A preparation method of a drug sustained-release microsphere

Abstract

The present invention provides a method for preparing a drug - sustained - release microsphere. The preparation method includes the following steps: providing amorphous nano - hydroxyapatite; dispersing nHAP in an inorganic salt solution as the internal aqueous phase, dissolving PLGA and PDLLA - PEG - PDLLA in ethyl acetate as the oil phase, emulsifying to prepare a W / O emulsion, and then emulsifying the W / O emulsion with a polyvinyl alcohol solution to obtain a W / O / W emulsion; etching and freeze - drying the W / O / W emulsion to obtain nHAP - PLGA porous microspheres; dissolving the nHAP - PLGA porous microspheres and dopamine in a buffer solution, and freeze - drying to obtain PDA - modified nHAP - PLGA porous microspheres; dispersing the PDA - modified nHAP - PLGA porous microspheres in a phosphate buffer solution, and adding a recombinant human type II tumor necrosis factor receptor antibody fusion protein (rhTNFR:Fc) solution, and freeze - drying to obtain rhTNFR:Fc - PLGA injectable drug - sustained - release microspheres. The solution of the present invention significantly improves the drug loading rate of the microspheres, and the loading is very stable, eliminating the burst - release effect of the microspheres.

Description

Technical Field

[0001] The present invention relates to the field of preparation of drug - sustained - release microspheres, and particularly to a method for preparing drug - sustained - release microspheres. Specifically, it relates to a method for preparing injectable recombinant human type II tumor necrosis factor receptor antibody fusion protein - poly(lactic - co - glycolic acid) drug - sustained - release microspheres that can effectively relieve rheumatoid arthritis. Background Art

[0002] Rheumatoid arthritis (RA) is a systemic inflammatory disease mainly characterized by chronic synovitis and joint structure destruction. The affected joints are mostly symmetric, progressive, and erosive polyarticular chronic swelling and pain. It causes chronic joint inflammation and the formation of pannus. The diseased synovium erodes the underlying cartilage and bone tissue, ultimately resulting in joint structure destruction, dysfunction, and even disability. RA is a multi - factor disease, and its basic pathological feature is synovitis and vasculitis caused by infiltration of inflammatory cells such as lymphocytes. Cytokines play a key role in the occurrence and development of the disease. It is relatively certain that the imbalance of the cytokine regulatory network is involved in the pathological process of RA. T and B cells can act on synovial cells by secreting a large number of cytokines, stimulating the formation of new blood vessels in the synovial tissue, synovial cell proliferation, and infiltration of monocytes / macrophages, and participating in RA synovial inflammation. At the same time, these cytokines can act on immune cells through a complex cytokine network, further expanding the autoimmune response. In addition to immune cells, macrophage - like synovial cells and fibroblast - like synovial cells in the synovial tissue can also produce various cytokines such as TNF - a, IL - 1, IL - 6, IL - 12, etc. Among the numerous cytokines involved in RA synovial inflammation, TNF - a plays an extremely crucial role. It causes synovial proliferation, cartilage erosion, and systemic inflammatory responses through mechanisms such as stimulating the proliferation of synovial fibroblasts, inducing the production of IL - 6, GM - CSF, chemokines, matrix metalloproteinases, and prostaglandins.

[0003] Recombinant human tumor necrosis factor receptor type II - antibody fusion protein for injection (rhTNFR:Fc) is a fusion protein composed of the human p75 tumor necrosis factor - α (TNF - α) receptor and the Fc segment of human IgG1. The time for Fc to reach a steady state in the human body is 408 ± 20 h. The Fc segment only includes the hinge region, CH2 region, and CH3 region, and has good non - immunogenicity and tolerance. Due to its structure containing 2 soluble TNF - α receptors, compared with the cell - surface TNF - α receptor, it has a stronger specific binding ability to TNF - α, thus blocking the interaction between the cell - surface TNF - α receptor and TNF - α and reducing its biological activity. Existing drugs all require multiple injections for patients in a short period of time. How to solve the problems of low existing bioavailability and low patient compliance caused by multiple injections urgently needs to be addressed. Summary of the Invention

[0004] The inventors of the present invention found that poly (lactic - co - glycolic acid) polymer (PLGA) is a biodegradable functional high - molecular organic compound randomly polymerized from monomers of lactic acid and glycolic acid. Microsphere preparations have many advantages, such as avoiding the phenomenon of peak - valley blood drug concentration, reducing adverse reactions, and improving the bioavailability of drugs. PLGA has excellent properties, good film - forming and encapsulation properties, non - toxicity, and convenient preparation, and can be used as a sustained - release carrier for various drugs. PLGA microspheres have the advantages of protecting drugs from being destroyed, targeting drugs to certain special tissues, delaying / controlling drug release, prolonging the drug action time, reducing drug toxicity and irritation, etc.

[0005] Polydopamine (PDA) is a mussel - biomimetic material that can be self - polymerized from dopamine (DA) in a weakly alkaline environment and has good adhesion, stability, and biocompatibility. The structure of PDA contains a large number of catechols and primary and secondary amines, making it almost adhere to any surface and having good optical and electrical properties. Therefore, it has been widely studied in medicine and materials science. PDA adsorbed on the material surface can be regarded as a "bridge" for reactions. Through Michael addition or Schiff - base reaction, it reacts with reagents containing nucleophilic groups, thereby introducing other functional groups on the material surface to achieve surface modification of the material. At the same time, PDA is the main component of melanin, which is distributed in various parts of the human body, so it has good biocompatibility.

[0006] An object of the present invention is to improve the drug loading rate of microspheres, enhance the stability of the loading, and eliminate the burst - release effect of microspheres, thereby avoiding multiple injections for patients in a short period of time, improving bioavailability, and avoiding the problem of low patient compliance caused by multiple injections.

[0007] A further object of the present invention is to improve the biocompatibility and stability of nano-hydroxyapatite.

[0008] In particular, the present invention provides a method for preparing a drug sustained-release microsphere, comprising the following steps:

[0009] Providing amorphous nano-hydroxyapatite (nHAP);

[0010] Dispersing the nHAP in an inorganic salt solution as the internal aqueous phase, dissolving PLGA and PDLLA-PEG-PDLLA in ethyl acetate as the oil phase, emulsifying to prepare a W / O emulsion, and then emulsifying the W / O emulsion with a polyvinyl alcohol solution to obtain a W / O / W emulsion;

[0011] Stirring and evacuating the W / O / W emulsion, centrifuging to obtain microspheres, and etching and freeze-drying the microspheres to obtain nHAP-PLGA porous microspheres;

[0012] Dissolving the nHAP-PLGA porous microspheres and dopamine in a buffer solution, stirring, centrifuging and freeze-drying to obtain PDA-modified nHAP-PLGA porous microspheres;

[0013] Dispersing the PDA-modified nHAP-PLGA porous microspheres in a phosphate buffer solution, and applying a recombinant human type II tumor necrosis factor receptor antibody fusion protein (rhTNFR:Fc) solution, and freeze-drying to obtain rhTNFR:Fc-PLGA injectable drug sustained-release microspheres.

[0014] Optionally, in the step of dissolving the nHAP-PLGA porous microspheres and dopamine in a buffer solution, stirring, centrifuging and freeze-drying to obtain PDA-modified nHAP-PLGA porous microspheres, the mass ratio of the nHAP-PLGA porous microspheres to the dopamine is any value in the range of 5-10:1;

[0015] In the solution of the nHAP-PLGA porous microspheres, dopamine and buffer solution, the mass fraction of the nHAP-PLGA porous microspheres is any value in the range of 0.1-1%;

[0016] Optionally, the buffer solution is tris(hydroxymethyl)aminomethane hydrochloride buffer solution, and the pH value of the buffer solution is any value in the range of 8-9.

[0017] Optionally, in the step of dissolving the nHAP-PLGA porous microspheres and dopamine in a buffer solution, stirring, centrifuging, and freeze-drying to obtain PDA-modified nHAP-PLGA porous microspheres, before stirring, centrifuging, and freeze-drying to obtain PDA-modified nHAP-PLGA porous microspheres after dissolving the nHAP-PLGA porous microspheres and dopamine in the buffer solution, the following steps are further included: stirring and reacting in the dark for 12-16 h, and performing water washing.

[0018] Optionally, in the step of dispersing the PDA-modified nHAP-PLGA porous microspheres in a phosphate buffer solution, the pH value of the phosphate buffer solution is any value in the range of 7-8;

[0019] In the solution of the PDA-modified nHAP-PLGA porous microspheres and the phosphate buffer solution, the mass fraction of the PDA-modified nHAP-PLGA porous microspheres is any value in the range of 1-10%;

[0020] In the step of dispersing the PDA-modified nHAP-PLGA porous microspheres in a phosphate buffer solution and adding a recombinant human type II tumor necrosis factor receptor antibody fusion protein (rhTNFR:Fc) solution, the rhTNFR:Fc solution is a solution of a phosphate buffer solution containing rhTNFR:Fc, and in the rhTNFR:Fc solution, the mass percentage of rhTNFR:Fc is any value in the range of 0.05-0.5%;

[0021] Optionally, in the rhTNFR:Fc solution, the pH value of the phosphate buffer solution is any value in the range of 7-8;

[0022] Optionally, in the step of dispersing the PDA-modified nHAP-PLGA porous microspheres in a phosphate buffer solution, adding a recombinant human type II tumor necrosis factor receptor antibody fusion protein (rhTNFR:Fc) solution, and freeze-drying to obtain rhTNFR:Fc-PLGA injectable drug sustained-release microspheres, the following steps are further included before freeze-drying: stirring and reacting in the dark at a preset temperature for a preset time, and performing water washing;

[0023] The preset temperature is any value in the range of 30-40 °C, and the preset time is any value in the range of 12-16 h.

[0024] Optionally, the steps of stirring and evacuating the W / O / W emulsion, centrifuging to obtain microspheres, etching the microspheres, and freeze-drying to obtain nHAP-PLGA porous microspheres include the following steps:

[0025] Remove the organic reagent from the W / O / W emulsion and wash it with water to obtain microsphere particles;

[0026] Disperse the microsphere particles in ultrapure water, stir for 12 - 16 h, and perform centrifugal freeze-drying operation to obtain a solid powder;

[0027] Disperse the solid powder in an alkaline etching solution for etching, and then centrifuge and wash with water. After freeze-drying, the nHAP-PLGA porous microspheres are obtained.

[0028] Optionally, in the step of dispersing the nHAP in an inorganic salt solution as the internal aqueous phase, dissolving PLGA and PDLLA-PEG-PDLLA in ethyl acetate as the oil phase, emulsifying to prepare a W / O emulsion, and then emulsifying the W / O emulsion and a polyvinyl alcohol solution to obtain a W / O / W emulsion, the mass ratio of the inorganic salt in the inorganic salt solution is any value in the range of 0.5 - 5%, and the density of the nHAP in the internal aqueous phase is any value in the range of 10 - 50 mg / ml;

[0029] Optionally, the mass ratio of PLGA in the oil phase is any value in the range of 1 - 5%, and the mass ratio of PDLLA-PEG-PDLLA in the oil phase is any value in the range of 1 - 5%.

[0030] Optionally, the method for preparing the W / O emulsion includes the following steps:

[0031] Prepare an aqueous solution of polyvinyl alcohol with a mass percentage in the range of 0.5 - 10%;

[0032] Stir the oil phase and quickly add the internal aqueous phase to the oil phase to obtain the W / O emulsion;

[0033] Optionally, in the method for preparing the W / O emulsion, the volume ratio of the internal aqueous phase to the oil phase is any value in the range of 1:2 - 10.

[0034] Optionally, the method for preparing the W / O / W emulsion includes the following steps:

[0035] Stir an aqueous solution of polyvinyl alcohol with a volume 5 - 30 times that of the oil phase;

[0036] Place the W / O emulsion into the aqueous solution of polyvinyl alcohol to obtain the W / O / W emulsion.

[0037] Optionally, in the step of providing amorphous nano-hydroxyapatite (nHAP), the preparation method of the nHAP includes the following steps:

[0038] Apply the polyaspartic acid (PASP) solution to the calcium salt solution, and obtain solution A through a chelation reaction;

[0039] Apply the polyacrylic acid (PAA) solution to the disodium hydrogen phosphate dodecahydrate solution, and obtain solution B through a chelation reaction;

[0040] Separate the solution A and the solution B into a high gravity machine for treatment and then perform centrifugal water washing to obtain the nHAP.

[0041] Optionally, in the solution A, the volume ratio of the PASP solution to the calcium salt solution is any value within the range of 1:5 - 25;

[0042] In the solution B, the volume ratio of the PAA solution to the disodium hydrogen phosphate dodecahydrate solution is any value within the range of 1:5 - 25.

[0043] According to the solution of the present invention, by emulsifying to prepare a W / O emulsion, the nHAP can be effectively dispersed inside the dissolved oil phase. Then, by emulsifying the W / O emulsion and the polyvinyl alcohol solution, the W / O emulsion can be dispersed in the internal water phase in the form of droplets, and then spherical particles are precipitated by solvent evaporation. Moreover, by compounding the nHAP in the PLGA microspheres, it is possible to effectively buffer the acidic environment during degradation when the PLGA microspheres degrade, thereby reducing the impact on the released protein drug. At the same time, the prepared microspheres have a porous structure, which greatly increases the surface area. Through the in-situ polymerization of dopamine on the surface of the microspheres, a PDA layer is formed, significantly increasing the drug loading rate of the microspheres. And because it binds to protein drugs through chemical bonds, the loading is very stable, eliminating the burst release effect of the microspheres.

[0044] Based on the following detailed description of the specific embodiments of the present invention in conjunction with the accompanying drawings, those skilled in the art will more clearly understand the above and other objects, advantages, and features of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Some specific embodiments of the present invention will be described in detail hereinafter with reference to the accompanying drawings in an exemplary but not restrictive manner. The same reference numerals in the drawings denote the same or similar components or parts. Those skilled in the art should understand that these drawings are not necessarily drawn to scale. In the drawings:

[0046] Figure 1 Shows a schematic flowchart of a method for preparing a drug sustained-release microsphere according to an embodiment of the present invention;

[0047] Figure 2 Shows a schematic flowchart of a method for preparing amorphous nano-hydroxyapatite according to an embodiment of the present invention;

[0048] Figure 3 Shows a schematic flowchart of a method for preparing a W / O emulsion according to an embodiment of the present invention;

[0049] Figure 4 Shows a schematic flowchart of a method for preparing a W / O / W emulsion according to an embodiment of the present invention;

[0050] Figure 5 Shows a schematic flowchart of a method for stirring and evacuating a W / O / W emulsion, centrifuging to obtain microspheres, etching the microspheres, and freeze-drying to obtain nHAP-PLGA porous microspheres according to an embodiment of the present invention;

[0051] Figure 6 Shows a Fourier transform infrared spectroscopy diagram of nano-hydroxyapatite according to an embodiment of the present invention;

[0052] Figure 7 Shows an XRD diagram of nano-hydroxyapatite according to an embodiment of the present invention;

[0053] Figure 8 Shows a transmission electron microscope diagram of nano-hydroxyapatite according to an embodiment of the present invention;

[0054] Figure 9 Shows a scanning electron microscope diagram of microspheres obtained by freeze-drying a W / O / W emulsion;

[0055] Figure 10 Shows a scanning electron microscope diagram of nHAP-PLGA porous microspheres according to an embodiment of the present invention;

[0056] Figure 11 Shows a scanning electron microscope diagram of PDA-modified nHAP-PLGA porous microspheres according to an embodiment of the present invention;

[0057] Figure 12 Shows Fourier transform infrared spectroscopy diagrams of dopamine, nHAP-PLGA porous microspheres, and PDA-modified nHAP-PLGA porous microspheres;

[0058] Figure 13 Shows a pH change curve diagram of solutions of nHAP-PLGA porous microspheres according to an embodiment of the present invention and PLGA microspheres without nHAP composite in the comparative example after accelerated degradation at 70°C;

[0059] Figure 14 Shows a comparison diagram of the cumulative drug release curves of injectable drug sustained-release microspheres without dopamine modification within 15 days and rhTNFR:Fc-PLGA injectable drug sustained-release microspheres according to an embodiment of the present invention;

[0060] Figure 15 shows Figure 14 the curve graph of the first 12 hours therein;

[0061] Figure 16 shows the comparative curve graph of the pharmacokinetic results of rhTNFR:Fc raw drug and rhTNFR:Fc-PLGA sustained-release microspheres in rats;

[0062] Figure 17 shows the picture after staining the sections of the main organs of rats when using rhTNFR:Fc-PLGA injectable drug sustained-release microspheres to treat collagen-induced rheumatoid arthritis (CIA) rats according to an embodiment of the present invention;

[0063] Figure 18 shows the level of TNF-α in the serum of rats after injecting rhTNFR:Fc-PLGA microspheres;

[0064] Figure 19 shows the level of IL-6 in the serum of rats after injecting rhTNFR:Fc-PLGA microspheres;

[0065] Figure 20 shows the level of IL-1β in the serum of rats after injecting rhTNFR:Fc-PLGA microspheres;

[0066] Figure 21 shows the level of TNF-α in the homogenate of rat synovial tissue after injecting rhTNFR:Fc-PLGA microspheres;

[0067] Figure 22 shows the level of IL-6 in the homogenate of rat synovial tissue after injecting rhTNFR:Fc-PLGA microspheres;

[0068] Figure 23 shows the level of IL-1β in the homogenate of rat synovial tissue after injecting rhTNFR:Fc-PLGA microspheres. Detailed implementation manners

[0069] The following describes the implementation manners of the present application through specific specific examples. Those skilled in the art can easily understand other advantages and effects of the present application from the content disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. The present application can also be implemented or applied through other different specific implementation manners. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present application. It should be noted that, without conflict, the following embodiments and the features in the embodiments can be combined with each other. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present application without creative efforts belong to the scope protected by the present application.

[0070] Figure 1 FIG. shows a schematic flowchart of a method for preparing a drug - sustained - release microsphere according to an embodiment of the present invention. As Figure 1 shown, the preparation method includes:

[0071] Step S100: Provide amorphous nano - hydroxyapatite (nHAP);

[0072] Step S200: Disperse nHAP in an inorganic salt solution as the internal aqueous phase, dissolve PLGA and PDLLA - PEG - PDLLA in ethyl acetate as the oil phase, emulsify to prepare a W / O emulsion, and then emulsify the W / O emulsion with a polyvinyl alcohol solution to obtain a W / O / W emulsion;

[0073] Step S300: Stir and evacuate the W / O / W emulsion, centrifuge to obtain microspheres, and etch and freeze - dry the microspheres to obtain nHAP - PLGA porous microspheres;

[0074] Step S400: Dissolve the nHAP - PLGA porous microspheres and dopamine in a buffer solution, stir, centrifuge and freeze - dry to obtain PDA - modified nHAP - PLGA porous microspheres;

[0075] Step S500: Disperse the PDA - modified nHAP - PLGA porous microspheres in a phosphate buffer solution, and apply a solution of recombinant human type II tumor necrosis factor receptor antibody fusion protein (rhTNFR:Fc), and then freeze - dry to obtain rhTNFR:Fc - PLGA injectable drug - sustained - release microspheres.

[0076] According to the solution of the present invention, by emulsifying to prepare a W / O emulsion, nHAP can be effectively dispersed inside the dissolved oil phase. Then, by emulsifying the W / O emulsion and a polyvinyl alcohol solution, the W / O emulsion can be dispersed in the inner aqueous phase in the form of droplets, and then spherical particles are precipitated by solvent evaporation. Moreover, by compounding nHAP in PLGA microspheres, the acidic environment during degradation can be effectively buffered when the PLGA microspheres degrade, thereby reducing the impact on the released protein drug. At the same time, the prepared microspheres have a porous structure, which greatly increases the surface area. By in-situ polymerization of dopamine on the surface of the microspheres, a PDA layer is formed, significantly improving the drug loading rate of the microspheres. And because it binds protein drugs through chemical bonds, the loading is very stable, eliminating the burst release effect of the microspheres.

[0077] Figure 2 FIG. shows a schematic flow chart of a method for preparing amorphous nano-hydroxyapatite according to an embodiment of the present invention. As Figure 2 shown, the preparation method includes:

[0078] Step S110, applying a polyaspartic acid (PASP) solution to a calcium salt solution, and performing a chelation reaction to obtain solution A;

[0079] Step S120, applying a polyacrylic acid (PAA) solution to a disodium hydrogen phosphate dodecahydrate solution, and performing a chelation reaction to obtain solution B;

[0080] Step S130, separately placing solution A and solution B in a high gravity machine for treatment and then performing centrifugal washing with water to obtain nHAP.

[0081] In this step S110, the volume ratio of the PASP solution to the calcium salt solution is 1:5, 1:10, 1:15, 1:20 or 1:25, and can also be any other value within the range of 1:5 - 25. The PASP solution can be, for example, a solution prepared by dispersing PASP in a Tris buffer solution. The pH value of the Tris buffer solution can be, for example, 7, 7.3, 7.6, 7.8 or 8, and can also be any value within 7 - 8. The concentration of the Tris buffer solution can be, for example, 0.5 mM, 0.8 mM, 1 mM, 1.2 mM or 1.5 mM, and can also be any other value within 0.5 - 1.5 mM. The density of the PASP solution can be, for example, 10 mg / ml, 20 mg / ml, 40 mg / ml, 60 mg / ml, 80 mg / ml, 90 mg / ml or 100 mg / ml, and can also be any other value within 10 - 10 mg / ml.

[0082] The calcium salt solution can be, for example, calcium chloride (CaCl2), Ca(OH)2, or CaHPO4·2H2O. The calcium salt solution can be, for example, a solution prepared by dissolving a calcium salt in Tris buffer, where the pH value of the Tris buffer can be, for example, 7, 7.3, 7.6, 7.8, or 8, or any value between 7 and 8. The concentration of the Tris buffer can be, for example, 0.5 mM, 0.8 mM, 1 mM, 1.2 mM, or 1.5 mM, or any other value between 0.5 and 1.5 mM. The concentration of the calcium salt solution can be, for example, 0.05 mol / l, 0.1 mol / l, 0.15 mol / l, or 0.2 mol / l, or any other value between 0.05 and 0.2 mol / l.

[0083] In step S110, the chelation reaction time can be, for example, 5 min, 10 min, 20 min, 30 min, 40 min, 50 min, or 60 min, or any time value between 5 and 60 min.

[0084] In step S120, the volume ratio of the PAA solution to the disodium hydrogen phosphate dodecahydrate solution is 1:5, 1:10, 1:15, 1:20, or 1:25, or any other value in the range of 1:5 - 25. The PAA solution can be, for example, a solution prepared by dissolving PAA in HEPES buffer salt solution, where the pH value of the HEPES buffer salt solution can be, for example, 6.5, 6.8, 7, 7.1, 7.3, or 7.5, or any value between 6.5 and 7.5. The concentration of the HEPES buffer salt solution can be, for example, 45 mM, 48 mM, 50 mM, 52 mM, or 55 mM, or any other value between 45 and 55 mM. The density of the PAA solution can be, for example, 10 mg / ml, 15 mg / ml, 20 mg / ml, 25 mg / ml, or 30 mg / ml, or any other value between 10 and 30 mg / ml.

[0085] The disodium hydrogen phosphate dodecahydrate solution can be, for example, a solution prepared by dissolving disodium hydrogen phosphate dodecahydrate in HEPES buffer salt solution, where the pH value of the HEPES buffer salt solution can be, for example, 6.5, 6.8, 7, 7.1, 7.3, or 7.5, or any value between 6.5 and 7.5. The concentration of the HEPES buffer salt solution can be, for example, 45 mM, 48 mM, 50 mM, 52 mM, or 55 mM, or any other value between 45 and 55 mM. The concentration of the disodium hydrogen phosphate dodecahydrate solution can be, for example, 0.05 mol / l, 0.1 mol / l, 0.15 mol / l, or 0.2 mol / l, or any other value between 0.05 and 0.2 mol / l.

[0086] In this step S120, the chelation reaction time can be, for example, 5 min, 10 min, 20 min, 30 min, 40 min, 50 min or 60 min, or any time value within 5 - 60 min.

[0087] Specifically, in this step S130, solution A and solution B are respectively placed at the feed inlet of a high gravity machine, and the feed flow rate ratio is set to their volume ratio so that the two solutions are processed simultaneously. The rotation speed of the high gravity machine is set to any value within 1000 - 2500 r. A solution is obtained at the discharge outlet, then centrifuged, washed with water, and dispersed in ultrapure water and stored at 4°C.

[0088] According to the solution of the embodiment of the present invention, by modifying to generate nHAP, its biocompatibility can be good, stable and amorphous. And by adopting the high gravity method of separately placing the said solution A and the said solution B in the high gravity machine for treatment, nanoparticles can be generated quickly.

[0089] This step S200 is to prepare a W / O / W emulsion by the double emulsion method. The mass ratio of the inorganic salt in the inorganic salt solution is 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4% or 5%, or any other value within the range of 0.5 - 5%. The inorganic salt solution can be, for example, sodium chloride solution, ammonium bicarbonate solution or polyethyleneimine solution. The density of nHAP in the inner aqueous phase is, for example, 10 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml or 50 mg / ml, or any other value within 10 - 50 mg / ml. The mass ratio of PLGA in the oil phase is, for example, 1%, 2%, 3%, 4% or 5%, or any other value within the range of 1 - 5%. The mass ratio of PDLLA - PEG - PDLLA in the oil phase is, for example, 1%, 2%, 3%, 4% or 5%, or any other value within the range of 1 - 5%.

[0090] Figure 3 The schematic flowchart showing the method for preparing a W / O emulsion according to an embodiment of the present invention is as Figure 3 shown, and the method for preparing the W / O emulsion includes:

[0091] Step S210, preparing an aqueous solution of polyvinyl alcohol with a mass percentage in the range of 0.5 - 10%;

[0092] Step S220, stirring the oil phase and quickly adding the inner aqueous phase to the oil phase to obtain a W / O emulsion.

[0093] In this step S210, the mass percentage of the polyvinyl alcohol aqueous solution can be, for example, 0.5%, 1%, 2%, 4%, 6%, 8%, 9% or 10%, or any other value within 0.5 - 10%. In this step S220, the oil phase is placed in a homogenizer and stirred. The rotation speed in the homogenizer can be, for example, 3000r, 5000r, 8000r, 10000r or 12000r, or any other value within 3000 - 12000r. The volume ratio of the inner water phase to the oil phase can be, for example, 1:2, 1:4, 1:6, 1:8, 1:9 or 1:10, or any other value within the range of 1:2 - 10.

[0094] Figure 4 FIG. shows a schematic flowchart of a method for preparing a W / O / W emulsion according to an embodiment of the present invention. As Figure 4 shown, the preparation method includes:

[0095] Step S230, stirring a polyvinyl alcohol aqueous solution with a volume 5 - 30 times that of the oil phase;

[0096] Step S240, placing the W / O emulsion into the polyvinyl alcohol aqueous solution to obtain a W / O / W emulsion.

[0097] In this step S230, the volume of the polyvinyl alcohol aqueous solution is 5 times, 10 times, 15 times, 20 times, 25 times or 30 times that of the oil phase, or any other value within 5 - 30 times. The stirring is carried out in a homogenizer, and the stirring speed can be, for example, 10000r, 12000r, 15000r, 18000r, 20000r or 25000r, or any other value within 10000 - 25000r.

[0098] In this step S200, nHAP is dispersed in PLGA microspheres by homogenizer emulsification, which can effectively buffer the acidic environment during the degradation of PLGA microspheres.

[0099] Figure 5 FIG. shows a schematic flowchart of a method for stirring and evacuating a W / O / W emulsion, centrifuging to obtain microspheres, and etching and freeze-drying the microspheres to obtain nHAP-PLGA porous microspheres according to an embodiment of the present invention. As Figure 5 shown, the method includes:

[0100] Step S310, removing the organic reagent from the W / O / W emulsion and washing with water to obtain microsphere particles;

[0101] Step S320, dispersing the microsphere particles in ultrapure water, stirring for 12 - 16h, and performing centrifugation and freeze-drying operations to obtain a solid powder;

[0102] Step S330: Disperse the solid powder in an alkaline etching solution for etching, followed by centrifugation, washing with water, and freeze-drying to obtain nHAP-PLGA porous microspheres.

[0103] In this step S310, the following method can be used to remove the organic reagent in the W / O / W emulsion: quickly pour the W / O / W emulsion into a three-necked flask equipped with a stirring paddle, stir at a stirring speed of 300 - 600 r for 20 - 40 min, and then stir while evacuating for 1 - 4 h to remove the organic reagent. The way of washing with water can be, for example: centrifuge and wash the solution in the flask at a centrifugal speed of 8500 r for 5 - 30 min, and repeat the washing process multiple times.

[0104] In this step S320, the volume of ultrapure water is 1 / 10 - 1 / 2 of the volume of the solution in the three-necked flask. The stirring time can be, for example, 12 h, 14 h, 15 h, or 16 h, or any other value within 12 - 16 h.

[0105] In this step S330, the alkaline etching solution can be, for example, sodium hydroxide etching solution, potassium hydroxide etching solution, sodium carbonate etching solution, or sodium bicarbonate etching solution. The concentration of this alkaline etching solution can be, for example, 0.05 mol / l, 0.1 mol / l, 0.2 mol / l, 0.3 mol / l, 0.4 mol / l, or 0.5 mol / l, or any other value within 0.05 - 0.5 mol / l. The mass percentage of the solid powder in the solution formed by dispersing the solid powder in the alkaline etching solution can be, for example, 0.1%, 0.5%, or 1%, or any other value within 0.1 - 1%. The etching time can be, for example, 4 min, 6 min, 8 min, or 10 min, or any other value within 4 - 10 min.

[0106] In this step S400, the mass ratio of nHAP-PLGA porous microspheres to dopamine can be, for example, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1, or any other value within the range of 5 - 10:1. The buffer solution in this step is tris(hydroxymethyl)aminomethane hydrochloride buffer solution, and the pH value of the buffer solution can be, for example, 8, 8.5, 8.8, or 9, or any other value within the range of 8 - 9. In the solution of nHAP-PLGA porous microspheres, dopamine, and buffer solution, the mass fraction of nHAP-PLGA porous microspheres is 0.1%, 0.5%, 0.8%, or 1%, or any other value within 0.1 - 1%.

[0107] In step S400, before dissolving nHAP-PLGA porous microspheres and dopamine in a buffer solution, and before stirring, centrifuging, and freeze-drying to obtain PDA-modified nHAP-PLGA porous microspheres, the following steps are further included: stirring and reacting in the dark for 12-16 h, and performing water washing. The time for stirring and reacting in the dark can be, for example, 12 h, 13 h, 15 h, or 16 h, or any other value within 12-16 h. The method of water washing is centrifugal water washing, and it can be washed multiple times.

[0108] In step S500, the pH value of the phosphate buffer solution can be, for example, 7, 7.2, 7.5, 7.8, or 8, or any other value within the range of 7-8. In the solution of PDA-modified nHAP-PLGA porous microspheres and the phosphate buffer solution, the mass fraction of PDA-modified nHAP-PLGA porous microspheres can be, for example, 1%, 3%, 5%, 7%, 9%, or 10%, or any other value within the range of 1-10%. The rhTNFR:Fc solution is a solution of phosphate buffer containing rhTNFR:Fc. In the rhTNFR:Fc solution, the mass percentage of rhTNFR:Fc can be, for example, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%, or any other value within the range of 0.05-0.5%. In the rhTNFR:Fc solution, the pH value of the phosphate buffer solution can be, for example, 7, 7.2, 7.5, 7.8, or 8, or any other value within the range of 7-8.

[0109] In this step S500, before freeze-drying, the following steps are further included: stirring and reacting in the dark for a preset time at a preset temperature, and performing water washing. The preset temperature can be, for example, 30 °C, 34 °C, 38 °C, or 40 °C, or any other value within the range of 30-40 °C. The preset time can be, for example, 12 h, 14 h, 15 h, or 16 h, or any other value within the range of 12-16 h.

[0110] Figure 6 Shows the Fourier infrared spectrogram of nano-hydroxyapatite according to an embodiment of the present invention. As Figure 6 shown, 3500 cm -1 and 1600 cm -1 are the characteristic absorption peaks of OH - , 600 cm -1 and 1100 cm -1 are the characteristic absorption peaks of PO4 3- , indicating the successful synthesis of hydroxyapatite. Figure 7 Shows the XRD pattern of nano-hydroxyapatite according to an embodiment of the present invention. From Figure 7It can be seen that there is a large peak around 25°, indicating that nHAP is amorphous. Figure 8 The transmission electron microscope image of nano-hydroxyapatite according to an embodiment of the present invention is shown. Figure 8 It can be seen that the nano-hydroxyapatite is spherical particles with a particle size of about 10 nm.

[0111] Figure 9 The scanning electron microscope image of the microspheres obtained by freeze-drying the W / O / W emulsion is shown. Figure 10 The scanning electron microscope image of nHAP-PLGA porous microspheres according to an embodiment of the present invention is shown. Figure 11 The scanning electron microscope image of PDA-modified nHAP-PLGA porous microspheres according to an embodiment of the present invention is shown. Figures 9 to 11 It can be seen that after surface etching with sodium hydroxide, a porous structure is formed on the surface of the microspheres. Then, after in-situ surface polymerization modification with PDA, the surface becomes rough, which is due to the uneven polymerization of dopamine resulting in local particle sense.

[0112] Figure 12 The Fourier transform infrared spectra of dopamine, nHAP-PLGA porous microspheres, and PDA-modified nHAP-PLGA porous microspheres are shown. In the figure, 1 represents dopamine, 2 represents nHAP-PLGA porous microspheres, and 3 represents PDA-modified nHAP-PLGA porous microspheres. It can be seen from the comparison in the figure that at 3300 cm -1 and 1550 cm -1 After modification with dopamine, new peaks appear in the nHAP-PLGA microspheres, which correspond to the O-H peak and N-H peak of dopamine, indicating that PDA has been successfully modified on nHAP-PLGA.

[0113] Figure 13 The pH change curve graph of the solutions of nHAP-PLGA porous microspheres according to an embodiment of the present invention and PLGA microspheres without nHAP composite in the comparative example after accelerated degradation at 70°C is shown. Figure 13 It can be seen that on the 3rd, 5th, and 7th days, the pH of the nHAP-PLGA group is significantly higher than that of the PLGA group, proving the effective buffering effect of nHAP.

[0114] Figure 14 The cumulative release curve comparison graph of injectable drug sustained-release microspheres without dopamine modification and rhTNFR:Fc-PLGA injectable drug sustained-release microspheres according to an embodiment of the present invention within 15 days is shown. Figure 15 shows Figure 14 the curve graph in the first 12 hours. Figure 14 and Figure 15It can be seen that the burst release effect of the modified PDA microspheres is eliminated, and their sustained release effect is better.

[0115] Figure 16 The comparative curve graph of the pharmacokinetic results of rhTNFR:Fc raw drug and rhTNFR:Fc-PLGA sustained release microspheres in rats is shown. From Figure 16 It can be known that after loading rhTNFR:Fc on PLGA microspheres, the burst release effect of the raw drug can be eliminated and the long-acting sustained release effect can be maintained.

[0116] Figure 17 The picture after staining the sections of the main organs of rats is shown when rhTNFR:Fc-PLGA injectable drug sustained release microspheres according to an embodiment of the present invention are used to treat collagen-induced rheumatoid arthritis (CIA) rats. Figure 17 Among them, the Sham group represents healthy rats, the CIA group represents rats with rheumatoid arthritis without drug treatment, the rhTNFR:Fc group represents rats with rheumatoid arthritis treated with the raw drug rhTNFR:Fc, and the rhTNFR:Fc-PLGA group represents rats with rheumatoid arthritis treated with rhTNFR:Fc-PLGA. Scale bar = 100μm. From Figure 18 It can be seen that there is no abnormality in the main organs of the drug administration group, indicating the biosafety of rhTNFR:Fc-PLGA sustained release microspheres.

[0117] Figure 18 The level of TNF-α in the serum of rats is shown after injecting rhTNFR:Fc-PLGA microspheres. Figure 19 The level of IL-6 in the serum of rats is shown after injecting rhTNFR:Fc-PLGA microspheres. Figure 20 The level of IL-1β in the serum of rats is shown after injecting rhTNFR:Fc-PLGA microspheres. Figure 21 shows the level of TNF-α in the homogenate of rat synovial tissue after injecting rhTNFR:Fc-PLGA microspheres. Figure 22 The level of IL-6 in the homogenate of rat synovial tissue is shown after injecting rhTNFR:Fc-PLGA microspheres. Figure 23 The level of IL-1β in the homogenate of rat synovial tissue is shown after injecting rhTNFR:Fc-PLGA microspheres. From Figures 18 to 23 It can be known that after treatment with rhTNFR:Fc-PLGA, the levels of TNF-α, IL-6 and IL-1β in rats are all lower than those in the rhTNFR:Fc group, indicating that after loading rhTNFR:Fc with the microspheres prepared by us, the treatment effect has been significantly improved.

[0118] To study the effect of dopamine obtained in the experiment, a comparative experiment on the drug loading capacity and encapsulation efficiency of nHAP-PLGA porous microspheres and the PDA-modified nHAP-PLGA porous microspheres (labeled as nHAP-PLGA-PDA) obtained in step S400 was carried out. Table 1 below shows the drug loading capacity and encapsulation efficiency of nHAP-PLGA porous microspheres and nHAP-PLGA-PDA microspheres. For each type of microsphere, three drug loading experiments were conducted, and thus three drug loading capacities and encapsulation efficiencies were obtained for each microsphere.

[0119]

[0120] As can be seen from Table 1, the drug loading capacity and encapsulation efficiency of the PDA-modified microspheres have been significantly improved under the same conditions.

[0121] The following will be described in detail by way of specific embodiments:

[0122] Example 1:

[0123] The embodiment of the present invention provides a method for preparing a drug sustained-release microsphere, including the preparation of nHAP, the preparation of nHAP-PLGA porous microspheres, and the preparation of rhTNFR:Fc-PLGA injectable drug sustained-release microspheres.

[0124] The preparation of nHAP includes the following steps: Dissolve 2.220 g of calcium chloride in 200 ml of a pH = 7.6, 1 mM Tris solution, dissolve 0.15 g of PASP solution in 20 ml of a pH = 7.6, 1 mM Tris solution, dissolve 7.163 g of disodium hydrogen phosphate dodecahydrate in 200 ml of a pH = 7.1, 50 mM HEPES salt solution, and dissolve 0.3 g of PAA in 40 ml of a pH = 7.1, 50 mM HEPES salt solution; Slowly drop the PASP solution into the calcium chloride solution and chelate for 30 minutes with a volume ratio of 1:10 to obtain solution A; Slowly drop the PAA solution into the disodium hydrogen phosphate dodecahydrate solution and chelate for 30 minutes with a volume ratio of 1:5 to obtain solution B; Place solutions A and B at the inlets of a high gravity machine respectively, with a feed flow rate ratio of 11:12, adjust the rotation speed to 2500 r / min, obtain a solution at the outlet, then centrifuge and wash three times with 8500 r for 10 min, and disperse in ultrapure water and store at 4°C.

[0125] The preparation of nHAP-PLGA porous microspheres includes the following steps: Disperse nHAP in 0.9% sodium chloride solution with an nHAP content of 30 mg / ml to obtain the aqueous phase; dissolve 0.5 g of PLGA and 0.5 g of PDLLA-PEG-PDLLA in 20 ml of ethyl acetate to obtain the oil phase; prepare a 2% PVA aqueous solution to obtain another aqueous phase; stir the oil phase with a homogenizer, and after reaching 5000 r, quickly add the aqueous phase to the oil phase. After 3 min, stop stirring to obtain a water-in-oil emulsion; then quickly place 400 ml of 1% PVA aqueous solution under the homogenizer. After reaching 10,000 r, pour the previous water-in-oil emulsion into the 2% PVA aqueous solution. After a period of time, a water-in-oil-in-water emulsion is obtained, which is quickly poured into a three-necked flask and stirred at 400 r for 30 min, and then stirred while evacuating for 2 h to remove the organic reagent; centrifuge the solution in the flask at 8500 r for 10 min and wash three times with water, disperse it in 100 ml of ultrapure water, stir for 12 h, centrifuge and then freeze-dry; weigh 200 mg of the dried microspheres and disperse them in 40 ml of 0.3 M sodium hydroxide solution, stir at 500 r on a stirring table for 8 min, and centrifuge and wash three times with water at 8500 r for 10 min, and then freeze-dry to obtain PLGA porous microspheres.

[0126] The preparation of rhTNFR:Fc-PLGA injectable drug sustained-release microspheres includes the following steps: Dissolve 100 mg of PLGA porous microspheres and 20 mg of dopamine in 20 ml of Tris-HCl solution with a pH of 8.5, stir and react in the dark for 12 h, then centrifuge and wash three times with water at 8500 r for 20 min and freeze-dry; weigh 5 mg of the dried microspheres and disperse them in 500 μl of 1×PBS solution with a pH of 7.8, and add 500 μl of 1×PBS solution containing 1 mg of rhTNFR:Fc with a pH of 7.8 to it, stir and react at 37 °C in the dark at 1000 r for 12 h, then centrifuge and wash three times with water at 10000 r for 5 min, and freeze-dry the centrifuged microspheres to obtain rhTNFR:Fc-PLGA porous microspheres.

[0127] Example 2:

[0128] The difference between the second embodiment and the first embodiment lies in the different preparation of the rhTNFR:Fc-PLGA injectable drug sustained-release microspheres. In this second embodiment, the preparation of the rhTNFR:Fc-PLGA injectable drug sustained-release microspheres includes the following steps: Dissolve 100 mg of PLGA porous microspheres and 10 mg of dopamine in 10 ml of Tris-HCl solution with a pH of 8.5, stir and react for 12 h in the dark, then centrifuge three times with water washing at 8500 r for 10 min, and freeze-dry; Weigh 5 mg of the dried microspheres and disperse them in 500 μl of 1×PBS solution with a pH of 7.8, and add 500 μl of 1×PBS solution containing 1 mg of rhTNFR:Fc to it, stir and react at 37 °C in the dark at 1000 r for 12 h, then centrifuge three times with water washing at 10000 r for 5 min, and freeze-dry the microspheres obtained by centrifugation to obtain rhTNFR:Fc-PDA-PLGA porous microspheres.

[0129] Example 3:

[0130] The difference between the third embodiment and the first embodiment lies in the different preparation of the nHAP-PLGA porous microspheres and the rhTNFR:Fc-PLGA injectable drug sustained-release microspheres. In this third embodiment, the preparation of the nHAP-PLGA porous microspheres includes the following steps: Disperse nHAP in 0.5% wt sodium chloride solution with an nHAP content of 40 mg / ml to obtain an aqueous phase; Dissolve 0.5 g of PLGA and 0.5 g of PDLLA-PEG-PDLLA in 20 ml of ethyl acetate to obtain an oil phase; Prepare a 1% PVA aqueous solution to obtain another aqueous phase; Stir the oil phase under a homogenizer, and when it reaches 7500 r, quickly add 5 ml of the aqueous phase to the oil phase. After 2 min, stop stirring to obtain a water-in-oil emulsion; Then quickly place 200 ml of 1% PVA aqueous solution under the homogenizer, and when it reaches 15000 r, pour the previous water-in-oil emulsion into the 1% PVA aqueous solution. After a period of time, obtain a water-in-oil-in-water emulsion, quickly pour it into a three-necked flask and stir at 400 r for 20 min, then stir while evacuating for 1 h to remove the organic reagent; Centrifuge and wash the solution in the flask three times with water at 8500 r for 10 min, disperse it in 50 ml of ultrapure water, stir for 12 h, centrifuge and then freeze-dry; Weigh 200 mg of the dried microspheres and disperse them in 40 ml of 0.2 M sodium hydroxide solution, stir at 500 r on a stirring table for 5 min, and centrifuge and wash three times with water at 8500 r for 5 min, and freeze-dry to obtain PLGA porous microspheres.

[0131] The preparation of rhTNFR:Fc-PLGA injectable sustained-release microspheres comprises the following steps: Dissolve 100 mg of PLGA porous microspheres and 20 mg of dopamine in 20 ml of Tris-HCl solution with pH = 8.5, stir and react in the dark for 12 h, then centrifuge at 8500 r for 20 min, wash three times with water, and freeze-dry; Weigh 3 mg of the dried microspheres and disperse them in 500 μl of 1×PBS solution with pH = 7.8, and add 500 μl of 1×PBS solution containing 1 mg of rhTNFR:Fc with pH = 7.8 thereto, stir and react at 37 °C in the dark at 1000 r for 12 h, then centrifuge at 10000 r for 5 min, wash three times with water, and freeze-dry the microspheres obtained by centrifugation to obtain rhTNFR:Fc-PLGA porous microspheres.

[0132] Example 4:

[0133] The difference between this Example 4 and Example 1 lies in the preparation of nHAP and the preparation of nHAP-PLGA porous microspheres. In this Example 4, the preparation of nHAP comprises the following steps: Dissolve 1.110 g of calcium chloride in 100 ml of 1 mM Tris solution with pH = 7.6, dissolve the solution containing 0.15 g of PASP in 10 ml of 1 mM Tris solution with pH = 7.6, dissolve 2.149 g of disodium hydrogen phosphate dodecahydrate in 100 ml of 50 mM HEPES salt solution with pH = 7.1, dissolve 0.3 g of PAA in 20 ml of 50 mM HEPES salt solution with pH = 7.1; Slowly drop the PASP solution into the calcium chloride solution, chelate for 30 minutes, with a volume ratio of 1:10, to obtain Solution A; Slowly drop the PAA solution into the disodium hydrogen phosphate dodecahydrate solution, chelate for 30 minutes, with a volume ratio of 1:5, to obtain Solution B; Place Solutions A and B at the inlets of a high gravity machine respectively, with a feed flow rate ratio of 11:12, adjust the rotation speed to 2500 r / min, obtain a solution at the outlet, then centrifuge at 8500 r for 10 min, wash three times with water, and disperse in ultrapure water and store at 4 °C.

[0134] The preparation of nHAP-PLGA porous microspheres includes the following steps: Disperse nHAP in 0.9% sodium chloride solution with an nHAP content of 50 mg / ml to obtain the aqueous phase; dissolve 0.5 g of PLGA and 0.5 g of PDLLA-PEG-PDLLA in 20 ml of ethyl acetate to obtain the oil phase; prepare 1% PVA aqueous solution to obtain another aqueous phase; stir the oil phase with a homogenizer, and after reaching 7500 r, quickly add the aqueous phase to the oil phase. After 2 min, stop stirring to obtain a water-in-oil emulsion; then quickly place 200 ml of 1% PVA aqueous solution under the homogenizer, and after reaching 15000 r, pour the previous water-in-oil emulsion into the 1% PVA aqueous solution. After a period of time, obtain a water-in-oil-in-water emulsion, quickly pour it into a three-neck flask and stir at 400 r for 30 min, then stir while evacuating for 4 h to remove the organic reagent; centrifuge and wash the solution in the flask three times at 8500 r for 10 min, disperse it in 50 ml of ultrapure water, stir for 12 h, centrifuge and then freeze-dry; weigh 200 mg of the dried microspheres and disperse them in 40 ml of 0.2 M sodium hydroxide solution, stir at 500 r on a stirring platform for 10 min, and centrifuge and wash three times at 8500 r for 10 min, then freeze-dry to obtain PLGA porous microspheres.

[0135] Example Five:

[0136] The difference between this Example Five and Example One lies in the different preparation of nHAP. In this Example Five, the preparation of nHAP includes the following steps: Dissolve 2.220 g of calcium chloride in 200 ml of pH = 7.6, 1 mM Tris solution, dissolve 0.15 g of PASP solution in 20 ml of pH = 7.6, 1 mM Tris solution, dissolve 7.163 g of disodium hydrogen phosphate dodecahydrate in 200 ml of pH = 7.1, 50 mM HEPES salt solution, and dissolve 1 g of PAA in 40 ml of pH = 7.1, 50 mM HEPES salt solution; slowly drop the PASP solution into the calcium chloride solution and chelate for 60 minutes with a volume ratio of 1:10 to obtain Solution A; slowly drop the PAA solution into the disodium hydrogen phosphate dodecahydrate solution and chelate for 60 minutes with a volume ratio of 1:5 to obtain Solution B; place Solutions A and B at the inlets of a high-gravity machine respectively, with a feed flow rate ratio of 11:12, adjust the rotation speed to 2000 r / min, obtain a solution at the outlet, then centrifuge and wash three times at 8500 r for 10 min, and disperse it in ultrapure water and store it at 4°C.

[0137] At this point, those skilled in the art should recognize that although numerous exemplary embodiments of the present invention have been shown and described in detail herein, many other variations or modifications consistent with the common principles of the present invention can still be directly determined or derived from the content disclosed in the present invention without departing from the spirit and scope of the present invention. Therefore, the scope of the present invention should be understood and determined to cover all such other variations or modifications.

Claims

1. A preparation method of a drug sustained-release microsphere, characterized in that It includes the following steps: Provide amorphous nano-hydroxyapatite (nHAP); Disperse the nHAP in an inorganic salt solution as the internal aqueous phase, dissolve PLGA and PDLLA-PEG-PDLLA in ethyl acetate as the oil phase, emulsify to prepare a W / O emulsion, and then emulsify the W / O emulsion and a polyvinyl alcohol solution to obtain a W / O / W emulsion; Stir and evacuate the W / O / W emulsion, centrifuge to obtain microspheres, and etch and freeze-dry the microspheres to obtain nHAP-PLGA porous microspheres; Dissolve the nHAP-PLGA porous microspheres and dopamine in a buffer solution, stir, centrifuge and freeze-dry to obtain PDA-modified nHAP-PLGA porous microspheres; Disperse the PDA-modified nHAP-PLGA porous microspheres in a phosphate buffer solution, and apply a solution of recombinant human type II tumor necrosis factor receptor antibody fusion protein (rhTNFR:Fc), and freeze-dry to obtain rhTNFR:Fc-PLGA injectable drug sustained-release microspheres; The mass ratio of PLGA in the oil phase is any value in the range of 1-5%, and the mass ratio of PDLLA-PEG-PDLLA in the oil phase is any value in the range of 1-5%; In the step of dissolving the nHAP-PLGA porous microspheres and dopamine in a buffer solution, stirring, centrifuging and freeze-drying to obtain PDA-modified nHAP-PLGA porous microspheres, the mass ratio of the nHAP-PLGA porous microspheres to the dopamine is any value in the range of 5-10:

1.

2. The preparation method according to claim 1, wherein In the solution of the nHAP-PLGA porous microspheres, dopamine and buffer solution, the mass fraction of the nHAP-PLGA porous microspheres is any value in the range of 0.1-1%.

3. The preparation method according to claim 2, wherein The buffer solution is tris(hydroxymethyl)aminomethane hydrochloride buffer solution, and the pH value of the buffer solution is any value in the range of 8-9.

4. The preparation method according to claim 1, characterized in that, In the step of dissolving the nHAP-PLGA porous microspheres and dopamine in a buffer solution, stirring, centrifuging and freeze-drying to obtain PDA-modified nHAP-PLGA porous microspheres, after dissolving the nHAP-PLGA porous microspheres and dopamine in the buffer solution and before stirring, centrifuging and freeze-drying to obtain PDA-modified nHAP-PLGA porous microspheres, the following steps are further included: stir and react in the dark for 12-16 h and perform water washing.

5. The preparation method according to claim 1, characterized in that, In the step of dispersing the PDA-modified nHAP-PLGA porous microspheres in a phosphate buffer solution, the pH value of the phosphate buffer solution is any value in the range of 7-8; In the solution of the PDA-modified nHAP-PLGA porous microspheres and the phosphate buffer solution, the mass fraction of the PDA-modified nHAP-PLGA porous microspheres is any value in the range of 1-10%. In the step of dispersing the PDA-modified nHAP-PLGA porous microspheres in phosphate buffer solution and adding a solution of recombinant human type II tumor necrosis factor receptor antibody fusion protein (rhTNFR:Fc), the rhTNFR:Fc solution is a solution of phosphate buffer solution containing rhTNFR:Fc. In the rhTNFR:Fc solution, the mass percentage of rhTNFR:Fc is any value in the range of 0.05 - 0.5%.

6. The preparation method according to claim 5, wherein in the rhTNFR:Fc solution, the pH value of the phosphate buffer solution is any value in the range of 7 - 8.

7. The preparation method according to claim 6, wherein in the step of dispersing the PDA-modified nHAP-PLGA porous microspheres in phosphate buffer solution, adding a solution of recombinant human type II tumor necrosis factor receptor antibody fusion protein (rhTNFR:Fc), and obtaining rhTNFR:Fc-PLGA injectable drug sustained-release microspheres after freeze-drying, the following steps are further included before freeze-drying: stirring and reacting in the dark at a preset temperature for a preset time, and performing water washing; the preset temperature is any value in the range of 30 - 40 °C, and the preset time is any value in the range of 12 - 16 h.

8. The preparation method according to any one of claims 1-5, characterized in that, The steps of stirring and evacuating the W / O / W emulsion, centrifuging to obtain microspheres, and etching and freeze-drying the microspheres to obtain nHAP-PLGA porous microspheres include the following steps: Removing the organic reagent in the W / O / W emulsion and performing water washing to obtain microsphere particles; Dispersing the microsphere particles in ultrapure water, stirring for 12 - 16 h, and performing centrifugal freeze-drying operation to obtain a solid powder; Dispersing the solid powder in an alkaline etching solution for etching, centrifuging and washing with water, and obtaining the nHAP-PLGA porous microspheres after freeze-drying.

9. The preparation method according to any one of claims 1-5, characterized in that, In the step of dispersing the nHAP in an inorganic salt solution as the internal aqueous phase, dissolving PLGA and PDLLA-PEG-PDLLA in ethyl acetate as the oil phase, emulsifying to prepare a W / O emulsion, and then emulsifying the W / O emulsion and a polyvinyl alcohol solution to obtain a W / O / W emulsion, the mass ratio of the inorganic salt in the inorganic salt solution is any value in the range of 0.5 - 5%, and the density of nHAP in the internal aqueous phase is any value in the range of 10 - 50 mg / ml.

10. The preparation method according to claim 9, characterized in that, The method for preparing a W / O emulsion includes the following steps: Preparing an aqueous polyvinyl alcohol solution with a mass percentage in the range of 0.5 - 10%; Stirring the oil phase and quickly adding the internal aqueous phase to the oil phase to obtain the W / O emulsion.

11. The preparation method according to claim 10, wherein In the method for preparing a W / O emulsion, the volume ratio of the internal aqueous phase to the oil phase is any value in the range of 1:2 - 10.

12. The preparation method according to claim 9, characterized in that, The method for preparing a W / O / W emulsion includes the following steps: Stirring an aqueous polyvinyl alcohol solution with a volume 5 - 30 times that of the oil phase; Placing the W / O emulsion into the aqueous polyvinyl alcohol solution to obtain the W / O / W emulsion.

13. The preparation method according to any one of claims 1-5 and 10-12, characterized in that, In the step of providing amorphous nano-hydroxyapatite (nHAP), the preparation method of the nHAP comprises the following steps: Applying a polyaspartic acid (PASP) solution to a calcium salt solution, and performing a chelation reaction to obtain solution A; Applying a polyacrylic acid (PAA) solution to a disodium hydrogen phosphate dodecahydrate solution, and performing a chelation reaction to obtain solution B; Respectively placing solution A and solution B into a high gravity machine for treatment, followed by centrifugation and water washing to obtain the nHAP.

14. The preparation method according to claim 13, wherein In solution A, the volume ratio of the PASP solution to the calcium salt solution is any value within the range of 1:5 - 25; In solution B, the volume ratio of the PAA solution to the disodium hydrogen phosphate dodecahydrate solution is any value within the range of 1:5 - 25.

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