A microfluidic device and a method for preparing bile salt microparticles using the same

Abstract

The application discloses a micro-fluidic device and a method for preparing bile salt microparticles by using the device, and belongs to the technical field of functional materials. The micro-fluidic chip comprises a micro-fluid channel, four dispersed phase inlets, one continuous phase inlet and one outlet. The bile salt microparticles are prepared by using the micro-fluidic chip, non-simultaneous mixing of multi-phase solutions is realized, the microparticles can be formed in situ to form bile salt particles, the size of the prepared bile salt microparticles is adjustable, the shape is uniform, and the slow-release time can reach at least 5 days.

Description

Technical Field

[0001] The present invention relates to the technical field of functional materials, and in particular to a microfluidic device and a method for preparing bile salt particles using the device. Background Art

[0002] Bile salts are naturally occurring surfactants that help solubilize lipids in the small intestine and regulate liver, biliary, and intestinal function. Bile acids and their salts have been proposed as therapeutic agents for the treatment of various diseases, including bile synthesis and peroxisomal disorders, primary biliary cirrhosis, gallstones and bile duct stones, nonalcoholic fatty liver disease, type 2 diabetes, cancer, and for the local removal of unwanted fat.

[0003] Given the societal need, the activity of bile salts in promoting adipocyte lysis has high commercial value. Body image disturbances and unwanted fat deposits may be the underlying causes of various psychiatric disorders, such as body dysmorphic disorder and depression. Fat contouring treatments have been shown to improve self-esteem and interpersonal relationships, provide mood stability, and alleviate depression and anxiety. Liposuction is currently the most commonly used method for removing unwanted fat deposits. However, the invasive nature, high cost, and associated long recovery time limit patients from seeking this treatment. Therefore, alternative, non-invasive treatments are needed.

[0004] Kybella is an FDA-approved injection for removing submental fat. Its main ingredient is deoxycholic acid (C24H40O4). It is used by subcutaneous injection, with 0.2 mL of injection solution spaced 1 cm apart until all planned treatment areas have been injected. The course of treatment is 2-4 times. Between 1-2 months after surgery, the amount of submental fat in the subjects can be reduced by at least 10%. However, during the treatment process, adverse reactions such as swollen lymph nodes, urticaria at the injection site, neck pain and bruising may occur, which weaken the effect of Kybella and are limited to reducing submental fat. In China, this drug can only be used in regular cosmetic hospitals in Hong Kong and Taiwan. For a long time, controlled or targeted drug delivery has been used as an alternative to taking drugs to improve specificity while reducing side effects.

[0005] Bile salts are sodium or potassium salts formed by the combination of bile acids secreted by hepatocytes with glycine or taurine. They are highly soluble in water. Forming them into tablets or microparticles can prolong their sustained-release effect, extending the duration of drug action and reducing the number of doses. Prior art discloses sodium cholate tablets, such as CN201610729276.2. The sodium cholate tablets provided in this invention are obtained by tablet compression and sugar coating, and are not sustained-release tablets.

[0006] Therefore, fabricating bile salt microparticle-based controlled-release systems may address the limitations of the use of bile acids and their salts as therapeutic agents. Summary of the Invention

[0007] The present invention provides a microfluidic device and a method for preparing bile salt particles using the device. The microfluidic device of the present invention is specifically used for preparing bile salt particles. The prepared bile salt particles have adjustable size and uniform particle size.

[0008] The present invention first provides a microfluidic chip, comprising a microchannel, four dispersed phase inlets, a continuous phase inlet and an outlet;

[0009] The dispersed phase inlet 1 and the dispersed phase inlet 2 are connected by a microchannel and merge into a microchannel a. The angle between the microchannel intersection of the dispersed phase inlet 1 and the dispersed phase inlet 2 is 62°.

[0010] The dispersed phase inlet 3 is divided into two microchannels and intersects with the microchannel a to form a microchannel b; the intersection angles of the two microchannels divided by the dispersed phase inlet 3 and the microchannel a are 60 degrees respectively;

[0011] The dispersed phase inlet 4 is divided into two microchannels and intersects with the microchannel b to form a microchannel c; the intersection angles of the two microchannels divided by the dispersed phase inlet 4 and the microchannel b are 60 degrees respectively;

[0012] The continuous phase inlet 5 is divided into two microchannels and intersects with the microchannel c to form a microchannel d; the microchannel d is connected to the outlet;

[0013] The angles between the two microchannels divided by the continuous phase inlet 5 and the intersection of the microchannel c are 60 degrees.

[0014] In the above-mentioned microfluidic chip, the cross-section of the microchannel is a square with a side length of 50 to 200 μm, specifically 100 μm; the inner wall surface of the microchannel is hydrophobicized; specifically, the hydrophobic agent of the hydrophobic treatment is Pico-Glide, Aquapel or TurtleWax;

[0015] The material of the microfluidic chip is selected from polydimethylsiloxane, polymethyl methacrylate, cyclic olefin copolymer, polycarbonate, polystyrene, quartz, borosilicate glass or single crystal silicon.

[0016] Second, the present invention provides a microfluidic device for preparing bile salt particles, which includes the above-mentioned microfluidic chip.

[0017] Specifically, the microfluidic device also includes a peristaltic pump or a syringe pump, an optical microscope, a syringe, an input tube, an output tube and a collection tube; the syringe is placed on the peristaltic pump or the syringe pump, one end of the input tube is connected to the syringe, and the other end is connected to the inlet of the microfluidic chip; one end of the output tube is connected to the outlet of the microfluidic chip, and the other end is connected to the collection tube; the microfluidic chip is placed under the optical microscope.

[0018] Third, the present invention provides a method for preparing bile salt particles using the above-mentioned microfluidic device, comprising the following steps:

[0019] (1) Prepare dispersed phase solution: prepare sodium citrate solution, chloroauric acid solution and bile salt solution respectively;

[0020] (2) Preparation of continuous phase solution: Preparation of oil phase containing surfactant;

[0021] (3) Using the microfluidic device described in claim 3, the sodium citrate solution is injected from the dispersed phase inlet 1 of the microfluidic chip; the chloroauric acid solution is injected from the dispersed phase inlet 2 of the microfluidic chip; water is injected from the dispersed phase inlet 3 of the microfluidic chip; the bile salt solution is injected from the dispersed phase inlet 4 of the microfluidic chip; the oil phase containing the surfactant is injected from the continuous phase inlet 5 of the microfluidic chip; and monodispersed oil-in-water droplets are collected from the outlet;

[0022] (4) heating the water-in-oil droplet system obtained in step (3), and then allowing the bile salt particles to grow; and obtaining the bile salt particles.

[0023] In the above method, the concentration of the sodium citrate solution is 0.5-3.0 wt%, specifically 2.0 wt%; the concentration of the chloroauric acid solution is 0.4-2.0 wt%, specifically 1.0 wt%; the concentration of the bile salt solution is 4.0-10.0 wt%, specifically 4.0 wt%, 6.0 wt%, etc.

[0024] In the oil phase containing a surfactant, the concentration of the surfactant is 0.5 to 2.0 wt %, specifically 0.5 wt %;

[0025] The solvent of the oil phase containing the surfactant is one of fluorinated oil, mineral oil, vegetable oil, olive oil, silicone oil and petroleum ether;

[0026] The surfactant is sorbitan laurate, polyoxyethylene sorbitan trioleate, poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), sorbitan monooleate, polyoxyethylene (10) oleyl ether, polyoxyethylene (2) oleyl ether or Krytox-PEG-Krytox;

[0027] The bile salt is sodium cholate or sodium deoxycholate.

[0028] In the above method, in step (3), the injection flow rate of the sodium citrate solution is 50-200 μL / h, specifically 50 μL / h; the injection flow rate of the chloroauric acid solution is 50-200 μL / h, specifically 50 μL / h; in the injection of water, the flow rate of the two microchannels is 50-200 μL / h, specifically 100 μL / h; in the injection of the bile salt solution, the flow rate of the two microchannels is 50-200 μL / h, specifically 50 μL / h; in the injection of the oil phase containing the surfactant, the flow rate of the two microchannels is 500-1500 μL / h, specifically 800 μL / h.

[0029] In the above method, the monodisperse water-in-oil droplets are collected in an oil phase containing a surfactant;

[0030] Preferably, the concentration of the surfactant in the oil phase containing the surfactant is 0.5 to 2.0 wt %, specifically 0.5 wt %.

[0031] In the oil phase containing the surfactant, the solvent can be one of fluorinated oil, mineral oil, vegetable oil, olive oil, silicone oil and petroleum ether; the surfactant can be sorbitan laurate, polyoxyethylene sorbitan trioleate, poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), anhydrous sorbitan monooleate, polyoxyethylene (10) oleyl ether, polyoxyethylene (2) oleyl ether or Krytox-PEG-Krytox.

[0032] In the above method, in step (4), the heating temperature is 40-60° C., specifically 45° C.; the heating time is 0.5-4 h, specifically 1 h;

[0033] The placing temperature is room temperature; the placing time is 12 to 24 hours.

[0034] In the above method, in step (4), after the placing, the mixture is washed with a solution containing 1H,1H,2H,2H-perfluorooctanol and water respectively, and then dried;

[0035] Preferably, the concentration of the solution containing 1H,1H,2H,2H-perfluorooctanol is 15-30 wt %, specifically 20 wt %.

[0036] The solvent of the solution containing 1H,1H,2H,2H-perfluorooctanol is one of fluorinated oil, mineral oil, vegetable oil and olive oil.

[0037] The drying temperature is 50-80° C.; specifically, the drying is performed at 65° C. for 2 hours.

[0038] Finally, the present invention provides bile salt microparticles prepared by the above method;

[0039] Preferably, the particle size of the bile salt particles is 4 to 20 μm.

[0040] In the present invention, the room temperature is well known to those skilled in the art, and is generally 15 to 35°C.

[0041] The primary mechanism for in situ bile salt microparticle formation in this invention involves doping the aqueous phase with Au(III) ions and sodium citrate as precursors for gold nanoparticle formation. Heating reduces the gold precursors encapsulated within the droplets to nanoparticles. The presence of the oil-water interface and the reduction of gold ions promote the assembly of bile acid ions, generating bile salt microparticles. The stable bile salt microparticles are then separated from the free gold nanoparticles by low-speed centrifugation.

[0042] The present invention has the following beneficial effects:

[0043] The microfluidic device of the present invention utilizes a fluid focusing design to construct multi-layer concentric axis fluids, and then obtains water-in-oil droplets by shearing a continuous phase, thereby achieving continuous sampling and high-throughput, rapid production of monodisperse water-in-oil droplets with precisely controllable size. Furthermore, bile salt microparticles prepared using the microfluidic device achieve non-simultaneous mixing of multiphase solutions, and the obtained droplets can form bile salt particles in situ. The prepared bile salt microparticles have adjustable size and uniform shape, with a yield of 70-80% and a sustained release time of at least 5 days. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Schematic diagram of the structure of the microfluidic chip of the present invention;

[0045] Figure 2 Schematic diagram of the structure of the microfluidic device of the present invention; in the figure, 1 is a peristaltic pump or a syringe pump, 2 is a syringe, 3 is an input tube, 4 is a microfluidic chip, 5 is an optical microscope, 6 is an output tube, and 7 is a collection tube;

[0046] Figure 3 This is an optical microscope image of the sodium cholate microparticles prepared in Example 2;

[0047] Figure 4 This is an optical microscope image of the sodium cholate microparticles prepared in Example 3;

[0048] Figure 5 This is a scanning electron micrograph of the sodium cholate microparticles prepared in Example 3;

[0049] Figure 6 This is the EDS analysis chart of the sodium cholate microparticles prepared in Example 3;

[0050] Figure 7This is a graph showing the quantitative release of cholate from the sodium cholate microparticles prepared in Example 3 after incubation at different time points;

[0051] Figure 8 This is an optical microscope image of the sodium deoxycholate microparticles prepared in Example 4;

[0052] Figure 9 This is an optical microscope image of the sodium deoxycholate microparticles prepared in Example 5;

[0053] Figure 10 This is a scanning electron micrograph of the sodium deoxycholate microparticles prepared in Example 5;

[0054] Figure 11 This is the EDS analysis of the sodium deoxycholate microparticles prepared in Example 5;

[0055] Figure 12 Schematic diagram of the structure of the microfluidic chip 2 used in Comparative Example 1;

[0056] Figure 13 Schematic diagram of the structure of the microfluidic chip 3 used in Comparative Example 2;

[0057] Figure 14 This is an optical microscope image of the sodium cholate microparticles prepared in Comparative Example 3. DETAILED DESCRIPTION

[0058] The present invention will be further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention, not for limiting the scope of the present invention.

[0059] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0060] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0061] Example 1

[0062] The schematic diagram of the microfluidic chip structure of the present invention is as follows Figure 1 As shown, the microfluidic chip includes a microchannel, four dispersed phase inlets (1, 2, 3 and 4), a continuous phase inlet (5), and an outlet (6); the substrate of the microchannel chip is an ordinary glass sheet.

[0063] The dispersed phase inlet 1 and the dispersed phase inlet 2 are connected by a microchannel and merge into a microchannel a. The intersection angle of the microchannels of the dispersed phase inlet 1 and the dispersed phase inlet 2 is 62°; the dispersed phase inlet 3 is divided into two microchannels and merges with the microchannel a to form a microchannel b; the intersection angles of the two microchannels divided by the dispersed phase inlet 3 and the microchannel a are 60° respectively; the dispersed phase inlet 4 is divided into two microchannels and merges with the microchannel b to form a microchannel c; the intersection angles of the two microchannels divided by the dispersed phase inlet 4 and the microchannel b are 60° respectively; the continuous phase inlet 5 is divided into two microchannels and merges with the microchannel c to form a microchannel d, and the microchannel d is connected to the outlet 6; the intersection angles of the two microchannels divided by the continuous phase inlet 5 and the microchannel c are 60° respectively.

[0064] The cross section of the microchannel is a square with a side length of 100 μm, and the microchannels are all on the same horizontal plane.

[0065] The microchannel material of this embodiment is specifically polydimethylsiloxane;

[0066] The inner wall surfaces of the microchannels are all subjected to hydrophobic treatment, and the specific steps are as follows:

[0067] (1) Use a syringe to draw in a hydrophobic agent (Pico-Glide, Aquapel, TurtleWax are all acceptable, and Aquapel is specifically used in this embodiment), then connect a needle and connect the polytetrafluoroethylene tube;

[0068] (2) Fill the microchannels with a hydrophobic agent using a syringe. When all microchannels are filled with the hydrophobic agent, cover the microfluidic device with tape to prevent evaporation.

[0069] (3) Place the microfluidic chip at room temperature for at least 5 minutes;

[0070] (4) Fluorinated oil is introduced to replace the hydrophobic agent in the microchannel.

[0071] The structural diagram of the microfluidic device used in the present invention is as follows Figure 2 As shown, it includes a peristaltic pump or a syringe pump 1, an optical microscope 5, a microfluidic chip 4, a syringe 2, an input tube 3, an output tube 6 and a collection tube 7.

[0072] Place the syringe on the syringe pump, connect one end of the input tube to the syringe and the other end to the inlet of the microfluidic chip, connect one end of the output tube to the outlet of the microfluidic chip and the other end to the collection tube, and place the microfluidic chip under an optical microscope to observe the generation of droplets.

[0073] The inner diameter of the input and output tubes is 0.5 mm and the material is polytetrafluoroethylene (PTFE).

[0074] Example 2

[0075] (1) preparing dispersed phase and continuous phase solutions, wherein the dispersed phase comprises 2.0 wt% sodium citrate solution, 1.0 wt% chloroauric acid solution, and 4.0 wt% sodium cholate solution; and the continuous phase comprises fluorinated oil containing 0.5 wt% surfactant (Krytox-PEG-Krytox);

[0076] (2) Using the microfluidic device of Example 1, sodium citrate solution was injected from inlet 1 of the microfluidic chip at a flow rate of 50 μL / h; chloroauric acid solution was injected from inlet 2 of the microfluidic chip at a flow rate of 50 μL / h; pure water was injected from inlet 3 of the microfluidic chip at a flow rate of 100 μL / h in both microchannels; sodium cholate solution was injected from inlet 4 of the microfluidic chip at a flow rate of 50 μL / h in both microchannels; fluorinated oil containing 0.5 wt% surfactant was injected from inlet 5 of the microfluidic chip at a flow rate of 800 μL / h in both microchannels. Monodisperse oil-in-water droplets were formed at the "cross" outlet 6;

[0077] (3) When the flow rate stabilizes and the generated monodisperse water-in-oil droplets are observed to be uniform in size, droplet collection is initiated. The prepared droplets are collected in fluorinated oil containing 0.5 wt% surfactant (Krytox-PEG-Krytox), then heated in a 45°C metal bath for 1 h. They are then left at room temperature for 12 h to allow the sodium cholate microparticles to grow.

[0078] (4) Adding fluorinated oil containing 20 wt% PFO (1H,1H,2H,2H-perfluorooctanol) to the system obtained in step (3), shaking, and then standing to separate the layers, and removing the lower fluorinated oil layer. This step is to wash away the surfactant; then adding water to the upper aqueous phase, shaking, and finally centrifuging at 600 rpm. The resulting precipitate is dried at 65°C for 2 h to obtain sodium cholate microparticles.

[0079] The morphology and size of the sodium cholate microparticles obtained in Example 2 were observed under an optical microscope. Figure 3 As shown by Figure 3 It can be seen that the sodium cholate microparticles prepared in this example are in the shape of rice grains and are uniform in size, with a size of 4.8±0.3 μm.

[0080] Example 3

[0081] (1) preparing dispersed phase and continuous phase solutions: dispersed phase: 2.0 wt% sodium citrate solution, 1.0 wt% chloroauric acid solution, 6.0 wt% sodium cholate solution; continuous phase: fluorinated oil containing 0.5 wt% surfactant (Krytox-PEG-Krytox);

[0082] (2) Using the microfluidic device of Example 1, sodium citrate solution was injected from inlet 1 of the microfluidic chip at a flow rate of 50 μL / h; chloroauric acid solution was injected from inlet 2 of the microfluidic chip at a flow rate of 50 μL / h; pure water was injected from inlet 3 of the microfluidic chip at a flow rate of 100 μL / h in both microchannels; sodium cholate solution was injected from inlet 4 of the microfluidic chip at a flow rate of 50 μL / h in both microchannels; fluorinated oil containing 0.5 wt% surfactant was injected from inlet 5 of the microfluidic chip at a flow rate of 800 μL / h in both microchannels. Monodisperse oil-in-water droplets were formed at the "cross" outlet 6;

[0083] (3) When the flow rate stabilizes and the generated monodisperse water-in-oil droplets are observed to be uniform in size, droplet collection is initiated. The prepared droplets are collected in fluorinated oil containing 0.5 wt% surfactant (Krytox-PEG-Krytox), then heated in a 45°C metal bath for 1 h. They are then left at room temperature for 16 h to allow the sodium cholate microparticles to grow.

[0084] (4) Adding fluorinated oil containing 20 wt% PFO to the system obtained in step (3), shaking, and then standing to separate the layers, and removing the lower fluorinated oil layer. This step is to wash away the surfactant; then adding water to the upper aqueous phase, shaking, and finally centrifuging at 600 rpm. The resulting precipitate is dried at 65°C for 2 h to obtain sodium cholate microparticles.

[0085] The sodium cholate microparticles obtained in this example were characterized by optical microscopy, scanning electron microscopy and EDS elemental analysis. Figure 4 and Figure 5 As shown, the sodium cholate microparticles prepared in this example are in the shape of rice grains, with a size of 6.8±0.7 μm. Figure 6 This is the EDS analysis diagram of the sodium cholate microparticles of Example 2. Figure 6 It can be seen that the surface of the particles is mainly composed of carbon and oxygen - carbon and oxygen account for 90%, with a small amount of Na and Cl, and the gold content is very small and harmless to the human body.

[0086] The sodium cholate microparticles were subjected to an in vitro sustained release study. The dried sodium cholate particles were resuspended in deionized water (concentration of 10 mg / mL) and incubated at 37°C. At the desired time point, the particle suspension was spun down and the supernatant was collected. The amount of bile salt released was measured using a bile salt ELISA (enzyme-linked immunosorbent assay) kit. The experiment was repeated n = 3 times, and the release curve is shown in Figure 7 .Depend on Figure 7 It can be seen that the release curve is close to linear and the release time can reach 5 days.

[0087] Example 4

[0088] (1) preparing dispersed phase and continuous phase solutions, wherein the dispersed phase comprises 2.0 wt% sodium citrate solution, 1.0 wt% chloroauric acid solution, and 4.0 wt% sodium deoxycholate solution; and the continuous phase comprises fluorinated oil containing 0.5 wt% surfactant (Krytox-PEG-Krytox);

[0089] (2) Using the microfluidic device of Example 1, sodium citrate solution was injected from inlet 1 of the microfluidic chip at a flow rate of 50 μL / h; chloroauric acid solution was injected from inlet 2 of the microfluidic chip at a flow rate of 50 μL / h; pure water was injected from inlet 3 of the microfluidic chip at a flow rate of 100 μL / h in both microchannels; sodium deoxycholate solution was injected from inlet 4 of the microfluidic chip at a flow rate of 50 μL / h in both microchannels; fluorinated oil containing 0.5 wt% surfactant was injected from inlet 5 of the microfluidic chip at a flow rate of 800 μL / h in both microchannels. Monodisperse oil-in-water droplets were formed at the "cross" outlet 6;

[0090] (3) When the flow rate stabilizes and the generated monodisperse water-in-oil droplets are observed to be uniform in size, droplet collection is initiated. The prepared droplets are collected in fluorinated oil containing 0.5 wt% surfactant (Krytox-PEG-Krytox), then heated in a 45°C metal bath for 1 h. They are then left at room temperature for 12 h to allow the growth of sodium deoxycholate microparticles.

[0091] (4) Adding fluorinated oil containing 20 wt% PFO to the system obtained in step (3), shaking, and then standing to separate the layers, and removing the lower fluorinated oil layer. This step is to wash away the surfactant; then adding water to the upper aqueous phase, shaking, and finally centrifuging at 600 rpm. The resulting precipitate is dried at 65°C for 2 h to obtain sodium deoxycholate microparticles.

[0092] The morphology and size of the sodium deoxycholate microparticles obtained in this example were observed under an optical microscope. Figure 8 As shown by Figure 8 Analysis showed that the sodium deoxycholate microparticles prepared in this example were in cluster shape and had a size of 5.5±0.7 μm.

[0093] Example 5

[0094] (1) preparing dispersed phase and continuous phase solutions: dispersed phase: 2.0 wt% sodium citrate solution, 1.0 wt% chloroauric acid solution, 6.0 wt% sodium deoxycholate solution; continuous phase: fluorinated oil containing 0.5 wt% surfactant (Krytox-PEG-Krytox);

[0095] (2) Using the microfluidic device of Example 1, sodium citrate solution was injected from inlet 1 of the microfluidic chip at a flow rate of 50 μL / h; chloroauric acid solution was injected from inlet 2 of the microfluidic chip at a flow rate of 50 μL / h; pure water was injected from inlet 3 of the microfluidic chip at a flow rate of 100 μL / h in both microchannels; sodium deoxycholate solution was injected from inlet 4 of the microfluidic chip at a flow rate of 50 μL / h in both microchannels; fluorinated oil containing 0.5 wt% surfactant was injected from inlet 5 of the microfluidic chip at a flow rate of 800 μL / h in both microchannels. Monodisperse oil-in-water droplets were formed at the "cross" outlet 6;

[0096] (3) When the flow rate stabilizes and the generated monodisperse water-in-oil droplets are observed to be uniform in size, droplet collection is initiated. The prepared droplets are collected in fluorinated oil containing 0.5 wt% surfactant (Krytox-PEG-Krytox), then heated in a 45°C metal bath for 1 h. They are then left at room temperature for 16 h to allow the growth of sodium deoxycholate microparticles.

[0097] (4) Adding fluorinated oil containing 20 wt% PFO to the system obtained in step (3), shaking, and then standing to separate the layers, and removing the lower fluorinated oil layer. This step is to wash away the surfactant; then adding water to the upper aqueous phase, shaking, and finally centrifuging at 600 rpm. The resulting precipitate is dried at 65°C for 2 h to obtain sodium deoxycholate microparticles.

[0098] The sodium deoxycholate microparticles obtained in this example were characterized by optical microscopy, scanning electron microscopy and EDS elemental analysis. Figure 9 and Figure 10 As shown, the sodium deoxycholate microparticles prepared in this example are in cluster shape and have a size of 13.2±0.8 μm. Figure 11 This is the EDS analysis of the sodium deoxycholate microparticles prepared in Example 5; Figure 11 Analysis shows that the sample's particles are mainly composed of carbon, oxygen and gold - carbon and oxygen account for 90%, and the rest is gold. The gold accounts for a trace amount and is harmless to the human body.

[0099] Comparative Example 1

[0100] The microfluidic device used in this comparative example is the same as that in Example 1, except that the microfluidic chip used is different. The structural diagram of the microfluidic chip 2 used in this comparative example is shown in FIG. Figure 12As shown, the structure of the microfluidic chip 2 differs from that of the microfluidic chip of Example 1 in that the dispersed phase inlet 3 is divided into two microchannels, the dispersed phase inlet 4 is divided into two microchannels, and the continuous phase inlet 5 is divided into two microchannels, which simultaneously intersect with the microchannel a at one point to form a microchannel, and then connect to the outlet 6; the two microchannels of the dispersed phase inlet 3 are at an angle of 50° with the microchannel a at the intersection; the two microchannels of the dispersed phase inlet 4 are at an angle of 70° with the microchannel a at the intersection; and the two microchannels of the continuous phase inlet 5 are at an angle of 90° with the microchannel a at the intersection;

[0101] The preparation method of this comparative example is the same as that of Example 3.

[0102] Compared with Example 3 and Comparative Example 1, the four dispersed phases directly intersected with the continuous phase. It was found that the microfluidic chip 2 could not form monodisperse droplets of uniform size and could not generate sodium cholate microparticles.

[0103] Comparative Example 2

[0104] The microfluidic device used in this comparative example is the same as that in Example 1, except that the microfluidic chip used is different. The structural diagram of the microfluidic chip 3 used in this comparative example is shown in FIG. Figure 13 As shown, the structure of the microfluidic chip 3 is different from that of the microfluidic chip of Example 1 in that the dispersed phase inlet 3 is divided into two microchannels and intersects with the microchannel a to form a microchannel b, and the length of the microchannel b is 300 μm; the dispersed phase inlet 4 is divided into two microchannels, and the continuous phase inlet 5 is divided into two microchannels and intersects with the microchannel b at one point to form a microchannel, which is then connected to the outlet 6; the two microchannels of the dispersed phase inlet 3 are at an angle of 50° with the microchannel a at the intersection; the two microchannels of the dispersed phase inlet 4 are at an angle of 70° with the microchannel b at the intersection; the two microchannels of the continuous phase inlet 5 are at an angle of 20° with the dispersed phase inlet 4 at the intersection.

[0105] It was found that microfluidic chip 3 could not form monodisperse droplets of uniform size and could not generate sodium cholate microparticles.

[0106] The preparation method of this comparative example is the same as that of Example 3.

[0107] Comparative Example 3

[0108] (1) preparing dispersed phase and continuous phase solutions, wherein the dispersed phase comprises 2.0 wt% sodium citrate solution, 1.0 wt% chloroauric acid solution, and 6.0 wt% sodium cholate solution; and the continuous phase comprises fluorinated oil containing 0.5 wt% surfactant (Krytox-PEG-Krytox);

[0109] (2) Using the microfluidic device of Example 1, sodium citrate solution was injected from inlet 1 of the microfluidic chip 1 at a flow rate of 50 μL / h; sodium cholate solution was injected from inlet 2 of the microfluidic chip 1 at a flow rate of 50 μL / h; pure water was injected from inlet 3 of the microfluidic chip 1, and the flow rates of both microchannels were 100 μL / h; chloroauric acid solution was injected from inlet 4 of the microfluidic chip 1, and the flow rates of both microchannels were 50 μL / h; fluorinated oil containing 0.5 wt% surfactant was injected from inlet 5 of the microfluidic chip 1, and the flow rates of both microchannels were 800 μL / h. Monodisperse oil-in-water droplets were formed at the "cross" outlet 6;

[0110] (3) When the flow rate stabilizes, droplet collection begins. The prepared droplets are collected into fluorinated oil containing 0.5 wt% surfactant (Krytox-PEG-Krytox), then heated in a 45°C metal bath for 1 h. The mixture is then left at room temperature for 16 h to allow the sodium cholate microparticles to grow.

[0111] (4) Add fluorinated oil containing 20% ​​PFO to the system obtained in step (3), shake, and then let it stand to separate the layers. Remove the lower fluorinated oil layer. This step is to wash away the surfactant. Then add water to the upper aqueous phase, shake, and finally centrifuge at 600 rpm. The resulting precipitate is dried at 65°C for 2 hours to obtain sodium cholate microparticles.

[0112] Comparative Example 3 was compared with Example 3. The inlet of the dispersed phase solution was changed. It was found that precipitation would occur at the intersection of the chips. The generated sodium cholate crystals were uneven in size and needle-shaped, such as Figure 14 .

Claims

1. A microfluidic chip comprising a microfluidic channel, four dispersed phase inlets, one continuous phase inlet, and one outlet; The dispersed phase inlet 1 and the dispersed phase inlet 2 are connected by a microchannel and merge into a microchannel a. The angle between the microchannel intersection of the dispersed phase inlet 1 and the dispersed phase inlet 2 is 62°. The dispersed phase inlet 3 is divided into two microchannels and intersects with the microchannel a to form a microchannel b; the intersection angles of the two microchannels divided by the dispersed phase inlet 3 and the microchannel a are 60 degrees respectively; The dispersed phase inlet 4 is divided into two microchannels and intersects with the microchannel b to form a microchannel c; the intersection angles of the two microchannels divided by the dispersed phase inlet 4 and the microchannel b are 60 degrees respectively; The continuous phase inlet 5 is divided into two microchannels and intersects with the microchannel c to form a microchannel d; the microchannel d is connected to the outlet; The angles between the two microchannels divided by the continuous phase inlet 5 and the intersection of the microchannel c are 60 degrees.

2. The microfluidic chip according to claim 1, wherein: The cross section of the microchannel is a square with a side length of 50 to 200 μm.

3. A microfluidic device for preparing bile salt particles, characterized in that: The microfluidic device comprises the microfluidic chip according to claim 1 or 2.

4. A method for preparing bile salt particles using the microfluidic device according to claim 3, comprising the following steps: (1) Prepare dispersed phase solution: prepare sodium citrate solution, chloroauric acid solution and bile salt solution respectively; (2) Preparation of continuous phase solution: Prepare the oil phase containing surfactant; (3) Using the microfluidic device according to claim 3, the sodium citrate solution is injected from the dispersed phase inlet 1 of the microfluidic chip; the chloroauric acid solution is injected from the dispersed phase inlet 2 of the microfluidic chip; Water is injected from the dispersed phase inlet 3 of the microfluidic chip; The bile salt solution is injected from the dispersed phase inlet 4 of the microfluidic chip; the oil phase containing the surfactant is injected from the continuous phase inlet 5 of the microfluidic chip; and monodispersed water-in-oil droplets are collected from the outlet; (4) heating the water-in-oil droplet system obtained in step (3) and then allowing it to stand to allow bile salt particles to grow; obtaining the bile salt particles.

5. The method according to claim 4, wherein: The concentration of the sodium citrate solution is 0.5-3.0 wt %; the concentration of the chloroauric acid solution is 0.4-2.0 wt %; the concentration of the bile salt solution is 4.0-10.0 wt %; In the oil phase containing a surfactant, the concentration of the surfactant is 0.5-2.0 wt %; The solvent of the oil phase containing the surfactant is one of fluorinated oil, mineral oil, vegetable oil, olive oil, silicone oil and petroleum ether; The surfactant is sorbitan laurate, polyoxyethylene sorbitan trioleate, poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), sorbitan monooleate, polyoxyethylene (10) oleyl ether, polyoxyethylene (2) oleyl ether or Krytox-PEG-Krytox; The bile salt is sodium cholate or sodium deoxycholate.

6. The method according to claim 4 or 5, characterized in that: In step (3), the injection flow rate of the sodium citrate solution is 50~200 μL / h; the injection flow rate of the chloroauric acid solution is 50~200 μL / h; in the injection of water, the flow rate of the two microchannels is 50~200 μL / h; in the injection of the bile salt solution, the flow rate of the two microchannels is 50~200 μL / h; in the injection of the fluorinated oil solution containing a surfactant, the flow rate of the two microchannels is 500~1500 μL / h.

7. The method according to claim 4 or 5, characterized in that: The monodisperse water-in-oil droplets are collected in the oil phase containing the surfactant.

8. The method according to claim 7, wherein: The concentration of the surfactant in the oil phase containing the surfactant is 0.5-2.0 wt%; the solvent of the oil phase containing the surfactant is one of fluorinated oil, mineral oil, vegetable oil, olive oil, silicone oil and petroleum ether; the surfactant is sorbitan laurate, polyoxyethylene sorbitan trioleate, poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), anhydrous sorbitan monooleate, polyoxyethylene (10) oleyl ether, polyoxyethylene (2) oleyl ether or Krytox-PEG-Krytox.

9. The method according to claim 4 or 5, characterized in that: In step (4), the heating temperature is 40-60°C; the heating time is 0.5-4 h; The placement temperature is room temperature; the placement time is 12 to 24 hours.

10. The method according to claim 4 or 5, characterized in that: In step (4), after the placement, the sample is washed with a solution containing 1H,1H,2H,2H-perfluorooctanol and water respectively, and then dried.

11. The method according to claim 10, wherein: The concentration of the solution containing 1H,1H,2H,2H-perfluorooctanol is 15-30 wt %; the solvent of the solution containing 1H,1H,2H,2H-perfluorooctanol is at least one of fluorinated oil, mineral oil, vegetable oil and olive oil.

12. Bile salt microparticles prepared according to the method of any one of claims 4 to 9.

13. The bile salt particles according to claim 12, wherein: The particle size of the bile salt microparticles is 4-20 μm.

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