Micro-fluidic chip and application thereof in preparation of lipidosome, cell membrane vesicles or cell membrane hybrid lipid nanoparticles

Abstract

The invention discloses a micro-fluidic chip and application of the micro-fluidic chip in preparation of lipidosome, cell membrane vesicles or cell membrane hybrid lipid nanoparticles. The device comprises four layers of structures: a bottom plate layer, a chip layer, a sealing layer and a top cover layer. The chip layer is divided into a first-stage mixing area, a second-stage mixing area and a collecting area, and the top cover layer is provided with five flow channel inlets and a flow channel outlet corresponding to the chip layer. The microfluidic chip provided by the invention can be used for respectively preparing lipidosome, cell membrane vesicles and cell membrane bionic lipidosome particles which are coated with water-soluble drugs and fat-soluble drugs, synthesis of various types of lipid nanoparticles can be completed by one chip, and corresponding solutions only need to be continuously introduced from an inlet, so that the synthesis of the lipid nanoparticles can be completed. The method has the advantages that the problem that the existing liposome preparation process is complicated is solved, the preparation time of the traditional membrane pushing method for preparing the cell membrane wrapping liposome is shortened, and the preparation process of the cell membrane bionic liposome is simplified.

Description

Technical Field

[0001] The invention relates to a microfluidic chip and application thereof in preparing liposomes, cell membrane vesicles or cell membrane hybrid lipid nanoparticles, belonging to the technical field of microfluidic chips. Background Art

[0002] As an emerging field of science and technology, microfluidics can integrate biological and chemical processes on a single platform while easily controlling the flow behavior of small volumes of fluid in channels. In the design of microfluidic chips, only factors such as the geometry of the channels, the materials used to construct the devices, and the compatibility of these materials with various solvents need to be considered. Microfluidic chip laboratory technology can "shrink" traditional experimental chemical systems to a few square centimeters. The main feature of this system is the miniaturization of the fluid environment, in which chemical substances can be brought together by pump pressure for synthesis, separation, or analysis.

[0003] Existing methods for preparing liposomes often use thin film dispersion, extrusion, and supercritical extraction. These methods are not only complex and tedious to prepare, but also expensive and cannot achieve continuous production. The preparation of drug-loaded cell membrane nanoparticles often uses a cell membrane extruder, a method that is not only low in yield and costly, but also takes a long time to prepare. Currently, some microfluidic chips can be used for the preparation of liposomes or cell membrane vesicles, but each chip can only be used for the preparation of a single nanoparticle. Summary of the Invention

[0004] Purpose of the invention: The main purpose of the present invention is to provide a microfluidic chip and its application in the preparation of liposomes, cell membrane vesicles or cell membrane hybrid lipid nanoparticles, which can complete the synthesis of different types of lipid nanoparticles in one step through a single chip, so as to solve the problems of the current liposome preparation process being cumbersome, the preparation process being complex, the yield being low, and the inability to achieve continuous production.

[0005] Technical solution: The present invention provides a microfluidic chip, which includes a top cover layer, a sealing layer, a chip layer, and a bottom plate layer; the chip layer contains 5 inlet holes, 1 outlet hole, a primary mixing area, and a secondary mixing area; the second inlet hole and the first inlet hole intersect at a first mixing point through their respective channels, the first mixing point is connected to one end of the primary mixing area, and the other end of the primary mixing area intersects with the third to fifth inlet holes through their respective channels at a second mixing point; one end of the secondary mixing area is also connected to the second mixing point; and the other end of the secondary mixing area extends to the outlet hole.

[0006] The distance from the first inlet hole to the first mixing point is equal to the distance from the second inlet hole to the first mixing point; the distance from the fourth inlet hole to the second mixing point is equal to the distance from the fifth inlet hole to the second mixing point.

[0007] Among them, the ratio of the distance from the first inlet hole to the first mixing point, the straight-line distance from the first mixing point to the second mixing point, the distance from the third inlet hole to the second mixing point, the distance from the fourth inlet hole to the second mixing point and the straight-line distance from the second mixing point to the outlet hole is 0.5-1:1-2:1-2:1-2:1-3.

[0008] Wherein, the channels in the primary mixing area and the secondary mixing area are both tortuous curves.

[0009] Among them, the angle between the channel from the fourth inlet hole to the second mixing point and the channel from the fifth inlet hole to the second mixing point is 180°, the angle between the channel from the third inlet hole to the second mixing point and the channel from the fourth inlet hole to the second mixing point is 90°-150°, the angle between the line segment from the first mixing point to the second mixing point and the channel from the fourth inlet hole to the second mixing point is 20°-80°, and the angle between the channel from the first inlet hole to the first mixing point and the channel from the second inlet hole to the first mixing point is 20°-140°.

[0010] The diameters of the inlet and outlet holes are 0.5-0.8 mm, and the diameters of all passages are 100 μm-300 μm.

[0011] The second inlet hole is fed with a mixed solution of sodium deoxycholate and Trition X 100; the first inlet hole is fed with a pure water solution containing dissolved cell membranes or a phosphate buffer solution containing dissolved cell membranes; the third inlet hole is fed with an organic solvent containing dissolved phospholipid material; and the fourth and fifth inlet holes are fed with pure water or a phosphate buffer solution.

[0012] The concentration of Trition X 100 is 0-4 mM, and the concentration of sodium deoxycholate is 0-5 mM; the concentrations of the two solutions cannot be 0 mM at the same time.

[0013] The phospholipid materials are DSPC, cholesterol and DSPE-PEG2000, and the organic solvent is ethanol, methanol, a mixed solution of ethanol and pure water, or a mixed solution of methanol and pure water.

[0014] Among them, the flow rates of the liquid entering the fourth inlet hole and the fifth inlet hole are consistent; the flow rates of the liquid entering the first inlet hole and the second inlet hole are consistent; the flow rate ratio of the liquid entering the fourth inlet hole to the liquid entering the first inlet hole is 5:1-15:1, and the flow rate ratio of the liquid entering the fourth inlet hole to the liquid entering the third inlet hole is 5:1-15:1.

[0015] The base layer is made of transparent polymethyl methacrylate (PMMA), commonly known as acrylic, with a thickness of 1 mm to 5 mm. It primarily provides protection and support for the chip layer, and the transparent material facilitates observation of the mixing of different liquids within the chip layer.

[0016] The chip layer material can be a smooth flat plate made of one of polytetrafluoroethylene (PTFE), fluorinated ethylene propylene copolymer (FEP), perfluoroalkyl compound (PFA) and ethylene tetrafluoroethylene copolymer (ETFE), with a thickness of 1 mm to 2 mm. Laser etching is used to carve chip paths with a depth of 0.1 mm to 0.3 mm and a width of 0.1 mm to 0.3 mm on the surface of the material.

[0017] The chip layer material can be polydimethylsiloxane (PDMS), with a volume ratio of PDMS to crosslinker of 1:7-1:14. A male mold with chip channels 0.1 mm-0.3 mm in height and 0.1 mm-0.3 mm in width is prepared. A mixed solution of PDMS and crosslinker is poured into the mold and removed after curing to create a chip with channels.

[0018] The present invention also provides application of the microfluidic chip in preparing liposomes, cell membrane vesicles or cell membrane hybrid lipid nanoparticles.

[0019] The chip of the present invention can realize the preparation of liposomes, cell membrane vesicles and cell membrane vesicle biomimetic liposomes through a single chip, and can encapsulate not only water-soluble drugs but also fat-soluble drugs. In addition, the corresponding nanoparticles can be prepared in one step through the chip by simply introducing the corresponding solution into the chip inlet, which effectively simplifies the preparation process, reduces the preparation cost and realizes continuous production.

[0020] Beneficial effects: (1) The chip device provided by the present invention has a simple preparation process and low material cost. It only needs to change the type of liquid introduced into different inlets to achieve the preparation of liposomes, cell membrane vesicles and cell membrane biomimetic liposomes. (2) It only needs to change the flow rate and flow rate ratio of the fluid introduced into different inlets to control the particle size of the prepared nanoparticles. (3) Compared with the traditional method, the prepared nanoparticles have better uniformity between particles and improved dispersibility. (4) Compared with the traditional method, the preparation of cell membrane biomimetic liposomes by this device can effectively increase the yield while shortening the preparation time and simplifying the preparation process. (5) By connecting multiple chips, continuous production of nanoparticles can be achieved by only continuously introducing liquid. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1Figure 1 shows a physical image of the chip (A), a three-dimensional simulation image of the chip (B), a top view of the chip device's top cover layer (C), a top view of the sealing layer (D), a top view of the chip layer (E), and a top view of the bottom plate layer (F).

[0022] Figure 2 The changes in particle size and PDI of empty liposomes prepared under different total flow rates and flow rate ratios.

[0023] Figure 3 Figure 3 is the particle size distribution diagram and transmission electron microscopy image of simvastatin liposomes.

[0024] Figure 4 The particle size distribution diagram and transmission electron micrograph of MO-type macrophage membrane vesicles.

[0025] Figure 5 The particle size distribution diagram and transmission electron microscopy image of blank M2 macrophage membrane biomimetic liposomes.

[0026] Figure 6 The particle size distribution diagram and transmission electron microscopy image of simvastatin M2 macrophage membrane biomimetic liposomes.

[0027] Figure 7 These are the SDS-PAGE results of membrane proteins of M2 macrophages, M2 macrophage membranes, empty liposomes and blank M2 macrophage membrane biomimetic liposomes.

[0028] Figure 8 Figure 35-day stability study results of empty liposomes, simvastatin liposomes, blank M2 macrophage membrane biomimetic liposomes, and simvastatin M2 macrophage membrane biomimetic liposomes: (A) particle size, (B) PDI, (C) zeta potential, and (D) drug encapsulation efficiency of simvastatin liposomes and simvastatin M2 macrophage membrane biomimetic liposomes with drug loadings of 6%, 8%, and 10%, respectively. DETAILED DESCRIPTION

[0029] Example 1 A synthesis device for different types of cell membrane hybrid lipid nanoparticles like Figure 1 As shown in Figures 1A, 1B, and 1F, the chip device comprises a four-layer structure, including a top cover layer, a sealing layer, a chip layer, and a bottom plate layer. Each layer is provided with six interconnected screw holes 11 to 16. The top cover layer is used to protect the chip layer, and a steel needle is inserted at the corresponding inlet and outlet to connect the chip layer to the external silicone tube. Figure 1 As shown in C and 1D, the top cover layer is provided with inlet holes 1 to 5 and outlet hole 6 communicating with the sealing layer and the chip layer. The sealing layer is used to seal the chip channel and connect the chip layer and the top cover layer. Figure 1As shown in Figure E, the chip layer also has a primary mixing area 7 and a secondary mixing area 8. Inlet holes 1 and 2 intersect at a mixing point 9 through their respective channels. This mixing point 9 is connected to one end of the primary mixing area 7. The other end of the primary mixing area 7 intersects with inlet holes 3-5 through their respective channels at a mixing point 10. One end of the secondary mixing area 8 also communicates with the mixing point 10. The other end of the secondary mixing area 8 extends to the outlet hole 6. The distance from inlet hole 1 to the mixing point 9 is equal to the distance from inlet hole 2 to the mixing point 9, measuring 0.5-1 cm. The straight-line distance from the mixing point 9 to the mixing point 10 is 1-2 cm. The distance from inlet hole 3 to the mixing point 10 is 1-2 cm. The distances from inlet hole 4 to the mixing point 10 and from inlet hole 5 to the mixing point 10 are equal, measuring 1-2 cm. The straight-line distance from the mixing point 10 to the outlet hole 6 is 1-3 cm.

[0030] The angle between the channel from inlet hole 4 to mixing point 10 and the channel from inlet hole 5 to mixing point 10 is 180°, the angle between the channel from inlet hole 3 to mixing point 10 and the channel from inlet hole 4 to mixing point 10 is 90°-150°, and the angle between the line segment from mixing point 9 to mixing point 10 and the channel from inlet hole 4 to mixing point 10 is 20°-80°. The present invention controls the shear force of the water phase on the organic phase and the cell membrane solution by adjusting the size of the above-mentioned angles, thereby controlling the particle size of the prepared particles. The angle between the channel from inlet hole 1 to mixing point 9 and the channel from inlet hole 2 to mixing point 9 of the mixed solution is 20°-140°. By adjusting the size of the angle, the shear force of the liquid introduced into inlet hole 2 on the cell membrane solution introduced into inlet hole 1 is controlled, thereby making the cell membrane solution more uniform. The circular hole diameters of inlet holes 1-5 and outlet hole 6 are 0.5-0.8 mm. The diameters of all channels in the chip of the present invention are 100 μm-300 μm, and the channel depth is consistent with the circular hole depth, which is 50 μm-300 μm.

[0031] The channel in the primary mixing region 7 is a tortuous curve, which can make the cell membrane in the solution passed through the inlet hole 1 be sheared more evenly by the mixed solution of sodium deoxycholate and Trition X 100. The channel in the secondary mixing region 8 is also a curve, which can make the prepared nanovesicles continuously roll in the curve path, thereby making the prepared nanovesicles more uniform in size. The present invention can change the particle size of the nanovesicles by adjusting the number of bends and the angle of the bends, as well as the angle between the above-mentioned channels. Generally, the number of bends in the primary mixing region 7 is 5-10, the number of bends in the secondary mixing region 8 is 15-20, and the bend angle is 30°-90°.

[0032] A mixed solution of sodium deoxycholate and Trition X 100 flows from inlet hole 2 to the chip layer through a silicone tube. A pure water solution or a phosphate buffer solution containing dissolved cell membranes flows from inlet hole 1 to the chip layer. The two solutions then pass through their respective channels to meet at mixing point 9. The mixed solution then flows through primary mixing area 7 to mixing point 10. Furthermore, an organic solvent containing a dissolved phospholipid material flows from inlet hole 3, and pure water or a phosphate buffer solution flows from inlet holes 4 and 5, respectively, into the chip layer. The two solutions then pass through their respective channels to meet at mixing point 10. The mixed solution then flows into secondary mixing area 8 and outlet hole 6, and then flows out through the silicone tube. Among them, the flow rate of the liquid entering the inlet holes 4 and 5 is kept consistent; the flow rate of the liquid entering the inlet hole 1 and the inlet hole 2 is kept consistent; the flow rate of the liquid entering the inlet hole 4 (flow rate of inlet hole 4 + flow rate of inlet hole 5) / the flow rate of the liquid entering the inlet hole 1 or the inlet hole 2 is 5:1-15:1, and the flow rate of the liquid entering the inlet hole 3 (flow rate of inlet hole 4 + flow rate of inlet hole 5) is 5:1-15:1.

[0033] The base and cover layers are made of transparent polymethyl methacrylate (PMMA) with a thickness of 1 mm to 5 mm. Their length and width can be the same as or longer than the chip layer. They primarily provide protection and support for the chip layer. The transparent material facilitates observation of the mixing of different liquids within the chip layer. The chip layer can be made of a smooth, 1 mm to 2 mm thick flat plate made of polytetrafluoroethylene (PTFE), fluorinated ethylene propylene (FEP), perfluoroalkyl (PFA), or ethylene tetrafluoroethylene (ETFE). The chip and sealing layers can be made of polydimethylsiloxane (PDMS) and a crosslinker in a volume ratio of 1:7 to 1:14. After curing at 80°C for 2 h, ensure that their thickness is greater than the channel depth and their length and width are both longer than the chip channel, ensuring that the channel is entirely within the chip layer.

[0034] In the chip devices described in the following examples, the distance from inlet hole 1 to mixing point 9 is equal to the distance from inlet hole 2 to mixing point 9, measuring 0.7 cm. The linear distance from mixing point 9 to mixing point 10 is 2 cm. The distance from inlet hole 3 to mixing point 10 is 1.6 cm. The distances from inlet hole 4 to mixing point 10 and from inlet hole 5 to mixing point 10 are equal, measuring 1.5 cm. The linear distance from mixing point 10 to outlet hole 6 is 2 cm. The circular hole depth remains consistent with the chip via depth, at 200 μm. The angle between the channel from the inlet hole 4 to the mixing point 10 and the channel from the inlet hole 5 to the mixing point 10 is 180°, the angle between the channel from the inlet hole 3 to the mixing point 10 and the channel from the inlet hole 4 to the mixing point 10 is 150°, the angle between the line segment where the mixing point 9 is located and the channel from the inlet hole 4 to the mixing point 10 is 40°, the angle between the channel from the inlet hole 1 to the mixing point 9 and the channel from the mixed solution inlet hole 2 to the mixing point 9 is 90°, and by adjusting the angle, the shear force of the cell membrane solution introduced by the liquid in the inlet hole 2 to the cell membrane solution introduced by the inlet hole 1 is controlled, so that the cell membrane solution is more uniform. The circular hole diameter of the inlet hole 1 to 5 and the outlet hole 6 is 800 μm and the depth is 2 mm. The diameter of all passages of the present invention is 200 μm, and the depth is consistent with the circular hole depth.

[0035] The channel in the primary mixing zone 7 is a tortuous curve, which allows the cell membrane solution to be more evenly sheared by the mixed solution of sodium deoxycholate and Trition X 100. The channel in the secondary mixing zone 8 is also curved, which allows the prepared nanovesicles to tumble continuously along the curved path, thereby making the prepared nanovesicles more uniform in size. The primary mixing zone 7 has 9 curves, and the secondary mixing zone has 18 curves, with the angle of the curves being 45°.

[0036] The bottom and top layers are 2 mm thick, 4 cm long, and 3 cm wide. The chip layer is a smooth, 2 mm thick flat plate made of perfluoroalkyl (PFA). Laser etching is used to create chip pathways 200 μm deep and 200 μm wide. The sealing layer is made of polydimethylsiloxane (PDMS) with a volume ratio of 1:9. After curing at 80°C for 2 h, a 2 mm thick, 4 cm long, and 3 cm wide elastic membrane is formed.

[0037] Example 2: Preparation of empty liposomes under different flow rate conditions 10 mg of distearoylphosphatidylcholine (DSPC), 5 mg of cholesterol, and 2 mg of DSPE-PEG2000 (phospholipid-methoxypolyethylene glycol) were dissolved in 2 ml of anhydrous ethanol as stock solution No. 1. In the first step, stock solution No. 1 was introduced through inlet hole 3 of the chip. Second, 10 mL of pure water was introduced through inlet holes 4 and 5 of the chip, respectively. Third, because stock solution No. 1, which was introduced first, and the aqueous solution, which was introduced later, arrived at mixing point 10 at different times, the two solutions were not fully mixed. Therefore, it was necessary to wait until both stock solution No. 1 and water reached mixing point 10 before passing through secondary mixing zone 8. The prepared blank liposome solution was then collected at collection outlet hole 6 of the chip. Among them, the flow rates of inlet hole 3, inlet hole 4 and inlet hole 5 were changed, and the flow rate ratios of (inlet hole 4 flow rate + inlet hole 5 flow rate) / inlet hole 3 were set to 5:1, 10:1 and 15:1 respectively. Among them, the sum of the flow rates of inlet hole 3, inlet hole 4 and inlet hole 5 were 300 μL / mL, 500 μL / mL, 1000 μL / mL and 1500 μL / mL respectively, and the flow rate of inlet hole 4 was the same as that of inlet hole 5. Then, the prepared blank liposome solution was collected at the collection outlet hole 6. Finally, the particle size and PDI were characterized using a laser particle size analyzer. The results are shown in FIG. Figure 2 As shown, as the flow rate ratio (flow rate at organic solution inlet hole 3 / flow rate at aqueous solution inlet hole 4 + flow rate at aqueous solution inlet hole 5) increases, the particle size of the prepared empty liposomes gradually decreases, and the PDI value also gradually decreases. Under the same flow rate ratio, a higher total flow rate results in a smaller particle size of the prepared empty liposomes, with little effect on the PDI value. By varying the flow rate ratio and total flow rate, liposome particle size can be controlled. Therefore, this device can be used to prepare liposomes of a target size according to experimental requirements.

[0038] Example 3: Preparation of Simvastatin Liposomes 10 mg DSPC, 5 mg cholesterol, 2 mg DSPE-PEG2000 and 1.5 mg simvastatin were dissolved in 2 ml of anhydrous ethanol as reserve solution No. 1. In the first step, the reserve solution No. 1 was introduced from the inlet hole 3 of the chip, and the flow rate was set to 5 mL / h; in the second step, 10 mL of pure water was introduced from the inlet hole 4 and the inlet hole 5 of the chip respectively, and the flow rates of the aqueous solution inlet hole 4 and the aqueous solution inlet hole 5 were set to 25 mL / h. In the third step, when both the reserve solution No. 1 and the pure water reached the mixing point 10, they began to pass through the secondary mixing area 8 and collected the prepared fat-soluble drug-encapsulated liposomes at the collection outlet hole 6 of the chip. Its appearance was observed by transmission electron microscopy, and its particle size, potential and PDI were characterized by a laser particle size analyzer. The results are shown in FIG. Figure 3As shown, the average particle size of simvastatin liposomes prepared using the chip device is 187.5±2.34 nm, and the average PDI value is 0.242±0.003. Transmission electron microscopy results show that the prepared simvastatin liposomes have a smooth surface, a clear bilayer structure, good uniformity between particles, and good dispersion. Liposomes can be successfully prepared using this device.

[0039] Example 4: Preparation of M0 macrophage membrane vesicles Weigh 3.11 mg of sodium deoxycholate and 3.4 μL of TritionX-100 and dissolve them in 5 mL of pure water as the second stock solution. Prepare 2 mL of a macrophage membrane solution with a concentration of 200 μg / mL as the third stock solution. The first step is to pass the third stock solution through the inlet hole 1 of the chip and set the flow rate to 3 mL / h; the second step is to pass the second stock solution through the inlet hole 2 of the chip and set the flow rate to 3 mL / h; the third step is to pass 10 mL of pure water through the aqueous solution inlet hole 4 and the aqueous solution inlet hole 5 of the chip respectively and set the flow rate to 25 mL / h. The fourth step is that when the second stock solution and the third stock solution are mixed through the primary mixing area 7 and reach the mixing point 10, and the pure water reaches the mixing point 10, they start to pass through the secondary mixing area 8 and collect the prepared macrophage membrane vesicles at the collection outlet hole 6 of the chip. The appearance is observed by transmission electron microscopy, and the particle size, potential and PDI are characterized by a laser particle size analyzer. The results are as follows Figure 4 As shown in the figure, the average particle size of the M0 macrophage membrane vesicles prepared using the chip device is 137.37±0.125 nm, and the average PDI value is 0.247±0.003. Transmission electron microscopy results show that the prepared M0 macrophage membrane vesicles are spherical, with a smooth surface and good uniformity between particles. This device can be used to successfully prepare cell membrane vesicles.

[0040] Example 5: Preparation of blank M2 macrophage membrane biomimetic liposomes Dissolve 10 mg of DSPC, 5 mg of cholesterol, and 2 mg of DSPE-PEG-2000 in 2 mL of anhydrous ethanol as stock solution #1. Weigh 3.11 mg of sodium deoxycholate and 3.4 μL of TritionX-100 and dissolve them in 5 mL of pure water as stock solution #2. Prepare 2 mL of a 200 μg / mL macrophage membrane solution as stock solution #3. First, introduce stock solution #1 into the chip through inlet port 3 at a flow rate of 5 mL / h. Second, introduce stock solution #2 into the chip through inlet port 2 at a flow rate of 3 mL / h. Third, introduce stock solution #3 into the chip through inlet port 1 at a flow rate of 3 mL / h. Fourth, introduce 10 mL of pure water into inlet ports 4 and 5, respectively, at a flow rate of 25 mL / h. In the fifth step, when the No. 1 stock solution reaches the mixing point 10, the No. 2 and No. 3 stock solutions are mixed in the primary mixing area 7 and then reach the mixing point 10. When the pure water reaches the mixing point 10, it begins to pass through the secondary mixing area 8 and is collected at the collection outlet 6 of the chip. The appearance of the prepared blank M2 macrophage membrane biomimetic liposomes is observed by transmission electron microscopy, and the particle size, potential, and PDI are characterized using a laser particle size analyzer. The results are shown in FIG. Figure 5 As shown in the figure, the average particle size of the blank M2 macrophage membrane biomimetic liposomes prepared by the chip device is 157.42±1.364 nm, and the average PDI value is 0.217±0.021. The transmission electron microscopy results show that the prepared blank M2 macrophage membrane biomimetic liposomes are spherical, with a smooth surface, a clear bilayer structure, and good uniformity between the particles. The device can be used to successfully prepare cell membrane biomimetic liposomes.

[0041] Example 6: Preparation of Simvastatin M2 Macrophage Membrane Biomimetic Liposomes Dissolve 10 mg of DSPC, 5 mg of cholesterol, 2 mg of DSPE-PEG-2000, and 1.5 mg of simvastatin in 2 mL of anhydrous ethanol as stock solution #1. Weigh 3.11 mg of sodium deoxycholate and 3.4 μL of TritionX-100 and dissolve them in 5 mL of pure water as stock solution #2. Prepare 2 mL of a 200 μg / mL macrophage membrane solution as stock solution #3. First, introduce stock solution #1 into the chip through inlet port 3 at a flow rate of 5 mL / h. Second, introduce stock solution #2 into the chip through inlet port 2 at a flow rate of 3 mL / h. Third, introduce stock solution #3 into the chip through inlet port 1 at a flow rate of 3 mL / h. Fourth, introduce 10 mL of pure water into inlet ports 4 and 5, respectively, at a flow rate of 25 mL / h. In the fifth step, when the No. 1 stock solution reaches the mixing point 10, the No. 2 stock solution and the No. 3 stock solution are mixed in the primary mixing area 7 and then reach the mixing point 10. When the water reaches the mixing point 10, it begins to pass through the secondary mixing area 8. The prepared macrophage membrane biomimetic liposomes loaded with fat-soluble drugs are collected at the collection outlet hole 6 of the chip. The appearance of the macrophage membrane biomimetic liposomes is observed by transmission electron microscopy, and the particle size, potential and PDI are characterized by a laser particle size analyzer. The results are shown in FIG. Figure 6 As shown in the figure, the average particle size of simvastatin M2 macrophage membrane biomimetic liposomes prepared by the chip device is 161.9±1.364 nm, and the average PDI value is 0.251±0.017. Transmission electron microscopy results show that the prepared simvastatin M2 macrophage membrane biomimetic liposomes are spherical, with a smooth surface, a clear bilayer structure, and good uniformity between the particles. This device can be used to successfully prepare cell membrane biomimetic liposomes.

[0042] Example 7: SDS-PAGE results of the empty liposomes prepared in Example 2 and the blank M2 macrophage membrane biomimetic liposomes prepared in Example 5 M2 macrophages (5×10 5 / mL), M2 macrophage membrane (M2 macrophage membrane concentration is 300 μg / mL), empty liposomes prepared in Example 2 (phospholipid concentration is 5 mg / mL), and blank M2 macrophage membrane biomimetic liposomes prepared in Example 5 (M2 macrophage membrane concentration is 300 μg / mL) were dissolved in loading buffer, then boiled at 100°C for 5 minutes, 10 μL of sample was added to the precast gel, and the gel was run at 120V. After the end, protein band analysis was performed. The results are shown in Figure 2. Figure 7As shown, M2 macrophages, M2 macrophage membranes, and blank M2 macrophage membrane-mimetic liposomes all expressed distinct characteristic proteins near 35 kD, 40-50 kD, 70 kD, 100 kD, and 150 kD, and the bands of the three samples were highly similar, indicating that the extracted M2 macrophage membranes were derived from M2 macrophages and that the surface of the blank M2 macrophage membrane-mimetic liposomes contained M2 macrophage-specific proteins. In contrast, the empty liposome sample showed no protein expression. This demonstrates that the device successfully encapsulated / embedded M2 macrophage membranes on the liposome surface and that cell membrane-mimetic liposomes can be successfully prepared using this device.

[0043] Example 8: Stability Study Results of Empty Liposomes Prepared in Example 2, Simvastatin Liposomes Prepared in Example 3, Blank M2 Macrophage Membrane Biomimetic Liposomes Prepared in Example 5, and Simvastatin M2 Macrophage Membrane Biomimetic Liposomes Prepared in Example 6 Take the empty liposomes prepared in Example 2 (phospholipid concentration of 5 mg / mL), the simvastatin liposomes prepared in Example 3 (phospholipid concentration of 5 mg / mL), the blank M2 macrophage membrane biomimetic liposomes prepared in Example 5 (phospholipid concentration of 5 mg / mL), and the simvastatin M2 macrophage membrane biomimetic liposomes prepared in Example 6 (phospholipid concentration of 5 mg / mL), and store them in a stability test box at a temperature of 25 ± 2°C and a relative humidity of 40 ± 5% to investigate their storage stability for 35 days. Samples were taken out on the 1st, 2nd, 3rd, 6th, 10th, 14th, 21st, 28th and 35th day, and their particle size, PDI, zeta potential and EE were detected, and the relevant changes were recorded. The results are as follows. Figure 8 As shown, the empty liposomes, simvastatin liposomes, blank M2 macrophage membrane-mimetic liposomes, and simvastatin M2 macrophage membrane-mimetic liposomes prepared using this chip device showed excellent stability over 35 days. The particle size of the empty liposomes, blank M2 macrophage membrane-mimetic liposomes, and simvastatin M2 macrophage membrane-mimetic liposomes changed minimally, with a 35-day increase of approximately 20 nm. The PDI of the four formulations remained generally between 0.2 and 0.3, and the potential remained around -20 mV, indicating good dispersion and uniformity between particles. Over time, the drug encapsulation efficiency decreased from 90% to approximately 60%, with minimal drug leakage, demonstrating the excellent drug encapsulation capacity of this carrier.

[0044] In summary, the chip device can successfully prepare liposomes, cell membrane vesicles and cell membrane biomimetic liposomes, and the prepared nanoparticles have good stability.

Claims

1. A microfluidic chip, characterized in that: It comprises a top cover layer, a sealing layer, a chip layer, and a bottom plate layer; the chip layer comprises inlet holes (1) to (5), an outlet hole (6), a primary mixing area (7), and a secondary mixing area (8); the inlet hole (1) and the inlet hole (2) intersect at a mixing point (9) through respective channels, the mixing point (9) is connected to one end of the primary mixing area (7), and the other end of the primary mixing area (7) intersects with the inlet holes (3) to (5) through respective channels at a mixing point (10); one end of the secondary mixing area (8) is also connected to the mixing point (10); and the other end of the secondary mixing area (8) extends to the outlet hole (6).

2. The microfluidic chip according to claim 1, characterized in that: The distance from the inlet hole (1) to the mixing point (9) is equal to the distance from the inlet hole (2) to the mixing point (9); the distance from the inlet hole (4) to the mixing point (10) is equal to the distance from the inlet hole (5) to the mixing point (10).

3. The microfluidic chip according to claim 1, characterized in that: The ratio of the distance from the inlet hole (1) to the mixing point (9), the straight-line distance from the mixing point (9) to the mixing point (10), the distance from the inlet hole (3) to the mixing point (10), the distance from the inlet hole (4) to the mixing point (10), and the straight-line distance from the mixing point (10) to the outlet hole (6) is 0.5-1:1-2:1-2:1-2:1-3.

4. The microfluidic chip according to claim 1, characterized in that: The channels in the primary mixing area (7) and the secondary mixing area (8) are both tortuous bends.

5. The microfluidic chip according to claim 1, characterized in that: The angle between the channel from the inlet hole (4) to the mixing point (10) and the channel from the inlet hole (5) to the mixing point (10) is 180°, the angle between the channel from the inlet hole (3) to the mixing point (10) and the channel from the inlet hole (4) to the mixing point (10) is 90°-150°, the angle between the line segment from the mixing point (9) to the mixing point (10) and the channel from the inlet hole (4) to the mixing point (10) is 20°-80°, and the angle between the channel from the inlet hole (1) to the mixing point (9) and the channel from the mixed solution inlet hole (2) to the mixing point (9) is 20°-140°.

6. The microfluidic chip according to claim 1, characterized in that: The diameters of the circular holes of the inlet holes (1) to (5) and the outlet hole (6) are 0.5-0.8 mm; the diameters of all the passages are 100 μm-300 μm.

7. The microfluidic chip according to claim 1, characterized in that: A mixed solution of sodium deoxycholate and TritionX 100 is introduced into the inlet hole (2); a pure water solution in which cell membranes are dissolved or a phosphate buffer solution in which cell membranes are dissolved is introduced into the inlet hole (1); an organic solvent in which phospholipid material is dissolved is introduced into the inlet hole (3); and pure water or a phosphate buffer solution is introduced into the inlet hole (4) and the inlet hole (5).

8. The microfluidic chip according to claim 1, characterized in that: The flow rates of the liquid introduced through the inlet hole (4) and the inlet hole (5) are kept consistent; the flow rates of the liquid introduced through the inlet hole (1) and the inlet hole (2) are kept consistent; the ratio of the flow rate of the liquid introduced through the inlet hole (4) to the flow rate of the liquid introduced through the inlet hole (1) is 5:1-15:1, and the ratio of the flow rate of the liquid introduced through the inlet hole (4) to the flow rate of the liquid introduced through the inlet hole (3) is 5:1-15:

1.

9. The microfluidic chip according to claim 7, characterized in that: The concentration of Trition X 100 is 0-4 mM, and the concentration of sodium deoxycholate is 0-5 mM; the concentrations of the two solutions cannot be 0 mM at the same time.

10. Use of the microfluidic chip according to any one of claims 1 to 9 in preparing liposomes, cell membrane vesicles or cell membrane hybrid lipid nanoparticles.

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