Phosphate microfluidic hydrogel microspheres, lubricant, preparation method and application

Abstract

The invention relates to phosphate microfluidic hydrogel microspheres, a lubricant, a preparation method and application, and relates to the technical field of medicines and biological materials. Comprising the following steps: preparing a water-in-oil micro-fluidic microsphere from a water-phase solution and an oil-phase solution of a compound containing a phosphate group through a micro-fluidic technology; the water-in-oil micro-fluidic microspheres are subjected to curing and washing in sequence, and the phosphate micro-fluidic hydrogel microspheres are obtained. According to the phosphate microfluidic hydrogel microsphere lubricant provided by the invention, the defects that a hyaluronic acid lubricant is relatively high in degradation speed in vivo, needs to be repeatedly injected and the like are overcome, and meanwhile, the friction between joints is greatly reduced.

Description

Technical Field

[0001] The invention relates to the technical field of medicine and biomaterials, and in particular to a phosphate microfluidic hydrogel microsphere, a lubricant, and a preparation method and application thereof. Background Art

[0002] In the physiological functions of bone joints, synovial fluid plays a vital role as a lubricant. It is mainly composed of hyaluronic acid and other proteins, which can reduce friction between bones, reduce impact, and protect joint cartilage. However, with age and overuse of joints, the secretion of synovial fluid decreases, leading to joint pain, stiffness, and limited mobility. Therefore, finding an effective artificial lubricant to replace or enhance the function of natural synovial fluid has become a key topic in joint health research.

[0003] At present, commonly used artificial lubricants include hyaluronic acid, steroids, platelet-rich plasma (PRP) and other lubricants. Among them, hyaluronic acid is one of the most widely used intra-articular injection lubricants. It has good biocompatibility and lubricity, can temporarily replace the function of synovial fluid, reduce friction between joints, and relieve pain. However, hyaluronic acid degrades quickly in the body, and its effect can usually only last for a few weeks to a few months, and repeated injections are required. In addition, some patients do not respond well to hyaluronic acid, and may experience side effects such as joint swelling and increased pain.

[0004] With the development of materials science in recent years, researchers have begun to explore various new materials for the development of joint lubricants. For example, high molecular polymers, nanomaterials, natural biomaterials, etc. have become research hotspots. Although these new materials have shown certain advantages in lubrication effect, durability and biocompatibility, the products available on the market are still limited, and most new lubricants are still in the experimental research stage and have not yet been widely used in clinical practice. However, some medical bone and joint lubricants on the market currently still have problems in stability, ingredient safety and long-term effectiveness. For example, some products have problems such as phase separation, rapid degradation, and even immune response during use due to the addition of unstable ingredients or the use of inappropriate preparation processes. In order to overcome these problems, it is particularly important to develop a medical bone and joint lubricant that can be widely used in clinical practice and has excellent biocompatibility, lubrication effect, durability and safety. In view of this, the present invention provides a phosphate microfluidic hydrogel microsphere, a lubricant, a preparation method and an application. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a phosphate microfluidic hydrogel microsphere, a lubricant, a preparation method and an application thereof. The purpose is to provide a product with good biocompatibility, excellent lubrication effect and long-lasting durability, so as to improve the quality of life of patients with bone and joint diseases.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] In a first aspect, a method for preparing phosphate microfluidic hydrogel microspheres comprises the following steps: preparing an aqueous phase solution and an oil phase solution of a compound containing a phosphate group into oil-in-water microfluidic microspheres by microfluidic technology; and sequentially curing and washing the oil-in-water microfluidic microspheres to obtain phosphate microfluidic hydrogel microspheres.

[0008] Based on the above technical solution, the present invention can also be improved as follows.

[0009] Furthermore, washing is specifically as follows: the solidified water-in-oil microfluidic microspheres are sucked into a centrifuge tube, and repeatedly washed with PBS and deionized water to remove oil and surfactant.

[0010] Furthermore, the particle size of the phosphate microfluidic hydrogel microspheres is 95 μm-110 μm.

[0011] Further, the compound of the phosphate group includes at least one of 2-hydroxyethyl methacrylate phosphate, 2-methacryloyloxyethyl phosphorylcholine, phosphate ester of di[2-(methacryloyloxy)ethyl] phosphate, methacrylate phosphate (MAPO), vinyl phosphate, 2-hydroxyethyl acrylate (HEA), hydroxypropyl acrylate (HPA), and polymethacrylate (PMMA);

[0012] And / or, the solid content of the aqueous solution of the compound containing phosphate groups is 30%-70%, preferably 40%, 48%, 60%, etc.;

[0013] And / or, the oil phase solution comprises a surfactant and an oil solvent; the oil solvent comprises at least one of HF7500 and PEG-PFTE; the surfactant comprises at least one of sodium dodecyl sulfate (SDS), fatty acid salts (such as sodium salts), phosphate esters, cetyltrimethylammonium bromide (CTAB), benzalkonium chloride, polyoxyethylene (PEO), and polyoxyethylene-polyoxypropylene (POE-PPO) copolymers;

[0014] And / or, the surfactant accounts for 0.1%-2% of the mass of the oil phase solution.

[0015] Further, the compound of the phosphate group includes any two or more combinations of 2-hydroxyethyl methacrylate phosphate, 2-methacryloyloxyethyl phosphorylcholine, di[2-(methacryloyloxy)ethyl] phosphate, methacrylate phosphate (MAPO), vinyl phosphate, 2-hydroxyethyl acrylate (HEA), hydroxypropyl acrylate (HPA), and polymethacrylate (PMMA); for example, three combinations of 2-hydroxyethyl methacrylate phosphate, 2-methacryloyloxyethyl phosphorylcholine and di[2-(methacryloyloxy)ethyl] phosphate, and the mass ratio of the 2-hydroxyethyl methacrylate phosphate, the 2-methacryloyloxyethyl phosphorylcholine and the di[2-(methacryloyloxy)ethyl] phosphate is: 1-4:1-3:2-5, preferably 1:1:2, 2:1:3, 4:3:5, etc.

[0016] Furthermore, the curing of the water-in-oil microfluidic microspheres is carried out by introducing a photoinitiator and irradiating the microspheres under ultraviolet light for cross-linking;

[0017] And / or, the photoinitiator includes at least one of lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP), benzophenone, phenylbenzophenone, diphenylformyl (DMPA), and benzyl ketone.

[0018] And / or, the photoinitiator accounts for 0.1%-5% of the mass of the aqueous solution of the compound containing phosphate groups.

[0019] Furthermore, the microfluidic technology includes the following specific steps: using a microfluidic device, injecting the aqueous phase solution of the compound containing phosphate groups into the inner phase channel of the microfluidic device, and injecting the oil phase solution into the outer phase channel of the microfluidic device at the same time, controlling the pressure of the inner phase channel to 50mbar-100mbar, preferably 80mbar-90mbar, and controlling the pressure of the outer phase channel to 100mbar-150mbar, preferably 90mbar-100mbar; forming oil-in-water microfluidic microspheres at the intersection of the inner phase channel and the outer phase channel of the microfluidic device.

[0020] In a second aspect, a phosphate ester microfluidic hydrogel microsphere is provided, wherein the phosphate ester microfluidic hydrogel microsphere is prepared by the preparation method.

[0021] In a third aspect, a phosphate ester microfluidic hydrogel microsphere lubricant is provided, wherein the phosphate ester microfluidic hydrogel microsphere lubricant comprises the phosphate ester microfluidic hydrogel microsphere.

[0022] In a fourth aspect, a method for preparing a phosphate microfluidic hydrogel microsphere lubricant comprises the following steps: dispersing the phosphate microfluidic hydrogel microsphere in a carrier; the carrier comprises any one of a hyaluronic acid aqueous solution and PBS.

[0023] A fifth aspect provides an application of a phosphate ester microfluidic hydrogel microsphere lubricant, wherein the phosphate ester microfluidic hydrogel microsphere lubricant is used to prepare a drug for treating joint diseases.

[0024] The phosphate microfluidic hydrogel microsphere lubricant of the present invention is used to relieve the painful experience caused by joint degenerative lesions in patients and assist doctors in prescribing more humane and appropriate treatment plans. The phosphate microfluidic hydrogel microsphere lubricant of the present invention is suitable for the treatment of various joint diseases; its main indications include osteoarthritis (OA), rheumatoid arthritis (RA), synovitis, joint care after sports injuries, joint care after surgery, etc.; the lubricant can relieve pain and improve joint mobility by improving the fluidity of joint fluid and reducing intra-articular pressure.

[0025] The beneficial effects of the present invention are as follows:

[0026] (1) The present invention forms phosphate microfluidic hydrogel microspheres with controllable and uniform particle size through microfluidic technology;

[0027] (2) The phosphate microfluidic hydrogel microspheres of the present invention have an amphiphilic structure. The microspheres with this amphiphilic structure can form a layer of structure similar to a "hydrosynovial membrane" in the joint cavity. This lubricating membrane forms a hydration layer by attracting water molecules in the synovial fluid, thereby reducing direct contact between cartilages and reducing friction, thereby alleviating joint wear and pain.

[0028] (3) The phosphate groups on the surface of the phosphate microfluidic hydrogel microspheres of the present invention can coordinate with calcium ions in the joints, thereby increasing their retention time in the joint cavity and thus prolonging the duration of the lubrication effect.

[0029] (4) The phosphate microfluidic hydrogel microspheres of the present invention are used as lubricants. By adjusting the optimal particle size, it is beneficial to form a lubricating film with a uniform and stable size, providing a better friction effect. Microspheres with a particle size below the optimal range will precipitate in the synovial fluid and enter the articular cartilage, resulting in little lubrication effect.

[0030] (5) The MPC molecules used in the present invention have a structure similar to that of a biological membrane, which can effectively reduce the immune system's rejection of the lubricant and reduce the risk of inflammation and allergies.

[0031] (6) The phosphate microfluidic hydrogel microsphere lubricant of the present invention is injected into the affected joint, which can significantly reduce friction between joints, relieve pain, delay cartilage degeneration, and improve the patient's quality of life. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a diagram of the aqueous dispersion solution of microfluidic phosphate hydrogel microspheres of Example 1;

[0033] Figure 2 This is a diagram of the microfluidic phosphate hydrogel microspheres of Example 1;

[0034] Figure 3 Reaction flow chart for microsphere preparation;

[0035] Figure 4 The microfluidic phosphate hydrogel microsphere hyaluronic acid dispersion solution of Example 7;

[0036] Figure 5 The microscopic morphology of phosphate hydrogel microspheres; the left one is Example 1, the middle one is Example 5, and the right one is Comparative Example 1;

[0037] Figure 6 : is a particle size distribution diagram of phosphate hydrogel microspheres; wherein the left one is Example 1, the middle one is Example 5, and the right one is Control Example 1;

[0038] Figure 7 The machine for micro-ball friction test is UMT-8 universal friction tester;

[0039] Figure 8 is the friction coefficient of Examples 7, 8, 9, 10 and Comparative Examples 6 and 7;

[0040] Fig. 9 The friction coefficients of Example 8 and Comparative Examples 6 and 7;

[0041] Fig.10 The curves of the friction coefficients of Example 8 and Comparative Examples 6 and 7 changing with time;

[0042] Fig.11 The surface morphology of the microsphere solution after the experiment in Example 8;

[0043] Fig.12 The figure is a result diagram of the biocompatibility of microfluidic phosphate hydrogel microspheres; the left figure shows the cell survival rate at different concentrations, and the right figure shows the compound concentration at which the microspheres inhibit the cell biological activity (proliferation) by 50%;

[0044] Fig.13 This is a diagram showing the results of in vitro degradation experiments on microfluidic phosphate hydrogel microspheres. DETAILED DESCRIPTION

[0045] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0046] Example 1

[0047] This embodiment relates to a method for preparing phosphate microfluidic hydrogel microspheres, comprising the following steps:

[0048] (1) Preparation of phosphate microfluidic hydrogel microsphere aqueous solution:

[0049] Methacrylate-2-hydroxyethyl phosphate, 2-methacryloyloxyethyl phosphorylcholine and di[2-(methacryloyloxy)ethyl]phosphate were used as raw materials. Specifically, 0.48 g of methacrylate-2-hydroxyethyl phosphate and 0.72 g of di[2-(methacryloyloxy)ethyl]phosphate were weighed and mixed evenly, and 1.545 g of deionized water was added, vortexed and ultrasonically shaken. Then, 0.24 g of 2-methacryloyloxyethyl phosphorylcholine and 0.015 g of LAP (phenyl-2,4,6-trimethylbenzoylphosphonate) were weighed, with a mass ratio of 0.5%, and added to the prepared solution. The mixture was dissolved by ultrasonic. The ratio of 2-hydroxyethyl methacrylate phosphate, 2-methacryloyloxyethyl phosphorylcholine and di[2-(methacryloyloxy)ethyl]phosphate was 2:1:3. Finally, 3 g of aqueous solution of phosphate microfluidic hydrogel microspheres was prepared with a solid content of 48%. The solution was kept away from light. Figure 1 .

[0050] (2) Preparation of phosphate microfluidic hydrogel microspheres:

[0051] Through microfluidic technology, a chip with an oil phase coating an aqueous phase is used. The oil phase and the aqueous phase are compounded by shearing to form oil-in-water microfluidic microspheres. The oil-in-water microfluidic microspheres are solidified by ultraviolet light to form a phosphate hydrogel microsphere structure. Specifically: the oil phase uses HF7500 containing 2% surfactant, and the aqueous phase uses the solution prepared above, which are respectively introduced into the two-phase pipelines, and the pressures of the two-phase pipelines are adjusted to 80mbar and 90mbar, respectively. The oil-in-water microfluidic microspheres immersed in HF7500 are further transferred to a 10cm cell culture dish and cross-linked under ultraviolet light with a wavelength of 365nm for 30s. Then, the solidified microspheres are sucked into each centrifuge tube, and then repeatedly washed with PBS and deionized water to remove the oil and surfactant, such as Figure 2 The whole process is as follows Figure 3 .

[0052] Example 2

[0053] This embodiment relates to a method for preparing phosphate microfluidic hydrogel microspheres, comprising the following steps:

[0054] (1) Preparation of phosphate microfluidic hydrogel microsphere aqueous solution:

[0055] According to the method of Example 1, 0.6 g, 0.3 g, and 0.9 g of 2-hydroxyethyl methacrylate phosphate, 2-methacryloyloxyethyl phosphorylcholine, and di[2-(methacryloyloxy)ethyl] phosphate were weighed respectively in a ratio of 2:1:3, and 0.015 g of LAP (phenyl-2,4,6-trimethylbenzoylphosphonate lithium) was added, with a mass proportion of LAP of 0.5%. Finally, 3 g of an aqueous solution of phosphate microfluidic hydrogel microspheres was prepared with a solid content of 60%.

[0056] (2) Preparation of phosphate microfluidic hydrogel microspheres:

[0057] This step is the same as that used in Example 1.

[0058] Embodiment 3:

[0059] This embodiment relates to a method for preparing phosphate microfluidic hydrogel microspheres, comprising the following steps:

[0060] (1) Preparation of phosphate microfluidic hydrogel microsphere aqueous solution:

[0061] According to the method of Example 1, 0.36 g, 0.18 g, and 1.26 g of 2-hydroxyethyl methacrylate phosphate, 2-methacryloyloxyethyl phosphorylcholine, and bis[2-(methacryloyloxy)ethyl] phosphate were weighed respectively in a ratio of 2:1:7, 0.015 g of diphenylformyl (DMPA), and 0.0010 g of DMPA accounted for 0.3% by mass. Finally, 3 g of an aqueous phase solution of phosphate microfluidic hydrogel microspheres was prepared with a solid content of 60%.

[0062] (2) Preparation of phosphate microfluidic hydrogel microspheres:

[0063] This step is the same as that used in Example 1.

[0064] Example 4

[0065] This embodiment relates to a method for preparing phosphate microfluidic hydrogel microspheres, comprising the following steps:

[0066] (1) Preparation of phosphate microfluidic hydrogel microsphere aqueous solution:

[0067] According to the method of Example 1, 0.48 g, 0.24 g, and 0.72 g of 2-hydroxyethyl methacrylate phosphate, 2-methacryloyloxyethyl phosphorylcholine, and di[2-(methacryloyloxy)ethyl] phosphate were weighed respectively in a ratio of 2:1:3, and 0.006 g of LAP (phenyl-2,4,6-trimethylbenzoylphosphonate) was added, with a mass proportion of LAP of 0.2%. Finally, 3 g of an aqueous solution of phosphate microfluidic hydrogel microspheres was prepared with a solid content of 48%.

[0068] (2) Preparation of phosphate microfluidic hydrogel microspheres:

[0069] This step is the same as that used in Example 1.

[0070] Example 5

[0071] This embodiment relates to a method for preparing phosphate microfluidic hydrogel microspheres, comprising the following steps:

[0072] (1) Preparation of phosphate microfluidic hydrogel microsphere aqueous solution:

[0073] This step is the same as that used in Example 1.

[0074] (2) Preparation of phosphate microfluidic hydrogel microspheres:

[0075] Except that the two-phase pipeline pressure is adjusted to 80 mbar and 100 mbar, the remaining steps are the same as those adopted in Example 1.

[0076] Example 6

[0077] This embodiment relates to a method for preparing phosphate microfluidic hydrogel microspheres, comprising the following steps:

[0078] (1) Preparation of phosphate microfluidic hydrogel microsphere aqueous solution:

[0079] This step is the same as that used in Example 1.

[0080] (2) Preparation of phosphate microfluidic hydrogel microspheres:

[0081] Except that the two-phase pipeline pressure is adjusted to 80 mbar and 110 mbar, the remaining steps are the same as those used in Example 1.

[0082] Example 7

[0083] This embodiment relates to the preparation of acid ester microfluidic hydrogel microsphere lubricant, comprising the following steps:

[0084] The phosphate microfluidic hydrogel microspheres prepared in Example 1 were dispersed in a 5% hyaluronic acid aqueous solution to prepare a phosphate microfluidic hydrogel microsphere lubricant, wherein the concentration of the phosphate microfluidic hydrogel microspheres was 10 mg / ml. Figure 4 .

[0085] Example 8

[0086] This embodiment relates to the preparation of acid ester microfluidic hydrogel microsphere lubricant, comprising the following steps:

[0087] Compared with Example 7, except that the concentration of the bone joint cavity phosphate hydrogel microspheres is 1 mg / ml, the rest is the same.

[0088] Example 9

[0089] This embodiment relates to the preparation of acid ester microfluidic hydrogel microsphere lubricant, comprising the following steps:

[0090] Compared with Example 7, except that the concentration of the bone joint cavity phosphate hydrogel microspheres is 5 mg / ml, the rest is the same.

[0091] Example 10

[0092] This embodiment relates to the preparation of acid ester microfluidic hydrogel microsphere lubricant, comprising the following steps:

[0093] Compared with Example 7, except that the concentration of the bone joint cavity phosphate hydrogel microspheres is 20 mg / ml, the rest is the same.

[0094] Embodiment 11

[0095] This embodiment relates to the preparation of acid ester microfluidic hydrogel microsphere lubricant, comprising the following steps:

[0096] Compared with Example 7, except that the concentration of the bone joint cavity phosphate hydrogel microspheres is 3 mg / ml, the rest is the same.

[0097] Comparative Example 1

[0098] Compared with Example 1, the raw materials used are 2-hydroxyethyl methacrylate phosphate and 2-methacryloyloxyethyl phosphorylcholine in a ratio of 4:3. The rest are the same.

[0099] Comparative Example 2

[0100] Compared with Example 1, the raw materials used are 2-hydroxyethyl methacrylate phosphate and 2-methacryloyloxyethyl phosphorylcholine in a ratio of 7:5. The rest are the same.

[0101] Comparative Example 3

[0102] Compared with Example 1, the raw materials used are 2-hydroxyethyl methacrylate phosphate and 2-methacryloyloxyethyl phosphorylcholine in a ratio of 4:3 and a solid content of 55%. The rest are the same.

[0103] Comparative Example 4

[0104] Compared with Example 1, the raw materials used are 2-methacryloyloxyethyl phosphorylcholine and di[2-(methacryloyloxy)ethyl]phosphate in a ratio of 1:1 and a solid content of 60%. The rest are the same.

[0105] Comparative Example 5

[0106] Compared with Example 1, the two-phase pipeline pressures were adjusted to 80 mbar and 120 mbar. The rest were the same.

[0107] Comparative Example 6

[0108] Compared with Example 7, only 3% of the hyaluronic acid aqueous solution was used.

[0109] Comparative Example 7

[0110] Compared with Example 7, only PBS solution was used.

[0111] Comparative Example 8

[0112] Compared with Example 7, the concentration of the phosphate hydrogel microspheres in the bone joint cavity was set at 30 mg / ml. The rest was the same.

[0113] Comparative Example 9

[0114] Compared with Example 7, the concentration of the phosphate hydrogel microspheres in the bone joint cavity was set at 50 mg / ml. The rest was the same.

[0115] Test example

[0116] 1. Morphology and particle size of phosphate microfluidic hydrogel microspheres

[0117] In order to intuitively display the size and distribution of microspheres, Example 1, Example 5 (pipeline pressure), and Control Example 1 were selected to observe the morphology of phosphate hydrogel microspheres under an optical microscope. Figure 5 Overall, it is feasible to achieve uniform and stable distribution of spheres through microfluidic technology. It can be seen that the particle size distribution is 100.05±2μm, 108.8±2μm and 161.6±2μm. The pressure difference in the control example is too large, resulting in a relatively large particle size of the microspheres. Figure 5 From the three groups of experimental examples and control examples, the essential difference between the three lies in the average particle size. According to the optimal principle of particle size, microspheres larger than 110μm have little effect in the application of bone joint cavity compared with microspheres with the optimal particle size. Figure 6 It can be seen that the particle size distribution of the phosphate hydrogel microspheres is uniform and the size is uniform. Compared with the control example 1, the examples 1 and 5 are more uniform and the distribution is concentrated.

[0118] 2. Friction properties of phosphate microfluidic hydrogel microsphere lubricants

[0119] In order to explore the friction performance of the phosphate microfluidic hydrogel microsphere lubricant of the present invention, Examples 7, 8, 9, 10, 11 and Comparative Examples 6 and 7 were selected and a UMT high-frequency reciprocating friction tester (UMT-Tribolab, Bruker, Germany, Figure 7 ) were used for reciprocating sliding friction tests. In all tests, a polyethylene ball with a diameter of 4 mm (elastic modulus: 1 GPa; Poisson's ratio: 0.4) was used as the top surface, and a 304 stainless steel disk (elastic modulus: 194 GPa; Poisson's ratio: 0.3) was used as the bottom surface. The amplitude in the test was 5 mm and the frequency was 1 Hz. The average load applied was 2 N, and the maximum Hertzian pressure generated was 32.5 MPa. This pressure is slightly higher than the upper limit that normal joints can withstand (25.8 ± 5.2 MPa) and is intended to simulate the pressure conditions under damaged and inflamed joints. Figure 8 It can be seen that the friction coefficients of Examples 7, 8, 9, and 10 are significantly lower than those of Comparative Examples 6 and 7, which intuitively reflects that the invention can effectively reduce friction and relieve the pressure in the cavity. Fig. 9 The COF value frequency statistics of Comparative Examples 6, 7 and Example 8 are shown in FIG. Fig. 9 It can be seen that compared with Control Example 6 (COF: 0.264) and Control Example 7 (COF: 0.251), the phosphate ester microsphere lubricant of Example 8 reduces COF to 0.176 through the microsphere effect. This indicates that the phosphate ester microspheres reduce the friction in the joint cavity and play a lubricating effect. Fig.10 The friction coefficient curves of control example 6, control example 7 and embodiment 8 that change with time are shown in FIG. Fig.10 It can be seen that as time goes by, the friction coefficient rises sharply and then maintains a stable basic compound kinematic law. Fig.11 It can be seen that after applying the same pressure as that in the bone joint cavity, after a long period of friction movement, the microsphere solution still presents its original form, indicating that its microspheres can continue to stably play their corresponding role in the joint cavity.

[0120] 3. Biocompatibility of phosphate microfluidic hydrogel microspheres

[0121] In order to evaluate the cytotoxicity of cells to phosphate hydrogel microspheres, Example 7, Control Example 7, and Control Example 8 were selected for testing. CCK-8 reagent and PBS were used as controls. The culture dish was incubated at 37°C, and 4 hours after CCK-8 reagent was added to the culture medium at different time points (24h and 48h) at a volume ratio of 1:10. Subsequently, the mixed solution was inoculated in a fresh 96-well plate, protected from light, and the absorbance at 450nm was measured by a microplate reader (Infinite F50, TECAN, Switzerland) after 20 minutes. According to the CCK-8 display results, the cell survival rate of Example 12 is above 98%, and it has good biocompatibility. The results are as shown Fig.12 .

[0122] 4. In vitro degradability of phosphate microfluidic hydrogel microspheres

[0123] In order to explore the in vitro degradability of phosphate microfluidic hydrogel microspheres, according to literature records, phosphate microfluidic hydrogel microspheres belong to photocrosslinking free radical polymerization reaction, and their degradation performance is directly related to the content of added LAP photoinitiator. Therefore, Example 1 was selected into 5 groups, and 80 mg of each group was added with 50 times the volume of PBS solution. It was placed in a shaker at 37°C and 70rpm to simulate the degradation environment. Subsequent data was recorded. Fig.13 As can be seen from the figure, after 10 days, the diameter of the microsphere solution increased from about 100 microns to 130 microns, which indirectly shows that the degradation cycle of the microspheres is long, delaying the time they stay in the bone joint cavity.

[0124] In summary, the phosphate ester microfluidic hydrogel microsphere lubricant provided by the present invention overcomes the shortcomings of hyaluronic acid lubricants such as rapid degradation in the body and the need for repeated injections, while significantly reducing friction between joints.

[0125] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for preparing phosphate microfluidic hydrogel microspheres, characterized in that: The method comprises the following steps: preparing water-in-oil microfluidic microspheres from an aqueous solution and an oil-phase solution of a compound containing a phosphate group by microfluidic technology; and sequentially curing and washing the oil-in-water microfluidic microspheres to obtain phosphate microfluidic hydrogel microspheres.

2. The method for preparing phosphate microfluidic hydrogel microspheres according to claim 1, characterized in that: The particle size of the phosphate microfluidic hydrogel microspheres is 95 μm-110 μm.

3. The method for preparing phosphate microfluidic hydrogel microspheres according to claim 1, characterized in that: The compound of the phosphate group includes at least one of 2-hydroxyethyl methacrylate phosphate, 2-methacryloyloxyethyl phosphorylcholine, phosphate ester of di[2-(methacryloyloxy)ethyl] phosphate, methacrylate phosphate, vinyl phosphate, 2-hydroxyethyl acrylate, hydroxypropyl acrylate, and polymethacrylate; and / or, the solid content of the aqueous solution of the compound containing phosphate groups is 30%-70%; And / or, the oil phase solution comprises a surfactant and an oil solvent; the oil solvent comprises at least one of HF7500 and PEG-PFTE; the surfactant comprises at least one of sodium dodecyl sulfate, fatty acid salt, phosphate ester, hexadecyltrimethylammonium bromide, benzalkonium chloride, polyoxyethylene, and polyoxyethylene-polyoxypropylene copolymer; And / or, the surfactant accounts for 0.1%-2% of the mass of the oil phase solution.

4. The method for preparing phosphate microfluidic hydrogel microspheres according to claim 3, characterized in that: The compound of the phosphate group includes any two or more combinations of three of 2-hydroxyethyl methacrylate phosphate, 2-methacryloyloxyethyl phosphorylcholine, di[2-(methacryloyloxy)ethyl] phosphate, methacrylate phosphate, vinyl phosphate, 2-hydroxyethyl acrylate, hydroxypropyl acrylate, and polymethacrylate.

5. The method for preparing phosphate microfluidic hydrogel microspheres according to claim 1, characterized in that: The curing of the water-in-oil microfluidic microspheres is carried out by introducing a photoinitiator to crosslink under the action of ultraviolet light; and / or, the photoinitiator comprises at least one of phenyl-2,4,6-trimethylbenzoylphosphonate lithium, benzophenone, phenylbenzophenone, diphenylformyl, and benzyl ketone; And / or, the photoinitiator accounts for 0.1%-5% of the mass of the aqueous solution of the compound containing phosphate groups.

6. The method for preparing phosphate microfluidic hydrogel microspheres according to any one of claims 1 to 5, characterized in that: The microfluidic technology includes the following specific steps: using a microfluidic device, injecting the aqueous phase solution of the compound containing phosphate groups into the inner phase channel of the microfluidic device, and injecting the oil phase solution into the outer phase channel of the microfluidic device at the same time, controlling the pressure of the inner phase channel to 50mbar-100mbar, and controlling the pressure of the outer phase channel to 100mbar-150mbar; forming oil-in-water microfluidic microspheres at the intersection of the inner phase channel and the outer phase channel of the microfluidic device.

7. A phosphate microfluidic hydrogel microsphere, characterized in that: The phosphate microfluidic hydrogel microspheres are prepared by the preparation method according to any one of claims 1 to 6.

8. A phosphate microfluidic hydrogel microsphere lubricant, characterized in that: The phosphate ester microfluidic hydrogel microsphere lubricant comprises the phosphate ester microfluidic hydrogel microsphere according to claim 7.

9. A method for preparing a phosphate microfluidic hydrogel microsphere lubricant according to claim 8, characterized in that: The method comprises the following steps: dispersing the phosphate microfluidic hydrogel microspheres in a carrier; the carrier comprises any one of a hyaluronic acid aqueous solution and PBS.

10. An application of a phosphate microfluidic hydrogel microsphere lubricant, characterized in that: The phosphate microfluidic hydrogel microsphere lubricant described in claim 8 is used in the preparation of drugs for the treatment of joint diseases to relieve the painful experience caused by joint degenerative lesions in patients and to assist doctors in prescribing more humane and appropriate treatment plans.

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