Microfluidic preparation method of leuprorelin acetate microspheres

Abstract

The invention relates to a microflow control preparation method of leuprorelin acetate microspheres, which comprises the following steps: (1) dissolving a hydrophilic-hydrophobic polymer in an organic solvent, adding a leuprorelin acetate aqueous solution, stirring, evaporating and self-assembling for 30 + / -15 minutes to form a W / O type polymer microemulsion; (2) adding the polymer microemulsion into the pregel component, stirring, mixing and volatilizing for 1-2 hours, and removing the organic solvent to form a microgel precursor; and (3) taking the solution in the step (2) as a dispersion phase A, the pre-gel component as a dispersion phase B and fluorinated oil containing 2% of a surfactant Pico-Surf as a continuous phase to be connected into a micro-droplet generation system to generate monodispersed micro-droplets, and performing cross-linking curing, washing out and freeze-drying to obtain the leuprorelin acetate microspheres. The prepared microspheres are uniform in size and good in quality consistency, have the advantages of being high in encapsulation efficiency and drug loading capacity, controllable in drug sustained release, free of obvious burst release and the like, and are more suitable for meeting clinical requirements.

Description

Technical Field

[0001] The invention relates to the field of chemical medicine microsphere preparations and preparation thereof, and in particular to a microfluidic preparation method of leuprorelin acetate microspheres. Background Art

[0002] Leuprorelin acetate is the first synthetic active analog of luteinizing hormone-releasing hormone (LH-RH). It contains 9 amino acids and can be used to treat sex hormone-dependent diseases such as prostate cancer, endometriosis, uterine fibroids and central precocious puberty. However, leuprorelin acetate is easily degraded by various digestive enzymes in the gastrointestinal tract and body fluids, and has a short half-life in the body and low bioavailability, so it is not suitable for oral preparations. Microsphere preparations are made by dissolving or dispersing drugs in a polymer matrix to form microsphere entities with smaller particle sizes. The effect of long-term and stable drug release can be achieved by adjusting various parameters of the microspheres, such as concentration, polymer type and solvent type. Among them, Takeda Chemical Pharmaceutical Company of Japan used degradable polymers as carrier materials in 1989 to develop the first leuprorelin acetate injection microspheres that can achieve a 1-month sustained release effect, and was approved for marketing by the US Food and Drug Administration (FDA). Subsequently, global biopharmaceutical development companies such as Abbvie in the United States have successively developed various types of sustained-release microsphere preparations.

[0003] At present, there are many methods for preparing microspheres, and the best method needs to be selected according to the carrier material, the properties of the drug and the actual needs. The most common methods for preparing microspheres in China include emulsification-solvent evaporation method and spray drying method. The emulsification method is to dissolve the raw materials and auxiliary materials in two immiscible solvents respectively, and then form emulsion droplets by stirring or ultrasonic emulsification. Finally, the solvent evaporates and solidifies into microspheres. This method is simple to operate, but it is easily affected by the physical and chemical properties of the encapsulated drug. The inkjet drying method can directly spray the solution mixed with the drug and the carrier polymer into an inert gas through a sprayer to form countless small droplets, and control the temperature to evaporate quickly into balls, but it is not suitable for preparing protein polypeptide microspheres that are easily deformed at high temperatures. The above-mentioned traditional drug-loaded microspheres require continuous mechanical stirring or oscillation to achieve droplet dispersion during preparation. Due to the instability of external forces, the droplet size distribution, drug content and bioactive components are greatly affected. Therefore, it is necessary to find a new method that can make up for the defects of traditional drug-loaded microspheres.

[0004] Microfluidics is a technology that can precisely manipulate microscale fluids. In micron-scale channels, the interaction of fluid shear force and surface tension between two or more immiscible fluids can be used to cut and form droplets ranging in size from nanoliters to picoliters. The particle size of the microspheres can be effectively controlled by adjusting the flow rates of the continuous and dispersed phases. Compared with conventional preparation methods, the microspheres prepared based on microfluidics are highly uniform and monodisperse, which can effectively reduce the differences between batches. With the in-depth research on biomedical materials, high-consistency drug carriers prepared in combination with microfluidics have become a research trend in microsphere preparations. With the biocompatibility, biodegradability and easy molding characteristics of the material, it is expected to better achieve drug controlled release, which is of great significance for controlling the stability of blood drug concentrations in the body, reducing toxic side effects, and improving drug utilization. Summary of the invention

[0005] The purpose of the present invention is to provide a leuprorelin acetate microsphere with high consistency, uniform particle size and small toxic and side effects. The invention can solve the problems of low drug encapsulation rate and uncontrollable size components, thereby improving drug utilization and achieving controlled sustained release of drugs.

[0006] The technical solution of the present invention is a microfluidic preparation method of leuprorelin acetate microspheres.

[0007] (1) dissolving a hydrophilic-hydrophobic polymer in an organic solvent, adding a leuprorelin acetate aqueous solution, stirring, and evaporating for self-assembly for 30 minutes to form a W / O type polymer microemulsion;

[0008] (2) adding the polymer microemulsion to the pre-gel component, stirring and mixing and volatilizing for 1-2 hours to remove the organic solvent to form a microgel precursor;

[0009] (3) The solution of (2) is used as dispersed phase A, the pre-gel component is used as dispersed phase B, and the fluorinated oil containing 2% of the surfactant Pico-Surf is used as the continuous phase to connect to the microdroplet generation system to generate monodisperse microdroplets, which are then cross-linked and solidified, washed out, and freeze-dried to obtain leuprolide acetate microspheres;

[0010] Preferably, the hydrophilic-hydrophobic polymer in step (1) includes one or more of polyethylene glycol-polylactic acid-co-glycolic acid (PEG-PLGA), polyethylene glycol-polyurethane block polymer (PEG-PEUPE), and polyethyleneimine-polylactic acid block copolymer (PEI-PLGA), and the organic solvent includes but is not limited to dichloromethane, acetonitrile, methanol, acetone and chloroform, and the stirring rate is 600-1000 rpm.

[0011] Preferably, the concentration of leuprolide acetate in the polymer microemulsion of step (1) is 1-1000 mg / mL, and the mass volume ratio (mg:μL) of the hydrophilic-hydrophobic polymer to the organic solvent is 1-20:100; in the embodiment, the total volume is 0.5-2 mL.

[0012] Preferably, the pre-gel component in step (2) includes one or more of methacrylated gelatin, sodium alginate, hyaluronic acid, silk fibroin, chitosan, dextran, chondroitin sulfate, polylysine; polyether F127 diacrylate; hyperbranched polyethylene glycol diacrylate (HB-PEG) and thiolated hyaluronic acid (SH-HA); four-arm polyethylene glycol acrylate, the degree of methacrylation is 30%-70%, and the mixing rate is 600-1500rpm.

[0013] Preferably, the concentration of the pre-gel component in step (2) is 5%-20%, and the solvent is water or PBS; wherein the volume ratio of the polymer microemulsion to the pre-gel component is 1-5:10.

[0014] Preferably, the microdroplet generation system in step (3) is composed of a syringe pump, a microfluidic chip and a high-speed camera, wherein the microfluidic chip includes a PDMS microfluidic chip, a capillary microfluidic chip and an injection-molded microfluidic chip.

[0015] Preferably, in step (3), the volume ratio of dispersed phase A to dispersed phase B is 1-5:1, the concentration of dispersed phase B is 2%-20%, and the solvent is water or PBS; the size of the generated monodisperse droplets is 1-1000 μm.

[0016] Preferably, the cross-linking and curing method in step (3) includes one or more of Michael addition, photocuring covalent cross-linking, and physical cross-linking.

[0017] Preferably, the photoinitiator in step (3) is 2959 or LAP, and the concentration of the photoinitiator is 0.1%-0.5%.

[0018] The leuprorelin acetate microspheres of the present invention are first formed into W / O type emulsion droplets by an emulsification stirring method, and after the amphiphilic polymer volatilizes and self-assembles for a certain period of time (the W / O type emulsion droplet system is stable), they are mixed with a pre-gel solution, and a monodisperse microsphere is formed by a micro-droplet generation system for solidification and cross-linking. Wherein, the pre-gel solution is selected from a naturally modified biocompatible material, which has no obvious cytotoxicity, and by adjusting the concentration of the material and the content of the functionalized group, it can have a certain controllable degradation characteristic, while ensuring the effective coating of the drug during the preparation process, and also greatly improving the bioavailability of the drug. Further, the monodisperse drug-loaded microspheres prepared by microfluidics can avoid the sudden release of the drug, and the shape and structure are controllable, which improves the batch consistency of the drug-loaded microspheres, and is more conducive to the production of high-quality, high-stability and high-value-added new drug-loaded microsphere preparations.

[0019] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0020] (1) The leuprorelin acetate microspheres of the present invention have uniform particle size, controllable size, and are monodisperse, which can effectively reduce toxic and side effects and ensure the quality stability of the microsphere preparation;

[0021] (2) After forming the primary W / O type drug-loaded emulsion droplets, the present invention also mixes a curable cross-linked pre-gel solution in the outer layer, which greatly improves the drug loading and encapsulation efficiency. By adjusting various parameters of the pre-gel solution, the controlled sustained release of the microspheres can be achieved;

[0022] (3) The preparation method provided by the present invention has mild conditions, and the selected biocompatible materials can maintain the high activity of bioactive drugs such as peptides and proteins, and is expected to be applied to more complex drug microsphere preparation systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the preparation process of leuprorelin acetate microspheres;

[0024] Figure 2 Schematic diagram of chemical synthesis and cross-linking curing of methacryloyl gelatin;

[0025] Figure 3 This is a real picture of the microdroplet generation system; the left photo corresponds to the process of preparing leuprore acetate microspheres by microfluidics, and the two photos on the right are both leuprore acetate microspheres;

[0026] Figure 4 is the particle size distribution diagram of microdroplets;

[0027] Figure 5 is the live-dead fluorescence intensity distribution diagram of the microgel;

[0028] Figure 6 To evaluate the cytotoxicity of microgels;

[0029] Figure 7 is the standard curve of leuprolide acetate HPLC;

[0030] Figure 8 This is a physical picture of the degradation of leuprorelin acetate microspheres over time; the four pictures correspond to 0, 3, 5, and 10 minutes respectively;

[0031] Fig. 9 For the NMR spectrum. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0033] Embodiment (1)

[0034] like Figure 1 As shown, the microfluidic preparation of leuprorelin acetate microspheres mainly includes three steps: preparation of polymer microemulsion, formation of microgel precursor and microdroplet generation system. The specific preparation method is as follows:

[0035] 1. Preparation of polymer microemulsion

[0036] First, 10 mg of PEG-PLGA was weighed and mixed with 1 mL of dichloromethane as solution A, and 2 mg of leuprorelin acetate was weighed and dissolved in 1 mL of water as solution B. At 1000 rpm, solution B was slowly added to solution A for stirring and emulsification to form a W / O type polymer microemulsion, which was evaporated and self-assembled at room temperature for 30 minutes.

[0037] 2. Formation of Microgel Precursors

[0038] 2.1 Preparation of methacryloyl gelatin

[0039] Weigh 1 g of gelatin and dissolve it in 10 mL of aqueous solution (10% w / v) at 50°C. After it is completely dissolved, slowly add 0.6 g of methacrylic anhydride (MA) and react for 1 hour. The reaction product is diluted with deionized water (1:5) and dialyzed with deionized water at 40°C for 24 hours. After dialyzing three times, it is freeze-dried and stored.

[0040] The NMR spectrum of the prepared methacryloyl gelatin is shown in Fig. 9 As shown: Compared with the gelatin raw material, the synthesized methacryloyl gelatin contains two characteristic peaks of double bonds at 5.5ppm, which characterizes the successful synthesis of the material. Among them, the methacryloyl content is 68% calculated based on the peak area of ​​the chemical group.

[0041] 2.2 Experimental procedures

[0042] Weigh 400 mg of freeze-dried methacryloyl gelatin and dissolve it in 4 mL of water at 37°C. Continue to add 10 mg of photoinitiator LAP until it is completely dissolved to prepare a 10 wt% pre-gel solution. Slowly add the above polymer microemulsion into the pre-gel solution at 37°C and 600 rpm and continue stirring for 1 hour.

[0043] 3 Construction of microdroplet generation system

[0044] Weigh 200 mg of freeze-dried methacryloyl gelatin and dissolve it in 2 mL of water at 37°C. Continue to add 5 mg of photoinitiator LAP until it is completely dissolved to prepare a 10 wt% pre-gel solution, wherein the prepared pre-gel solution is divided into two parts, and 37°C is continuously maintained as dispersed phase B, the prepared microgel precursor is used as dispersed phase A, and fluorinated oil containing 2% surfactant Pico-Surf is used as continuous phase. The above solution is connected to the PDMS microfluidic chip through an injection pump, wherein the flow rate ratio of dispersed phase A, dispersed phase B, and continuous phase is set to 1:2:10, collect the microspheres in a centrifuge tube, cure under 405 nm ultraviolet light for 1 min, then wash out, and replace with PBS multiple times to obtain leuprorelin acetate microspheres. Figure 4 As shown, the microspheres prepared in the PDMS microfluidic chip are uniform in size and monodisperse.

[0045] Embodiment (2)

[0046] The specific preparation method of the leuprorelin acetate microspheres is as follows:

[0047] 1. Preparation of polymer microemulsion

[0048] First, 10 mg of PEG-PEUPE was weighed and mixed with 800 μL of dichloromethane as solution A, and 2 mg of leuprorelin acetate was weighed and dissolved in 200 μL of water as solution B. At 600 rpm, solution B was slowly added to solution A for stirring and emulsification to form a W / O type polymer microemulsion, which was evaporated and self-assembled at room temperature for 30 minutes.

[0049] 2. Formation of Microgel Precursors

[0050] 2.1 Preparation of HB-PEG pregel solution

[0051] 200 mg of hyperbranched polyethylene glycol diacrylate (HB-PEGDA) was dissolved in 2 mL of 1×PBS at pH 7.4 to prepare a pre-gel solution with a concentration of 10% (w / v);

[0052] 2.2 Experimental procedures

[0053] The polymer microemulsion was slowly added into the pre-gel solution at 1000 rpm and stirred for 1 h.

[0054] 3 Construction of microdroplet generation system

[0055] 100 mg of hyperbranched polyethylene glycol diacrylate (HB-PEGDA) was dissolved in 1 mL of 1×PBS at pH = 7.4 to prepare a 10% (w / v) solution as dispersed phase B1; 30 mg of thiolated hyaluronic acid (SH-HA) was dissolved in 1 mL of 1×PBS at pH = 7.4, adjusted to neutrality with 0.1 M NaOH solution, and then prepared into a 5% (w / v) solution as dispersed phase B2. The prepared microgel precursor was used as dispersed phase A, and fluorinated oil containing 2% surfactant Pico-Surf was used as a continuous phase. The above solutions were connected to a PDMS microfluidic chip through a syringe pump, wherein the flow rate ratio of dispersed phase A, dispersed phase B1, and dispersed phase B2 continuous phase was set to 1:4:2:10, and the microspheres were collected in a centrifuge tube, left to stand for cross-linking at room temperature for 24 hours, then washed out, and PBS was replaced multiple times to obtain leuprorelin acetate microspheres.

[0056] Microfluidic generation system microsphere preparation channel Figure 3 As shown, the dispersed phase A in the middle is sheared into monodisperse microspheres. By regulating the flow rate, the size of the microspheres can be kept around 150 μm. At the same time, the microspheres contain self-assembled particles after the polymer-coated drugs are contained.

[0057] Embodiment (3)

[0058] The specific preparation method of the leuprorelin acetate microspheres is as follows:

[0059] 1. Preparation of polymer microemulsion

[0060] First, 5 mg PEI-PLGA was weighed and mixed with 300 μL dichloromethane as solution A, and 5 mg leuprorelin acetate was weighed and dissolved in 200 μL water as solution B. At 600 rpm, solution B was slowly added to solution A for stirring and emulsification to form a W / O type polymer microemulsion, which was evaporated and self-assembled at room temperature for 30 minutes.

[0061] 2. Formation of Microgel Precursors

[0062] 2.1 Preparation of methacryloyl gelatin

[0063] Weigh 1 g of gelatin and dissolve it in 10 mL of aqueous solution (10% w / v) at 50°C. After it is completely dissolved, slowly add 0.6 g of methacrylic anhydride (MA) and react for 1 hour. The reaction product is diluted with deionized water (1:5) and dialyzed with deionized water at 40°C for 24 hours. After dialyzing three times, it is freeze-dried and stored.

[0064] 2.2 Experimental procedures

[0065] Weigh 200 mg of freeze-dried methacryloyl gelatin and dissolve it in 2 mL of water at 37°C. Continue to add 5 mg of photoinitiator 2959 until it is completely dissolved to form a 10 wt% pre-gel solution. Slowly add the above polymer microemulsion into the pre-gel solution at 37°C and 600 rpm and continue stirring for 1 hour.

[0066] Synthesis and gelling mechanism of methacryloyl gelatin Figure 2 As shown, under certain conditions, the amino group in the gelatin molecule is introduced into a double bond, and by mixing with a photoinitiator, under the irradiation of ultraviolet rays, the double bonds of the modified side chains open and become entangled, forming a cross-linked network to become a hydrogel.

[0067] 3 Construction of microdroplet generation system

[0068] Weigh 200 mg of freeze-dried methacryloyl gelatin and dissolve it in 2 mL of water at 37° C. Continue to add 5 mg of photoinitiator 2959 until it is completely dissolved to prepare a 10 wt% pre-gel solution, wherein the prepared pre-gel solution is divided into two parts, and 37° C. is continuously maintained as dispersed phase B, the prepared microgel precursor is used as dispersed phase A, and fluorinated oil containing 2% surfactant Pico-Surf is used as a continuous phase. The above solution is connected to a capillary microfluidic chip through a syringe pump, wherein the flow rate ratio of dispersed phase A, dispersed phase B, and continuous phase is set to 2:5:10, and the microspheres are collected in a centrifuge tube, cured under ultraviolet light at 365 nm for 20 seconds, then washed out, and replaced with PBS multiple times to obtain leuprorelin acetate microspheres.

[0069] like Figure 8 The four photos in the middle correspond to 0, 3, 5, and 10 minutes respectively. The washed leuprorelin acetate microspheres are placed in a PBS solution containing 125U / mL collagenase. The microspheres can be slowly degraded by collagenase over time to release the drugs inside. In addition, the rate of drug release can be controlled by controlling the degree of cross-linking of the microspheres.

[0070] Embodiment (4)

[0071] The specific preparation method of the leuprorelin acetate microspheres is as follows:

[0072] 1. Preparation of polymer microemulsion

[0073] First, 10 mg of PEG-PLGA was weighed and mixed with 800 μL of dichloromethane as solution A, and 5 mg of leuprorelin acetate was weighed and dissolved in 200 μL of water as solution B. At 600 rpm, solution B was slowly added to solution A for stirring and emulsification to form a W / O type polymer microemulsion, which was evaporated and self-assembled at room temperature for 30 minutes.

[0074] 2. Formation of Microgel Precursors

[0075] 2.1 Preparation of four-arm polyethylene glycol acrylate

[0076] 100 mg of four-arm polyethylene glycol diacrylate was dissolved in 2 mL of 1×PBS at pH=7.4 to prepare a pre-gel solution with a concentration of 5% (w / v).

[0077] 2.2 Experimental procedures

[0078] The polymer microemulsion was slowly added into the pre-gel solution at 600 rpm and stirred for 1 h.

[0079] 3 Construction of microdroplet generation system

[0080] 200 mg of freeze-dried four-arm polyethylene glycol diacrylate was weighed and dissolved in 1 mL of PBS, and 10 mg of photoinitiator LAP was added to prepare a 20 wt% pre-gel solution, wherein the prepared pre-gel solution was divided into two parts as dispersed phase B, the prepared microgel precursor was used as dispersed phase A, and fluorinated oil containing 2% surfactant Pico-Surf was used as a continuous phase. The above solution was connected to a capillary microfluidic chip through a syringe pump, wherein the flow rate ratio of dispersed phase A, dispersed phase B, and continuous phase was set to 1:2:10, and the microspheres were collected in a centrifuge tube, cured under ultraviolet light at 405 nm for 2 minutes, then washed out, and replaced with PBS multiple times to obtain leuprorelin acetate microspheres.

[0081] The encapsulation efficiency and drug loading of leuprolide acetate microspheres were measured by high performance liquid chromatography (HPLC). Figure 7 The standard curve of leuprorelin acetate was displayed. Combined with the mass of the lyophilized microspheres and the peak area of ​​the drug in the corresponding microspheres, the encapsulation efficiency and drug loading were about 80%-90%.

[0082] Embodiment (5)

[0083] The specific preparation method of the leuprorelin acetate microspheres is as follows:

[0084] 1. Preparation of polymer microemulsion

[0085] First, 10 mg of PEG-PLGA was weighed and mixed with 1.5 mL of dichloromethane as solution A, and 100 mg of leuprorelin acetate was weighed and dissolved in 500 μL of water as solution B. At 600 rpm, solution B was slowly added to solution A for stirring and emulsification to form a W / O type polymer microemulsion, which was evaporated and self-assembled at room temperature for 30 minutes.

[0086] 2. Formation of Microgel Precursors

[0087] 2.1 Preparation of methacrylylated silk fibroin

[0088] 400 mg of freeze-dried methacryloylated silk fibroin was weighed and dissolved in 4 mL of water to prepare a pre-gel solution with a concentration of 10% (w / v).

[0089] 2.2 Experimental procedures

[0090] The polymer microemulsion was slowly added into the pre-gel solution at 600 rpm and stirred for 1 h.

[0091] 3 Construction of microdroplet generation system

[0092] 200 mg of freeze-dried methacrylylated silk fibroin was weighed and dissolved in 2 mL of PBS, and 2 mg of photoinitiator LAP was added to prepare a 10 wt% pre-gel solution, wherein the prepared pre-gel solution was divided into two parts as dispersed phase B, the prepared microgel precursor was used as dispersed phase A, and fluorinated oil containing 2% surfactant Pico-Surf was used as a continuous phase. The above solution was connected to a PDMS microfluidic chip through a syringe pump, wherein the flow rate ratio of dispersed phase A, dispersed phase B, and continuous phase was set to 1:4:10, and the microspheres were collected in a centrifuge tube, cured under ultraviolet light of 405 nm for 2 minutes, then washed out, and replaced with PBS multiple times to obtain leuprorelin acetate microspheres, and the obtained leuprorelin acetate had a particle size of 150 microns, a PDI index of 0.12, and was monodisperse.

[0093] Characterization of biocompatibility of materials:

[0094] (1) Live-dead staining was used to characterize the material encapsulating leuprolide acetate. Mouse L-929 cells were co-incubated with freeze-dried microgel materials. The initial cell seeding amount was 5w / mL, and the concentration gradient of the microgel was set to 0mg / mL, 50mg / mL, 100mg / mL, 250mg / mL, 500mg / mL and 1000mg / mL respectively; Figure 5 As shown, when the microgels were co-incubated with cells, the average fluorescence intensity of live cells in microgels of different concentrations was much higher than that of dead cells, indicating that the microgel material had good biocompatibility;

[0095] (2) The toxicity of the material coated with leuprolide acetate was characterized by the MTT method, such as Figure 6 As shown, when the microgels were co-incubated with cells, the cell viabilities of the microgels at different concentrations were all greater than 80%, indicating that the microgel material had good biocompatibility.

[0096] Embodiment (6)

[0097] The specific preparation method of the leuprorelin acetate microspheres is as follows:

[0098] 1. Preparation of polymer microemulsion

[0099] First, 10 mg PEI-PLGA was weighed and mixed with 1.5 mL acetone as solution A, and 100 mg leuprorelin acetate was weighed and dissolved in 500 μL water as solution B. At 600 rpm, solution B was slowly added to solution A for stirring and emulsification to form a W / O type polymer microemulsion, which was evaporated and self-assembled at room temperature for 30 minutes.

[0100] 2. Formation of Microgel Precursors

[0101] 2.1 Preparation of polyether F127 diacrylate

[0102] 800 mg of freeze-dried polyether F127 diacrylate was weighed and dissolved in 8 mL of water to prepare a pre-gel solution with a concentration of 10% (w / v).

[0103] 2.2 Experimental procedures

[0104] The polymer microemulsion was slowly added into the pre-gel solution at 600 rpm and stirred for 1 h.

[0105] 3 Construction of microdroplet generation system

[0106] 200 mg of freeze-dried polyether F127 diacrylate was weighed and dissolved in 2 mL of PBS, and 2 mg of photoinitiator LAP was added to prepare a 10 wt% pre-gel solution, wherein the prepared pre-gel solution was divided into two parts as dispersed phase B, the prepared microgel precursor was used as dispersed phase A, and fluorinated oil containing 2% surfactant Pico-Surf was used as a continuous phase. The above solution was connected to an injection-molded microfluidic chip through a syringe pump, wherein the flow rate ratio of dispersed phase A, dispersed phase B, and continuous phase was set to 1:4:10, and the microspheres were collected in a centrifuge tube, cured under ultraviolet light at 405 nm for 2 minutes, then washed out, and replaced with PBS multiple times to obtain leuprorelin acetate microspheres.

[0107] Example (7)

[0108] The specific preparation method of the leuprorelin acetate microspheres is as follows:

[0109] 1. Preparation of polymer microemulsion

[0110] First, 50 mg of PEG-PEUPE was weighed and mixed with 1 mL of methanol as solution A, and 100 mg of leuprolide acetate was weighed and dissolved in 500 μL of water as solution B. At 600 rpm, solution B was slowly added to solution A for stirring and emulsification to form a W / O type polymer microemulsion, which was evaporated and self-assembled at room temperature for 30 minutes.

[0111] 2. Formation of Microgel Precursors

[0112] 2.1 Preparation of methacryloylated chondroitin sulfate

[0113] 800 mg of freeze-dried methacryloylated chondroitin sulfate was weighed and dissolved in 8 mL of water to prepare a pre-gel solution with a concentration of 10% (w / v).

[0114] 2.2 Experimental procedures

[0115] The polymer microemulsion was slowly added into the pre-gel solution at 800 rpm and stirred for 1 h.

[0116] 3 Construction of microdroplet generation system

[0117] 200 mg of freeze-dried methacryloylated chondroitin sulfate was weighed and dissolved in 2 mL of PBS, and 2 mg of photoinitiator LAP was added to prepare a 10 wt% pre-gel solution, wherein the prepared pre-gel solution was divided into two parts as dispersed phase B, the prepared microgel precursor was used as dispersed phase A, and fluorinated oil containing 2% surfactant Pico-Surf was used as a continuous phase. The above solution was connected to an injection-molded microfluidic chip through a syringe pump, wherein the flow rate ratio of dispersed phase A, dispersed phase B, and continuous phase was set to 1:4:10, and the microspheres were collected in a centrifuge tube, cured under ultraviolet light at 405 nm for 2 minutes, then washed out, and replaced with PBS multiple times to obtain leuprorelin acetate microspheres.

[0118] Embodiment (8)

[0119] The specific preparation method of the leuprorelin acetate microspheres is as follows:

[0120] 1. Preparation of polymer microemulsion

[0121] First, 20 mg of PEG-PLGAE was weighed and mixed with 1.5 mL of chloroform as solution A, and 100 mg of leuprorelin acetate was weighed and dissolved in 500 μL of water as solution B. At 600 rpm, solution B was slowly added to solution A for stirring and emulsification to form a W / O type polymer microemulsion, which was evaporated and self-assembled at room temperature for 30 minutes.

[0122] 2. Formation of Microgel Precursors

[0123] 2.1 Preparation of methacrylylated polylysine

[0124] 600 mg of freeze-dried methacryloyl polylysine was weighed and dissolved in 6 mL of water to prepare a pre-gel solution with a concentration of 10% (w / v).

[0125] 2.2 Experimental procedures

[0126] The polymer microemulsion was slowly added into the pre-gel solution at 800 rpm and stirred for 1 h.

[0127] 3 Construction of microdroplet generation system

[0128] 200 mg of freeze-dried methacryloyl polylysine was weighed and dissolved in 2 mL of PBS, and 2 mg of photoinitiator LAP was added to prepare a 10 wt% pre-gel solution, wherein the prepared pre-gel solution was divided into two parts as dispersed phase B, the prepared microgel precursor was used as dispersed phase A, and fluorinated oil containing 2% surfactant Pico-Surf was used as a continuous phase. The above solution was connected to an injection-molded microfluidic chip through a syringe pump, wherein the flow rate ratio of dispersed phase A, dispersed phase B, and continuous phase was set to 1:4:10, and the microspheres were collected in a centrifuge tube, cured under ultraviolet light at 405 nm for 2 min, then washed out, and replaced with PBS multiple times to obtain leuprorelin acetate microspheres.

[0129] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A microfluidic preparation method of leuprolide acetate microspheres, characterized in that: The following steps are involved: (1) dissolving a hydrophilic-hydrophobic polymer in an organic solvent, adding a leuprolide acetate aqueous solution and stirring, evaporating and self-assembling for 30±15 min to form a W / O type polymer microemulsion; (2) adding the polymer microemulsion to the pre-gel component, stirring and mixing and volatilizing for 1-2 hours to remove the organic solvent to form a microgel precursor; (3) The solution of (2) is used as dispersed phase A, the pre-gel component is used as dispersed phase B, and the fluorinated oil containing 2% of the surfactant Pico-Surf is used as the continuous phase to connect to the microdroplet generation system to generate monodisperse microdroplets, which are then cross-linked and solidified, washed out, and freeze-dried to obtain leuprolide acetate microspheres; The hydrophilic-hydrophobic polymer in step (1) includes one or more of polyethylene glycol-polylactic acid-co-glycolic acid (PEG-PLGA), polyethylene glycol-polyurethane block polymer (PEG-PEUPE), polyethyleneimine-polylactic acid block copolymer (PEI-PLGA), and the organic solvent includes but is not limited to dichloromethane, acetonitrile, methanol, acetone and chloroform; The pre-gel component of step (2) includes one or more of methacrylated gelatin, sodium alginate, hyaluronic acid, silk fibroin, chitosan, dextran, chondroitin sulfate, polylysine; polyether F127 diacrylate; hyperbranched polyethylene glycol diacrylate (HB-PEG) and thiolated hyaluronic acid (SH-HA); four-arm polyethylene glycol acrylate, the degree of methacrylation is 30%-70%, the concentration of the pre-gel component is 5%-20%, and the solvent is water or PBS.

2. The microfluidic preparation method of leuprorelin acetate microspheres according to claim 1, characterized in that: The concentration of leuprorelin acetate in the polymer microemulsion is 1-1000 mg / mL, and the mass volume ratio (mg:μL) of the hydrophilic-hydrophobic polymer to the organic solvent is 1-20:

100.

3. The microfluidic preparation method of leuprorelin acetate microspheres according to claim 1 or 4, characterized in that: In step (2), the volume ratio of the polymer microemulsion to the pre-gel component is 1-5:

10.

4. The microfluidic preparation method of leuprorelin acetate microspheres according to claim 1, characterized in that: The micro-droplet generation system in step (3) is composed of a syringe pump, a microfluidic chip and a high-speed camera, wherein the microfluidic chip includes a PDMS microfluidic chip, a capillary microfluidic chip or an injection-molded microfluidic chip.

5. The microfluidic preparation method of the leuprolide acetate microspheres according to claim 1, characterized in that: In step (3), the volume ratio of dispersed phase A to dispersed phase B is 1-5:1, the concentration of dispersed phase B is 2%-20%, and the solvent is water or PBS; the size of the generated monodisperse droplets is 1-1000 μm.

6. The microfluidic preparation method of leuprorelin acetate microspheres according to claim 1, characterized in that: The crosslinking and curing method in step (3) includes one or more of Michael addition, photoinitiation, photocuring covalent crosslinking, and physical crosslinking.

7. The crosslinking and curing method according to claim 6, characterized in that: The photoinitiator is 2929 or LAP, and the concentration of the photoinitiator is 0.1%-0.5%.

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