 mmol of tyramine (Compound 1) for 30 min, and then 10 mL of trimethoxyphosphine solution (110 mmol) was added dropwise for 24 h. The reaction conditions were all stirred at room temperature. After the reaction, the reaction solution was extracted with saturated brine / ethyl acetate (50 mL / 200 mL). After extraction three times, excess anhydrous magnesium sulfate was added to the organic phase for drying, the solution was collected by filtration, and the organic solvent was rotary evaporated. Purification was performed by column chromatography using acetone as the solvent, and compound 2 was obtained after vacuum drying. The results are as follows Figure 2 shown.

[0088] (2) 5 mL of anhydrous tetrahydrofuran solution containing p-hydroxybenzaldehyde (86.25 mmol) was added dropwise to 50 mL of anhydrous tetrahydrofuran containing hexachlorocyclotriphosphazene (17.25 mmol) and anhydrous potassium carbonate (172.5 mmol), and the mixture was reacted at room temperature for 24 h. The reaction progress was monitored by thin layer chromatography. The precipitate was removed by filtration, and then purified by column chromatography (ethyl acetate and n-hexane, v / v=3 / 7) to obtain cyclotriphosphazene (compound 4) with 5 p-hydroxybenzaldehyde modifications. The results are shown in FIG. Figure 3 (3) 10 mL of anhydrous tetrahydrofuran solution containing compound 2 (1.84 mmol) was added dropwise to 30 mL of anhydrous tetrahydrofuran containing compound 4 (1.53 mmol) and anhydrous cesium carbonate (4.59 mmol), and the mixture was reacted at room temperature for 24 h. The reaction progress was monitored by thin layer chromatography. The precipitate was removed by filtration, and then purified by column chromatography (methanol and dichloromethane, v / v=1 / 24) to obtain compound 5. The result is shown in Figure 4 shown.

[0089] (4) Under ice bath conditions, compound 5 (1 μmol) was fully dissolved in 150 mL of anhydrous tetrahydrofuran / methanol (volume ratio 4:1), and sodium borohydride (10 μmol) was added dropwise thereto, and the reaction was stirred for 24 hours. After the reaction was completed, it was purified by extraction, and 200 mL of distilled water and ethyl acetate / tetrahydrofuran (volume ratio 3:2) were used for 4 extraction operations. Anhydrous magnesium sulfate was added and dried for at least 3 hours, and the organic solvent was rotary evaporated to obtain a white solid. 200 mL of chloroform / pentane (volume ratio 1:3) was added, stirred for 2 hours, filtered, rotary evaporated and vacuum dried to obtain compound 6. Figure 5 shown.

[0090] (5) Add 8 mL SOCl to 0.71 mmol of compound 6 under ice bath conditions 2 The solution was stirred for 24 hours. 10 mL of anhydrous tetrahydrofuran was added to dissolve it, and then 100 mL of pentane / ether (2:1) was added and stirred for 30 minutes. The precipitate was collected and dried under vacuum to obtain compound 7. Figure 6 shown.

[0091] (6) 81.83 μmol of compound 7 was dissolved in 5 mL of trimethoxyphosphine solution and refluxed at 123°C for 6 days. 1 H NMR and 31 The reaction progress was detected by PNMR. After the reaction was completed, the trimethoxyphosphine was removed by vacuum drying. After adding 10 mL of dichloromethane to dissolve, it was added dropwise to 100 mL of pentane solution and stirred for 15 min. The precipitate was collected and vacuum dried to obtain compound 8. Figure 7 shown.

[0092] (7) Compound 8 (0.13 mmol) was dissolved in 10 mL of tetrahydrofuran under ice bath conditions, 2.07 mmol of trimethylsilyl bromide was added dropwise thereto and stirred for 24 h, the organic solvent was rotary evaporated and dried in vacuo. The product was suspended in water, and then 0.1 M sodium hydroxide solution (8.8 mL, 0.88 mmol) was added dropwise, stirred for 30 min under ice bath conditions, and lyophilized to obtain an asymmetric phosphorus-containing dendrimer compound 9 carrying an azabisphosphonate terminal, which was recorded as G0.PD-ABP. Figure 8 shown.

[0093] During the synthesis process, the product was characterized by hydrogen and phosphorus nuclear magnetic resonance spectra:

[0094] The present invention uses a 400MHz nuclear magnetic resonance instrument to perform hydrogen spectrum ( 1 H NMR) and phosphorus spectra ( 31 PNMR) test, the results are as follows:

[0095]

[0096] 1 H NMR (400 MHz, CDCl 3 )δ=2.73(m,2H,C 1 5 -H), 3.04(m,2H,C 1 6 -H),3.26(d,4H,C 1 7 -H),3.75(d,12H,C 1 8 -H), 6.78(m,2H,C 1 2 -H),7.10(m,2H,C 1 3 -H), 8.33(s,1H,OH)ppm.

[0097] 31 PNMR (162MHz, CDCl 3 )δ=26.48(s,P)ppm.

[0098]

[0099] 1 H NMR (400 MHz, CDCl 3 )δ=7.29(m,10H,C 0 2 -H),7.75(m,10H,C 0 3-H),9.90(2s,5H,C 0 5 -H)ppm

[0100] 31 PNMR(162MHz,CDCl 3 )δ=5.42(2d,P),20.74(dd,P 0 )ppm.

[0101]

[0102] 1 H NMR(400 MHz,CDCl3)δ=2.79(t,2H,C 1 5 -H),3.05(t,2H,C 1 6 -H),3.23(d,4H,C 1 7 -H),3.76(d,12H,C 1 8 -H),6.90(d,2H,C 1 2 -H),7.08-7.18(m,12H,C 0 2 -H and C 1 3 -H),7.72-7.76(m,10H,C 0 3 -H),9.94,9.99(s,5H,C 0 5 -H)ppm.

[0103] 31 PNMR(162 MHz,CDCl 3 )δ=7.38(m,N 3 P 3 ),26.59(s,P 1 )ppm.

[0104]

[0105] 1 H NMR(400 MHz,DMSO)δ=2.70(t,C 1 5 -H),2.93(t,2H,C 1 6 -H),3.2(d,4H,C 1 7-H),3.64(d,12H,C 1 8 -H),4.47(s,10H,C 0 5 -H),5.22(s,5H,OH),7.13(d,2H,C 1 2 -H),6.75-6.82(m,12H,C 0 2 -H and C 1 3 -H),7.18-7.21(m,10H,C 0 3 -H)ppm.

[0106] 31 PNMR(162 MHz,DMSO)δ=8.93(s,N 3 P 3 ),26.98(s,P 1 )ppm.

[0107]

[0108] 1 H NMR(400 MHz,DMSO)δ=2.73(t,2H,C 1 5 -H),2.93(t,2H,C 1 6 -H),3.21(d,4H,C 1 7 -H),3.64(d,12H,C 1 8 -H),4.78(s,10H,C 0 5 -H),7.13(d,2H,C 1 2 -H),6.77-6.88(m,12H,C 0 2 -H andC 1 3 -H),7.32-7.37(m,10H,C 0 3 -H)ppm.

[0109] 31 PNMR(162 MHz,DMSO)δ=8.73(s,N 3 P 3 ),26.85(s,P 1 )ppm.

[0110]

[0111] 1 \(^{1}\)H NMR (400 MHz, DMSO) δ=2.78 (t, 2H, C 1 5 -H), 3.06 (t, 2H, C 1 6 -H), 3.16 (d, 10H, C 0 5 -H), 3.23 (d, 4H, C 1 7 -H), 3.64 (d, 12H, C 1 8 -H), 3.67 (d, 30H, C 0 6 -H), 7.11 (d, 2H, C 1 2 -H), 6.90 - 6.94 (m, 12H, C 0 2 -H and C 1 3 -H), 7.14 - 7.17 (m, 10H, C 0 3 -H) ppm.

[0112] 31 \(^{31}\)P NMR (162 MHz, CDCl 3 ) δ=8.31 (s, N 3 P 3 ), 26.86 (s, P 1 ), 28.47 (s, P 2 ) ppm.

[0113]

[0114] 1 \(^{1}\)H NMR (400 MHz, D 2 O / CD 3 CN=9:1) δ=2.7 - 2.81 (d, 10H, C 0 5 -H), 2.96 (t, 2H, C 1 5 -H), 3.44 (t, 2H, C 1 6 -H), 3.13 (d, 4H, C 1 7 -H), 7.21 (d, 2H, C 12 -H), 6.75-6.86(m,12H,C 0 2 -H,C 1 3 -H), 7.14-7.21(m,10H,C 0 3 -H)ppm.

[0115] 31 PNMR(162MHz,D 2 O / CD 3 CN=9:1)δ=7.14(s,N 3 P 3 ),9.5(m,P 2 ),20.28(s,P 1 )ppm.

[0116] Example 2

[0117] Bro, G0.PD-ABP prepared in Example 1, and control material G0.PD were dissolved in aqueous solution, and the two dendrimers were added dropwise to the Bro solution at a mass ratio of 12:1 between dendrimer and protein, and stirred at room temperature for 4 hours. The mixture solution was then collected for determination of the surface potential and hydrodynamic diameter of the two nanocomposites. The results are shown in Fig. 9 As shown in the figure, the hydration kinetic size distribution shows that both G0.PD / Bro and G0.PD-ABP / Bro have uniform hydration kinetic size distribution, and the sizes of their hydrated particle sizes are 190.7nm and 217.5nm, respectively, which are much smaller than the 298.2nm of Bro alone, which means that both dendrimers can form a tight nanocomposite with Bro ( Fig. 9 A). In addition, after the phosphorus-containing dendrimers G0.PD and G0.PD-ABP were combined with Bro, their surface potentials decreased from -4.9 mV of Bro to -16.1 mV and -27.7 mV, respectively ( Fig. 9 B), while the surface potentials of G0.PD and G0.PD-ABP were -49.4 mV and -56.6 mV, respectively.

[0118] Example 3

[0119] In order to further verify the successful loading of Bro in G0.PD / Bro and G0.PD-ABP / Bro nanocomplexes, the G0.PD / Bro or G0.PD-ABP / Bro nanocomplexes prepared in Example 2 were subjected to SDS-PAGE characterization. 5 μL of Marker was added to the first protein lane, and then 10 μL of Bro, G0.PD / Bro or G0.PD-ABP / Bro nanocomplex (Bro concentration was 0.5 mg / mL) was mixed with 2 μL of loading buffer and heated at 100°C for 5 min. Subsequently, 10 μL of each sample was added to the corresponding lane and run at 180 V for 45 min. Fig.10 As shown, both G0.PD / Bro and G0.PD-ABP / Bro groups have the same electrophoretic bands as the Bro group, but the bands of G0.PD-ABP / Bro are significantly darker than those of the G0.PD / Bro group, which means that this asymmetric phosphorus-containing dendrimer G0.PD-ABP has a higher Bro encapsulation efficiency. In order to verify the protein encapsulation ability of the two different phosphorus-containing dendrimers, the encapsulation efficiency and drug loading of G0.PD-ABP for Bro were quantitatively determined by the BCA kit to be 72.3% and 5.7%, respectively, which were significantly higher than 53.0% and 4.2% of G0.PD.

[0120] Example 4

[0121] The G0.PD / Bro or G0.PD-ABP / Bro nanocomposite prepared in Example 2 was prepared into a sample with a concentration of 0.1 mg / mL, and then dropped onto the surface of the copper mesh with the carbon film, and placed at room temperature to dry to obtain a sample. The sample was then examined using a JEM-2010F transmission electron microscope. TEM results showed that the sizes of the G0.PD / Bro and G0.PD-ABP / Bro nanocomposites were 137.4 nm and 148.4 nm, respectively ( Fig.11 ), the size change trend was consistent with the hydrodynamic diameter test results, but G0.PD-ABP / Bro showed a more evenly dispersed and dense spherical structure, which further indicated that the asymmetric G0.PD-ABP had a better protein compression and stacking effect.

[0122] Example 5

[0123] The CCK-8 method was used to evaluate the effects of Bro, G0.PD / Bro or G0.PD-ABP / Bro on the viability of RAW264.7 cells. RAW264.7 cells in the logarithmic growth phase were collected and cultured at 1×10 4 The cells were seeded in 96-well cell culture plates at a density of 10 cells per well and incubated at 37°C and 5% CO 2The cells were cultured in an environment for 24 hours, and then replaced with fresh culture medium containing LPS (2 μg / mL) for another 24 hours of stimulation. Subsequently, DMEM culture medium containing Bro, G0.PD / Bro or G0.PD-ABP / Bro (Bro concentrations were 0, 3.125, 6.25, 12.5, 25, and 50 μg / mL, respectively) was incubated with LPS-activated cells for 24 hours, the original culture medium was discarded, the cells were washed three times with PBS, and fresh serum-free culture medium containing 10% (v / v) CCK-8 was added, and the cells were incubated in an incubator for another 3 hours. The absorbance of each well was tested at a wavelength of 450 nm using a multifunctional microplate reader. Cells treated with PBS were used as the control, and cell viability was recorded as 100%. The results are shown in Fig.12 As shown, within the experimental concentration range, the viability of RAW264.7 cells treated with Bro, G0.PD / Bro or G0.PD-ABP / Bro was above 90%, indicating that the two prepared phosphorus-containing dendrimer nanocomplexes have good cell compatibility and can be used as a safe nanodrug in subsequent experiments.

[0124] Example 6

[0125] The fluorescent dye Cy5.5-NHS was added dropwise to the Bro aqueous solution at a molar ratio of 3:1. After stirring at room temperature in the dark for 24 hours, it was transferred to a dialysis bag with a molecular weight cutoff of 5000Da and dialyzed with ultrapure water for three days. After freeze-drying, Cy5.5-labeled Bro (Cy5.5-Bro) was obtained. RAW264.7 cells were used as a cell model to verify the intracellular uptake efficiency of G0.PD / Bro and G0.PD-ABP / Bro complexes. Cells in the logarithmic growth phase were collected and 1×10 5 The cells were seeded at a density of 10 cells per well in a 12-well cell culture plate and cultured at 5% CO2 and 37°C for 24 hours. Fresh culture medium containing LPS (2 μg / mL) was replaced for another 24 hours of stimulation. Subsequently, fresh serum-free DMEM culture medium containing Bro, G0.PD / Bro or G0.PD-ABP / Bro was replaced and incubated with the cells for 4 hours (Bro was labeled with Cy5.5 and the Bro concentration was 30 μg / mL). The culture medium was discarded, the cells were trypsinized, centrifuged, and collected. After washing three times with PBS, the intracellular fluorescence intensity was detected by flow cytometry. Fig.13 As shown in A, compared with the free Bro group, both G0.PD / Bro and G0.PD-ABP / Bro can improve the cellular uptake efficiency of Bro to a certain extent, but the fluorescence intensity of the G0.PD-ABP / Bro treatment group is higher than that of the G0.PD / Bro treatment group, which indicates that the asymmetric phosphorus-containing dendrimer has a higher Bro delivery effect.

[0126] To further study the effect of two phosphorus-containing dendrimers on Bro intracellular delivery, cells in the logarithmic growth phase were collected and cultured at 1×10 5 The cells were seeded at a density of 10 cells per well in a laser confocal microplate culture dish and placed in a 5% CO 2 , cultured at 37°C for 24h, and replaced with fresh medium containing LPS (2μg / mL) for another 24h. Subsequently, fresh serum-free DMEM medium containing Bro, G0.PD / Bro or G0.PD-ABP / Bro (Bro labeled with Cy5.5, Bro concentration of 30μg / mL) was replaced and incubated with cells for 4h. The culture medium was discarded, washed three times with PBS, 1mL 2.5% glutaraldehyde was added to fix at room temperature for 15min, washed three times with PBS, 1mL LDAPI was added to co-incubate with cells for 5min, and the changes in cell fluorescence intensity were observed under a laser confocal microscope after washing three times with PBS. The results showed that due to the poor membrane permeability of Bro, a weak intracellular fluorescence signal was exhibited. Importantly, both the G0.PD / Bro or G0.PD-ABP / Bro groups showed enhanced fluorescence signals and were evenly dispersed in the cells, which further demonstrated that phosphorus-containing dendrimers have the ability to enhance intracellular Bro delivery. In addition, the asymmetry of G0.PD-ABP endowed the dendrimer with a stronger delivery effect, which was manifested by the most significant red fluorescence signal in the G0.PD-ABP / Bro group ( Fig.13 B).

[0127] Example 7

[0128] To verify the effect of G0.PD-ABP / Bro nanocomplex on macrophage polarization, RAW264.7 cells in the logarithmic growth phase were collected and cultured at 1×10 5 Cells were seeded at a density of 10 cells per well in a 12-well plate and placed in a 5% CO 2 , cultured at 37°C for 24 hours, and then replaced with DMEM medium containing LPS (concentration of 2μg / mL) and incubated with the cells for 24 hours. PBS was added to the control group. Subsequently, fresh DMEM medium containing Bro, G0.PD / Bro or G0.PD-ABP / Bro (Bro concentration of 30μg / mL) was replaced and incubated with the cells for 24 hours. Subsequently, the culture medium was discarded, washed twice with PBS, and the cells were collected by trypsin digestion. The cell pellet was resuspended with 200μL PBS, and Anti-CD206-FITC and Anti-CD86-PE antibodies were added to the cells and incubated with the cells at 4°C in the dark for 30 minutes. After washing three times with PBS to remove the antibodies that were not bound to the cells, the cell pellet was resuspended with 300μL PBS and transferred to a flow tube. The effect of nanomaterials on macrophage polarization was evaluated by detecting changes in the expression levels of CD86 and CD206 in the cells. The results are shown in Fig.14 As shown, the cells highly expressed the M1-related marker CD86 after LPS treatment, which indicates that LPS can effectively cause macrophages to polarize to the M1 phenotype. Since Bro alone is difficult to cross the cell membrane barrier and enter the cell, it has no significant effect on the expression of CD86 and CD206 on the surface of LPS-activated macrophages. G0.PD / Bro and G0.PD-ABP / Bro can inhibit the proportion of M1 macrophages and increase the level of M2 macrophages because they can enhance the intracellular delivery efficiency of Bro and combine the immunomodulatory activity of the carrier itself. Importantly, since G0.PD-ABP / Bro has a better intracellular delivery effect of Bro, it can induce the highest level of M2 macrophages (15.7%) and the lowest level of M1 macrophages (21.3%), which indicates that the G0.PD-ABP / Bro nanocomplex has excellent anti-inflammatory potential.

[0129] Example 8

[0130] In order to verify the protective effect of G0.PD-ABP / Bro-mediated macrophage polarization on chondrocytes, RAW264.7 cells in the logarithmic growth phase were collected and cultured at 2×10 5 Cells were seeded at a density of 10 cells per well in a 6-well plate and placed in a 5% CO 2 The cells were cultured at 37°C for 24 h, and then replaced with DMEM medium containing LPS (at a concentration of 2 μg / mL) and incubated with the cells for 24 h. PBS was added to the control group. Subsequently, fresh DMEM medium containing Bro, G0.PD / Bro, and G0.PD-ABP / Bro (Bro concentration of 30 μg / mL) was replaced and incubated with the cells for 24 h. The macrophage supernatant after different sample treatments was collected for culturing chondrocytes. Chondrocytes were cultured at 1×10 5 Cells were seeded at a density of 10 cells per well in a 12-well plate and placed in a 5% CO 2 , and cultured at 37°C for 24 hours. Subsequently, the supernatant of macrophages treated with the above-collected different samples was replaced and incubated with chondrocytes for 24 hours. The supernatant and adherent cells were collected, and the cell pellet was collected by centrifugation. After washing with PBS three times, it was resuspended in 0.5 mL PBS buffer, Annexin V-FITC (5 μL) and PI (5 μL) were added and incubated in a dark environment for 10 minutes, and then analyzed by flow cytometry. The results are shown in Fig.15As shown, compared with the PBS group (9.2%), the supernatant from LPS-activated macrophages can induce apoptosis and necrosis in chondrocytes (18.9%). This is because LPS can induce macrophages to secrete pro-inflammatory cytokines, which activate the apoptotic signaling pathway in chondrocytes. However, the excellent macrophage M2 polarization performance of G0.PD-ABP / Bro can protect chondrocytes from the damage of pro-inflammatory cytokines, effectively reverse chondrocyte apoptosis and necrosis (9.8%), and restore it to a normal level (close to the PBS negative control group).

[0131] Example 9

[0132] All animal experiments were approved by the Science and Technology Ethics Committee of Donghua University and were carried out strictly in accordance with the standards of the Animal Protection Association. Six-week-old male BALB / c mice used in the experiments were purchased from Shanghai Leigen Biotechnology Co., Ltd. An osteoarthritis animal model was established by injecting sodium iodoacetate (5 mg / kg) into the knee joint cavity for 3 days, and the knee joint cavity of the normal group was injected with an equal amount of PBS as a negative control. The white mice were randomly divided into 5 groups (PBS, OA, Bro, G0.PD / Bro, G0.PD-ABP / Bro), with 5 mice in each group. The day of starting the treatment was recorded as day 0. During the entire administration period, all mice were administered once on days 0, 4, 8, 12, 16, and 20 (the dose of Bro was 5 mg / kg), and the normal group (PBS) and the model group (OA) were respectively injected with an equal amount of PBS. Starting from day 0, the body weights of the mice in each group were recorded every 4 days. On day 24, the mice were sacrificed and the sera of the mice in each group were collected, and the expression levels of various cytokines (TNF-α, IL-1β, IL-10) in the sera were detected by ELISA. The results are as Fig.16 shown. The body weights of the mice in each treatment group increased slightly, indicating that the G0.PD-ABP / Bro complex prepared in the present invention has good biosafety. Subsequently, the expression of pro-inflammatory cytokines in the sera of the mice in each group was detected by ELISA. The results are as Fig.17 shown. Compared with the normal group of mice (PBS), there were high levels of pro-inflammatory cytokines (TNF-α and IL-1β) in the sera of OA model mice, indicating that the joint injury site of OA mice suffered from a severe inflammatory response. After treatment with different materials, especially G0.PD-ABP / Bro, the levels of pro-inflammatory cytokines in the sera of the mice decreased significantly, and the level of the anti-inflammatory cytokine IL-10 increased, indicating that the G0.PD-ABP / Bro complex can effectively block the inflammatory progression of the lesions in OA mice and has good anti-inflammatory treatment effects.

[0133] Example 10

[0134] 24 days after treatment, mice in different treatment groups were euthanized and their knee joint tissues were collected. After immersion in tissue fixative for 24 hours, cartilage degradation in the inflammatory knee joints of mice was analyzed by safranin-O-fast green staining. Fig.18 As shown in the figure, compared with normal mice, the cartilage tissue of OA model mice has obvious uneven staining or light staining, which means that the cartilage tissue of the knee joint of OA mice is degraded. Bro and G0.PD / Bro treatment alone has limited effect on alleviating articular cartilage degradation, because the surface of its cartilage tissue still shows unstained areas similar to the OA group. In addition, the articular cartilage tissue of mice in the G0.PD-ABP / Bro treatment group is almost fully stained, with only a small part of the lightly stained area, which is due to the fact that the asymmetric phosphorus-containing dendrimer G0.PD-ABP has a better Bro intracellular delivery effect than G0.PD, thereby amplifying the Bro biological activity and enhancing its protective performance on chondrocytes.

[0135] Embodiment 11

[0136] 24 days after treatment, mice in different treatment groups were euthanized and their knee joint tissues were collected. After immersion in tissue fixative for 24 hours, small animal Micro-CT was used to analyze the bone changes in the knee joints of mice. Fig.19 As shown in A, compared with healthy mice (PBS), the OA model group showed severe bone erosion around the knee joint and rough cartilage surface, while the free Bro treatment group had limited improvement in these pathological features. The quantitative results of Micro-CT showed ( Fig.19 BD), the bone volume fraction (BV / TV), bone density (BMD) and trabecular thickness (Tb.Th) values ​​of the OA model group were significantly reduced, indicating that the bone catabolism in the knee joint of OA mice was greater than the bone anabolism, and the bone mass was reduced. In contrast, the knee cartilage tissue of the G0.PD-ABP / Bro group was intact and smooth after treatment, and BMD, BV / TV and Tb.Th were significantly increased, indicating that the treatment of G0.PD-ABP / Bro can effectively inhibit the degradation of articular cartilage and promote bone synthesis. These results prove that the G0.PD-ABP / Bro nanocomplex can effectively regulate the phenotype of macrophages, inhibit the expression of proinflammatory factors, prevent OA-mediated cartilage erosion and bone destruction, and achieve the purpose of safe and efficient treatment of osteoarthritis.

Claims

1. An asymmetric phosphorus-containing dendrimer, characterized in that: The structural formula of the asymmetric phosphorus-containing dendrimer is:

2. A method for preparing an asymmetric phosphorus-containing dendrimer, comprising: (1) Formaldehyde, compound 1 and solvent are mixed, reacted in an ice bath, and then trimethoxyphosphine solution is added, stirred at room temperature for reaction, purified, and dried to obtain dimethyl phosphite-modified tyramine, which is recorded as compound 2; wherein compound 1 is tyramine; (2) p-hydroxybenzaldehyde, compound 3, potassium carbonate, and a solvent are mixed, reacted at room temperature, and purified to obtain five p-hydroxybenzaldehyde-modified cyclotriphosphazenes, which are referred to as compounds 4; wherein compound 3 is hexachlorocyclotriphosphazene; (3) mixing tyramine modified with dimethyl phosphite, 5 cyclotriphosphazenes modified with p-hydroxybenzaldehyde, cesium carbonate, and a solvent, reacting at room temperature, and purifying to obtain compound 5; (4) Mixing sodium borohydride, compound 5, and a solvent under ice bath conditions, stirring for reaction, and purifying to obtain compound 6; (5) Mix thionyl chloride and compound 6 under ice bath conditions, stir to react, purify, and dry to obtain compound 7; (6) Compound 7 and trimethoxyphosphorus solution are mixed, heated to reflux for reaction, purified, and dried to obtain compound 8; (7) Compound 8, a solvent, and trimethylsilyl bromide are mixed, stirred for reaction, the organic solvent is removed by rotary evaporation, and the mixture is dried in vacuo. The mixture is then suspended in an aqueous solution, a sodium hydroxide solution is added dropwise thereto, the mixture is stirred for reaction in an ice bath, and the mixture is freeze-dried to obtain an asymmetric phosphorus-containing dendrimer.

3. The preparation method according to claim 2, characterized in that: In the step (1), the molar ratio of tyramine to trimethoxyphosphine is 1:1 to 1:4; the solvent is anhydrous tetrahydrofuran; The ice bath reaction time in step (1) is 30-60 minutes; the room temperature stirring reaction time in step (1) is 12-24 hours; the molar ratio of hexachlorocyclotriphosphazene, p-hydroxybenzaldehyde and potassium carbonate in step (2) is 1:3-7:8-12; the potassium carbonate is anhydrous potassium carbonate; and the solvent includes anhydrous tetrahydrofuran; The room temperature reaction in step (2) is carried out by stirring at room temperature for 12-24 hours.

4. The preparation method according to claim 2, characterized in that: In the step (3), the molar ratio of the five p-hydroxybenzaldehyde-modified cyclotriphosphazenes, the dimethyl phosphite-modified tyramine, and the cesium carbonate is 1:1-2:2-4; the cesium carbonate is anhydrous cesium carbonate; and the solvent includes anhydrous tetrahydrofuran; The room temperature reaction in step (3) is stirring at room temperature for 12-24 hours; In the step (4), the molar ratio of compound 5 to sodium borohydride is 1:5-10; the solvent comprises an anhydrous tetrahydrofuran / methanol mixture; In the step (4), the reaction is stirred for 12-24 hours.

5. The preparation method according to claim 2, characterized in that: In the step (5), the ratio of thionyl chloride to compound 6 is 6-10 mL: 0.5-1 mmol; in the step (5), the reaction is stirred for 12-24 hours; In the step (6), the concentration of compound 7 in the trimethoxyphosphine solution is 10-20 μmol / mL; the heating reflux reaction is carried out at 100-150° C. for 5-7 days.

6. The preparation method according to claim 2, characterized in that: In the step (7), the molar ratio of compound 8 to trimethylsilyl bromide is 1:15-20; the molar ratio of sodium hydroxide to compound 8 is 5-10:1; and the solvent includes acetonitrile; In the step (7), the reaction is stirred for 12-24 hours; and the reaction is stirred in the ice bath for 30-60 minutes.

7. An asymmetric phosphorus-containing dendrimer complex, characterized in that: The complex comprises a complex formed by the asymmetric phosphorus-containing dendrimer according to claim 1 and a protease.

8. A method for preparing an asymmetric phosphorus-containing dendrimer complex, comprising: The asymmetric phosphorus-containing dendrimer of claim 1, protease and solvent are mixed, stirred at room temperature, and centrifuged to obtain an asymmetric phosphorus-containing dendrimer complex.

9. The preparation method according to claim 12, characterized in that: The mass ratio of the asymmetric phosphorus-containing dendrimer to the protease is 10-14:1; the protease is bromelain Bro; The stirring time is 2-6 hours; the centrifugation is performed at 8000-15000 rpm for 10-30 minutes.

10. Use of the asymmetric phosphorus-containing dendrimer complex according to claim 7 or the asymmetric phosphorus-containing dendrimer complex prepared by the method according to claim 8 in preparing a drug for treating osteoarthritis.

Images