A microfluidic device and method for preparing composite biofilm-coated drug-loaded nanoparticles

Abstract

The present invention discloses a microfluidic device and method for preparing composite biofilm-coated drug-loaded nanoparticles. The device includes at least one cell membrane channel, and when there are two or more cell membrane channels, they intersect in front of a cell membrane mixing channel to form a cell membrane mixing channel; a method for synthesizing composite biofilm-coated drug-loaded nanoparticles in one step is also provided, and the fluid flow rate and the final output drug concentration and the size of the nanoparticles are precisely controlled by using a microfluidic chip. By changing the flow channel to avoid direct contact and mixing between the cell membrane solution and the organic reagent, the solution after the nanoparticles are precipitated contains only a small amount of organic solvent, and then coating the nanoparticles through a spiral channel can prevent the high concentration of organic solvent from damaging the cell membrane, and the cell membrane can be better coated on the nanoparticles.

Description

Technical Field

[0001] The invention belongs to the technical field of drug-carrying nanosystems, and in particular relates to a microfluidic device and a method for preparing composite biofilm-coated drug-carrying nanoparticles. Background Art

[0002] Cancer is the second largest disease threatening human life and health worldwide, and chemotherapy is an important method to fight cancer. In order to treat cancer more effectively, providing the best drug combination is an important challenge facing clinicians.

[0003] The process of chemotherapy is affected by the pathogenesis and causes of various tumors and the accompanying symptoms, which complicates the drug resistance of tumors. The drugs used in chemotherapy are mainly cytostatics, which have strong toxic side effects on the human body. In order to better deal with these problems, it is particularly important to develop new drugs and change the delivery method to reduce their toxic side effects, enhance the anti-tumor effect and overcome tumor resistance.

[0004] Nano-formulation of drugs can significantly improve the safety and effectiveness of anticancer drugs. The larger surface area to volume ratio and specific structural characteristics of nano-drugs can enhance their ability to circulate in the body. Compared with general anticancer drugs, nano-formulated anticancer drugs can remain in the blood circulation for a longer time.

[0005] Membrane proteins on natural biological cell membranes have many practical functions. Coating different cell membranes on drug-loaded nanoparticles can give the nanoparticles various functions. For example, coating red blood cell membranes can avoid being recognized by immune cells and thus not being eliminated, and coating the cell membranes of corresponding tumor cells can give the nanoparticles active targeting functions. Fusion of different cell membranes can further enhance the functionality of the membrane.

[0006] At present, the main method to promote membrane fusion is to ultrasonically treat the mixed membrane solution, destroy the membrane structure through the squeezing force of the ultrasonic field and make it reassemble itself to achieve the effect of membrane fusion. However, a large amount of heat is generated during the ultrasonic process, which may cause membrane protein denaturation during the fusion process and thus cause it to lose its original function. In addition, nanoparticles prepared by ultrasound often have large differences in batches and poor dispersion.

[0007] In order to overcome these problems, the focus is on the microfluidics technology for preparing nanoparticles. Microfluidics, as a sub-millimeter-level fluid engineering manipulation technology, can provide better reproducibility and controllability by leading the mixing with the flow in the channel. These characteristics can make the prepared nanoparticles have better dispersion and smaller batch-to-batch differences.

[0008] In the prior art, Chinese Patent Publication No. CN110560186A discloses a microfluidic chip that combines ultrasound to coat cell membranes with nanoparticles.

[0009] The microfluidic chip has the following problems:

[0010] First, the microfluidic chip directly combines the cell membrane solution and the polymer solution in the first mixing channel, and the direct mixing of the organic solution and the membrane solution will damage the cell membrane.

[0011] Second, the right-angle bend provided at the beginning of the second mixing channel of the microfluidic chip easily causes the nanoparticles to aggregate and precipitate, thereby causing the chip to be blocked.

[0012] Third, the microfluidic chip needs to be equipped with an additional acoustic field to assist the chip in cell membrane coating of the nanoparticles. The entire chip needs to be immersed in a bath sonicator. In addition to the necessary microinjection pump, additional instruments are required, which increases the complexity of the operation. Summary of the invention

[0013] In view of this, the purpose of the present invention is to provide a microfluidic device and method for preparing composite biofilm-coated drug-loaded nanoparticles, so as to overcome the problems in the prior art that natural biological cell membranes cannot be fused under a mild environment and the prepared nanoparticles have unstable dispersion.

[0014] To achieve the above object, the present invention provides the following technical solutions:

[0015] The present invention discloses a microfluidic device for preparing composite biofilm-coated drug-loaded nanoparticles, the device comprises at least one cell membrane channel, when there are two or more cell membrane channels, they intersect in front of a cell membrane mixing channel, and converge into a cell membrane mixing channel; at least one PBS channel, a polymer channel, a nanoparticle mixing channel formed by the convergence of at least one PBS channel and a polymer channel; a cell membrane-coated channel formed by the convergence of a cell membrane mixing channel and a nanoparticle mixing channel. The present invention provides a method for synthesizing composite biofilm-coated drug-loaded nanoparticles in one step, by using a microfluidic chip to precisely control the fluid flow rate and the final output drug concentration and the size of the nanoparticles.

[0016] As a further improvement, the device described in the present invention is specifically as follows: two cell membrane channels that pass natural biological cell membrane solutions perform cell membrane fusion in a cell membrane mixing channel, two PBS channels that pass PBS solutions and a polymer channel that passes an organic solution containing a polymer for encapsulating drugs and a specific drug perform nanoprecipitation in a nanoparticle mixing channel, the cell membrane solution fused in the cell membrane mixing channel and the nanoparticles precipitated in the nanoparticle mixing channel intersect in front of the cell membrane coating channel and are mixed in the cell membrane coating channel so that the fused cell membranes are coated with nanoparticles.

[0017] As a further improvement, the shape of the cell membrane mixing channel and the nanoparticle mixing channel described in the present invention is any one of a single helix, a double helix, and a wave shape, and the shape of the cell membrane coated channel is any one of a single helix and a double helix.

[0018] As a further improvement, the cell membrane channel, polymer channel and PBS channel described in the present invention have independent inlets respectively, and the cell membrane-coated channel has an outlet. An injection device is provided at each inlet, which can be used to accurately control the input of each solution at a specified flow rate.

[0019] The present invention also discloses a method for preparing composite biofilm-coated drug-loaded nanoparticles using a microfluidic device, which is characterized by comprising the following steps:

[0020] Step 1: introducing PBS solutions of two natural biological cell membranes into two cell membrane channels respectively, so that the membranes fuse in the cell membrane mixing channel;

[0021] Step 2: an organic solution containing a polymer for encapsulating drugs and a specific drug is introduced into the polymer channel, and a PBS solution is introduced into the PBS channels on both sides of the polymer channel, so that the solutions in the three channels are mixed in the nanoparticle mixing channel;

[0022] Step 3: The cell membrane solution mixed and fused through the cell membrane mixing channel and the drug-loaded polymer nanoparticle solution mixed and precipitated through the nanoparticle mixing channel intersect in front of the cell membrane coating channel and mix in the cell membrane coating channel, so that the fused cell membranes coat the nanoparticles;

[0023] Step 4: After the preparation is completed, the mixed solution containing the composite biofilm-coated drug-loaded nanoparticles is output from the last channel port to complete the preparation of the composite biofilm-coated drug-loaded nanoparticles.

[0024] As a further improvement, the polymer described in the present invention is any one of polylactic acid-glycolic acid copolymer, polylactic acid, and polyglycolic acid.

[0025] As a further improvement, the organic solvent described in the present invention is any one of dimethyl sulfoxide, dimethylformamide, methanol, ethanol, and acetonitrile.

[0026] As a further improvement, the specific drug described in the present invention is any one of fat-soluble anticancer drugs such as doxorubicin, taxane, camptothecin, curcumin, and daunorubicin.

[0027] As a further improvement, the natural biological cell membrane described in the present invention is any one of a blood cell membrane, a tumor cell membrane, a macrophage membrane, an exosome, and a bacterial membrane.

[0028] Microfluidic devices include: cell membrane channels, cell membrane mixing channels, polymer channels, PBS channels, nanoparticle mixing channels, and cell membrane coated channels according to fluid flow direction.

[0029] The cell membrane channel and the cell membrane mixing channel are used to transport the cell membrane solution.

[0030] The cell membrane mixing channel starts at the intersection of the cell membrane channels when there are multiple cell membrane channels. When there is only a single cell membrane channel, it is merged with the cell membrane mixing channel without distinction. It intersects with the nanoparticle mixing channel before the cell membrane coating channel to mix the fluid.

[0031] The polymer channel only includes a single inlet channel and a PBS channel intersecting before the nanoparticle mixing channel for delivering the polymer solution containing the drug.

[0032] The PBS channel at least comprises an inlet channel which is arranged in parallel with the polymer channel and intersects in front of the nanoparticle mixing channel for conveying the PBS solution.

[0033] The nanoparticle mixing channel starts at the intersection of the polymer channel and the PBS channel and intersects with the cell membrane mixing channel before the cell membrane coating channel to mix the fluids. The shape can be any one of single helix, double helix, and wave.

[0034] The cell membrane coated channel starts at the intersection of the cell membrane mixing channel and the nanoparticle mixing channel, and is used to mix the fluid, and the shape can be any one of a single helix and a double helix. Among them, the cell membrane channel, the polymer channel, and the PBS channel have independent entrances, and the cell membrane coated channel has an exit.

[0035] The present invention discloses a method for preparing composite biofilm-coated drug-loaded nanoparticles using a microfluidic chip, comprising:

[0036] Step 1: Introduce PBS solutions of two natural biological cell membranes into two cell membrane channels respectively, so that they can undergo membrane fusion in the cell membrane mixing channel.

[0037] Step 2: An organic solution containing a polymer for encapsulating drugs and a specific drug is introduced into the polymer channel, and PBS solution is introduced into the PBS channels on both sides of the polymer channel, so that the solutions in the three channels are mixed in the nanoparticle mixing channel.

[0038] Step 3: The cell membrane solution mixed and fused through the cell membrane mixing channel and the drug-loaded polymer nanoparticle solution mixed and precipitated through the nanoparticle mixing channel intersect in front of the cell membrane coating channel and are mixed in the cell membrane coating channel to coat the fused cell membrane nanoparticles.

[0039] Step 4: After the preparation is completed, the mixed solution containing the composite biofilm-coated drug-loaded nanoparticles is output from the last channel port to complete the preparation of the composite biofilm-coated drug-loaded nanoparticles.

[0040] The polymer includes any one of polylactic acid-glycolic acid copolymer, polylactic acid and polyglycolic acid.

[0041] The organic solvent is any one of dimethyl sulfoxide, dimethylformamide, methanol, ethanol and acetonitrile.

[0042] Specific drugs include fat-soluble anticancer drugs such as doxorubicin, taxanes, and camptothecin.

[0043] Natural biological cell membranes include any one of blood cell membranes, tumor cell membranes, macrophage membranes, exosomes, and bacterial membranes.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] The present invention applies microfluidic technology to membrane fusion, and provides a one-step method for preparing composite biofilm-coated drug-loaded nanoparticles.

[0046] By changing the flow channel to avoid direct contact and mixing between the cell membrane solution and the organic reagent, the solution after the nanoparticles are precipitated contains only a small amount of organic solvent. Then, coating the nanoparticles through the spiral channel can prevent high-concentration organic solvents from damaging the cell membrane and the cell membrane can be better coated on the nanoparticles.

[0047] Moreover, this method only uses microfluidic chips, does not require additional ultrasonic fields, and the channel will not produce backflow due to flow rate differences. By precisely controlling the concentration and flow rate of the material, the particle size and drug concentration of the prepared nanoparticles can be simply changed, and the particle morphology is stable and uniform. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Schematic diagram of the structure of the microfluidic chip of the present invention;

[0049] Figure 2 This is a particle size distribution diagram of B16F10 cell membrane nanoparticles in Example 1 of the present invention;

[0050] Figure 3 This is a transmission electron microscopy image of the B16F10 cell membrane nanoparticles in Example 1 of the present invention;

[0051] Figure 4 This is a transmission electron micrograph of the nanoparticles coated after the fusion of the B16F10 cell membrane and the red blood cell membrane in Example 2 of the present invention.

[0052] In the figure, 1 is the cell membrane channel, 2 is the nanoparticle mixing channel, 3 is the cell membrane mixing channel, 4 is the polymer channel, 5 is the PBS channel, and 6 is the cell membrane coated channel. DETAILED DESCRIPTION

[0053] The present invention discloses a microfluidic device for preparing composite biofilm-coated drug-loaded nanoparticles. Figure 1 The schematic diagram of the structure of the microfluidic chip of the present invention includes at least one cell membrane channel 1, when there are two or more cell membrane channels 1, they intersect in front of the cell membrane mixing channel 3 and converge into the cell membrane mixing channel 3; at least one PBS channel 5, a polymer channel 4, a nanoparticle mixing channel 2 formed by the convergence of at least one PBS channel 5 and a polymer channel 4; a cell membrane coating channel 6 formed by the convergence of the cell membrane mixing channel 3 and the nanoparticle mixing channel 2. The present invention provides a method for synthesizing drug-loaded nanoparticles coated with composite biofilms in one step, by using a microfluidic chip to accurately control the fluid flow rate and the final output drug concentration and the size of the nanoparticles. The shape of the cell membrane mixing channel 3 and the nanoparticle mixing channel 2 is any one of a single helix, a double helix, and a wave shape, and the shape of the cell membrane coating channel 6 is any one of a single helix and a double helix. The cell membrane channel 1, the polymer channel, and the PBS channel 5 have independent inlets respectively, and the cell membrane coating channel 6 is provided with an outlet. An injection device is provided at each inlet, which can be used to accurately control each solution to input at a specified flow rate.

[0054] The microfluidic device includes, according to the fluid flow direction: a cell membrane channel 1, a cell membrane mixing channel 3, a polymer channel, a PBS channel 5, a nanoparticle mixing channel 2, and a cell membrane coating channel 6;

[0055] The cell membrane channel 1 contains at least one inlet channel, and in the case of multiple channels, they converge in front of the cell membrane mixing channel 3 for transporting the cell membrane solution.

[0056] The cell membrane mixing channel 3 starts at the intersection of the cell membrane channel 1 when the cell membrane channel 1 has multiple channels. When the cell membrane channel 1 has only a single channel, it is merged with the cell membrane mixing channel 3 without distinction. It intersects with the nanoparticle mixing channel 2 before the cell membrane coating channel 6 to mix the fluid. The shape can be any one of a single helix, a double helix, and a wave.

[0057] The polymer channel only includes a single inlet channel and intersects with the PBS channel 5 before the nanoparticle mixing channel 2 for transporting the polymer solution containing the drug.

[0058] The PBS channel 5 comprises at least one inlet channel which is arranged in parallel with the polymer channel and intersects in front of the nanoparticle mixing channel 2 for conveying the PBS solution.

[0059] The nanoparticle mixing channel starts at the intersection of the polymer channel and the PBS channel 5 and intersects with the cell membrane mixing channel 3 before the cell membrane coating channel 6 to mix the fluids. The shape can be any one of single helix, double helix, and wave.

[0060] In this embodiment, the device described in the present invention is specifically as follows: two cell membrane channels 1 that pass natural biological cell membrane solutions perform cell membrane fusion in the cell membrane mixing channel 3, two PBS channels 5 that pass PBS solutions and a polymer channel that passes an organic solution containing a polymer for encapsulating drugs and a specific drug perform nanoprecipitation in the nanoparticle mixing channel 2, the cell membrane solution fused in the cell membrane mixing channel 3 and the nanoparticles precipitated in the nanoparticle mixing channel 2 intersect in front of the cell membrane coating channel 6 and are mixed in the cell membrane coating channel 6 to form fused cell membrane-coated nanoparticles.

[0061] The present invention also discloses a method for preparing composite biofilm-coated drug-loaded nanoparticles using a microfluidic chip, comprising the following steps:

[0062] Step 1: PBS solutions of two natural biological cell membranes are introduced into two cell membrane channels 1 respectively, so that the membranes are fused in the cell membrane mixing channel 3 .

[0063] Step 2: An organic solution containing a polymer for encapsulating drugs and a specific drug is introduced into the polymer channel, and PBS solution is introduced into the PBS channels 5 on both sides of the polymer channel, so that the solutions in the three channels are mixed in the nanoparticle mixing channel 2.

[0064] Step 3: The cell membrane solution mixed and fused through the cell membrane mixing channel 3 and the drug-loaded polymer nanoparticle solution mixed and precipitated through the nanoparticle mixing channel 2 intersect in front of the cell membrane coating channel 6 and are mixed in the cell membrane coating channel 6 to coat the fused cell membrane nanoparticles.

[0065] Step 4: After the preparation is completed, the mixed solution containing the composite biofilm-coated drug-loaded nanoparticles is output from the last channel port to complete the preparation of the composite biofilm-coated drug-loaded nanoparticles.

[0066] The polymer is any one of polylactic acid-glycolic acid copolymer, polylactic acid, and polyglycolic acid; the organic solvent is any one of dimethyl sulfoxide, dimethylformamide, methanol, ethanol, and acetonitrile; the specific drug is any one of fat-soluble anticancer drugs such as doxorubicin, taxane, camptothecin, curcumin, and daunorubicin; and the natural biological cell membrane is any one of a blood cell membrane, a tumor cell membrane, a macrophage membrane, an exosome, and a bacterial membrane.

[0067] The following describes the embodiments of the present invention with reference to the accompanying drawings. These embodiments are only used to explain the technical principles of the present invention and are not intended to limit the protection scope of the present invention.

[0068] like Figure 1 As shown, it is a schematic diagram of the structure of the microfluidic chip of the present invention. The chip includes two cell membrane channels 1, a cell membrane mixing channel 3, a polymer channel 4, two PBS channels 5, a nanoparticle mixing channel 2, and a cell membrane coating channel 6 according to the flow direction of the fluid.

[0069] Specifically, the two cell membrane channels 1 have independent entrances (cylindrical structure is the entrance for liquid in and out in the figure), converge to the cell membrane mixing channel 3 in a spiral shape, polymer channel 4 and two PBS channels 5 also have independent entrances, three channels converge to the nanoparticle mixing channel 2 in a spiral shape, cell membrane mixing channel 3 and nanoparticle mixing channel 2 converge to the cell membrane coating channel 6 and also in a spiral shape, and have a unique liquid outlet. Each entrance has an injection device, and the flow rate of each solution can be controlled by the injection device to input the microfluidic chip.

[0070] When making the microfluidic chip, the present invention selects PDMS (polydimethylsiloxane) as the material for making the microfluidic chip, the chip sealing surface is combined with soda-lime glass after plasma treatment, and plastic tubes are inserted at the entrances and exits of each channel. The manufacturing method can be made according to methods well known to technicians in the field of microfluidic chips. The channels on the microfluidic chip can be connected to the injection device through various connecting tubes.

[0071] When the microfluidic chip of the present invention is used, cell membrane solutions are respectively introduced into the two cell membrane channels 1 so that the cell membranes are fully mixed and fused in the cell membrane mixing channel 3, an organic solution containing a polymer and a drug is introduced into the polymer channel 4, and a PBS solution is introduced into the two PBS channels 5 so that nanoparticles are precipitated in the nanoparticle mixing channel 2. The mixed cell membranes and the precipitated nanoparticles are encapsulated in the cell membrane coating channel 6 and then flow out from the outlet to complete the preparation of fused membrane nanoparticles.

[0072] It can be understood by those skilled in the art that the polymer can be other types of polymers such as polylactic acid-co-glycolic acid (PLGA), polylactic acid (PLA), polyglycolic acid (PGA); the drug can be a fat-soluble drug such as doxorubicin, taxane, camptothecin, etc.; the organic solvent can be dimethyl sulfoxide, N,N-dimethylformamide, methanol, ethanol or other organic solvents that can dissolve the above polymers and drugs; the cell membrane can be a cell membrane such as a tumor cell membrane, a red blood cell membrane, a platelet membrane, a white blood cell membrane, etc. The injection device can be a syringe pump, a peristaltic pump or other instruments that can regulate the flow rate of the fluid when it is injected into the chip.

[0073] In order to make the purpose of the present invention clearer, the present invention is further described below in conjunction with embodiments; the specific embodiments described herein are only used to explain the present invention and are not limited to the present invention.

[0074] A microfluidic method for preparing composite biofilm-coated drug-loaded nanoparticles comprises the following steps:

[0075] Step 1: introducing different cell membrane solutions into two cell membrane channels 1 respectively so that the two cell membranes are mixed in the cell membrane mixing channel 3;

[0076] Step 2: introducing an organic solution containing a polymer and a drug into the polymer channel 4, and introducing a PBS solution into the two PBS channels 5, so that the drug-loaded nanoparticles are precipitated in the nanoparticle mixing channel 2;

[0077] Step 3: After all inlet channels are filled with the corresponding solution, the mixed cell membrane and the precipitated drug-loaded nanoparticles are mixed and loaded in the cell membrane coating channel 6;

[0078] Step 4: After the preparation is completed, the drug-loaded nanoparticles coated with the composite membrane will be output at the outlet of the cell membrane-coated channel 6 to complete the preparation.

[0079] Example 1

[0080] Using the microfluidic chip of the present invention to carry out B16F10 tumor cell membrane encapsulation of PLGA-DOX nanoparticles

[0081] After B16F10 cells were collected, they were washed three times by centrifugation at 1000g and 4°C with 1×PBS solution, and the centrifugation time was 10 min each time. 1 mL of lysis solution (0.25×PBS, 1mM PMSF) was added to every 20 million cells, and the cells were repeatedly frozen and thawed in liquid nitrogen for 5 times until the cells were completely broken. The obtained solution was centrifuged at 1500g and 4°C for 15 min, and the supernatant was carefully aspirated. The obtained supernatant was centrifuged at 20000g and 4°C for 30 min, the supernatant was discarded, and 1×PBS solution was added to resuspend the obtained cell membrane, and then centrifuged again at 20000g. Repeated washing 3 times to obtain a pure B16F10 cell membrane solution for use.

[0082] PLGA and DOX were dissolved in N,N-dimethylformamide to make the PLGA concentration 5 mg mL -1 , DOX concentration was 0.5 mg mL -1 , at 1 mL·h -1 The flow rate was 2 mL·h in the polymer channel, and the PBS channels on both sides were -1 PBS solution was introduced at a flow rate of 1.5 mmol / L and the tumor cell membrane solution was prepared into 0.1 mg mL-1 And 15mL·h -1 The flow rate was passed into two cell membrane channels, and finally the prepared nanoparticles were collected from the channel outlets.

[0083] Take 1 mL of the prepared nanoparticles and add them to a cuvette and use a DLS dynamic light scattering particle size analyzer to analyze the nanoparticles. The results are as follows: Figure 2 As shown, the particle size of the nanoparticles is about 100 nm and has good dispersibility.

[0084] Take 5 μL of nanoparticles and drop them onto the copper mesh of the supporting film. After standing for 5 minutes, drop 1% uranyl acetate solution on it and continue to stand for 5 minutes. Then use the edge of filter paper to carefully absorb the droplets on the copper mesh. After the copper mesh is dried in the shade, use a TEM transmission electron microscope to observe the particle morphology. The results are as follows: Figure 3 As shown, the purpose of encapsulating cell membrane on PLGA nanoparticles can be achieved by using microfluidic channels, and the structure of the polymer core and the cell membrane shell is clearly visible and has uniform morphology.

[0085] Example 2

[0086] Using the microfluidic chip of the present invention to fuse B16F10 tumor cell membrane with erythrocyte membrane and encapsulate PLGA nanoparticles

[0087] Red blood cells were obtained from 6-week-old male ICR mice. The obtained raw blood was placed in a centrifuge tube coated with sodium heparin for anticoagulation, and then washed three times by centrifugation at 1000g and 4°C with 1×PBS solution, with each centrifugation lasting 10 min. After washing, the white cell layer on the upper layer of the centrifuged cells was aspirated to obtain pure red blood cells, and then resuspended in 0.5×PBS and placed in liquid nitrogen for repeated freezing and thawing five times until the cells were completely broken, to obtain a dark red clear solution, which was washed three times by centrifugation at 20,000g and 4°C with 1×PBS solution, and then resuspended to obtain a light pink red blood cell membrane solution for use.

[0088] The obtained red blood cell membrane solution was centrifuged at 20,000 g and 4°C for 30 min, the supernatant solvent was discarded, the prepared DiI cell membrane red fluorescent dye was added, the membrane was blown evenly with a pipette and incubated at 32°C for 20 min, and then centrifuged at 20,000 g and 4°C for 30 min, the dye solution was removed and the solution was washed three times by centrifugation with PBS solution.

[0089] The obtained B16F10 tumor cell membrane solution is subjected to the above-mentioned operation in the same manner, except that the DiI cell membrane red fluorescent dye is replaced with the DiO cell membrane green fluorescent dye, thereby obtaining two cell membrane solutions stained with fluorescent dyes of different colors.

[0090] PLGA was dissolved in N,N-dimethylformamide to obtain 5 mg mL -1 PLGA solution, at 1 mL·h -1 The flow rate was 2 mL·h in the polymer channel, and the PBS channels on both sides were -1 PBS solution was introduced at a flow rate of 1.5 mmol / L, and the red blood cell membrane solution and B16F10 tumor membrane solution were prepared to 0.1 mg mL -1 And 15mL·h -1 The flow rate was passed into two cell membrane channels respectively, and the prepared nanoparticles were collected from the outlet of the channel. B16F10 tumor cells were seeded in a 6-well plate with a sterile cover glass at a density of 100,000 per well. After incubation for 24 hours, the culture medium was discarded, and the prepared nanoparticles and a 1:1 mixed solution of red blood cell membrane and B16F10 cell membrane were added to the two wells respectively and the cells were allowed to phagocytize for 2 hours, then the solution was discarded and the cells were fixed and sealed.

[0091] When observed under a fluorescence confocal microscope, the nanoparticles treated by the microfluidic channel of the present invention showed yellow fluorescence co-localization, and the untreated mixed cell membrane showed dispersed red and green fluorescence signals, proving that the microfluidic channel of the present invention can fuse two different cell membranes together without external ultrasound.

[0092] The fused membrane-coated nanoparticles were observed using a transmission electron microscope (TEM). Figure 4 As shown, the nanoparticles prepared in this example also have obvious core-shell structure and uniform morphology.

[0093] So far, the technical solution of the present invention has been described in conjunction with the preferred implementation method shown in the accompanying drawings. However, it is easy for those skilled in the art to understand that the protection scope of the present invention is not limited to these specific implementation methods. Without departing from the principle of the present invention, those skilled in the art can make equivalent changes and substitutions to the relevant technical features, and the technical solutions after these changes and substitutions should fall within the protection scope of the present invention.

[0094] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Various changes can be made to the present invention in form and detail without departing from the spirit and scope of the present invention as defined by the appended claims, and all should be within the scope of protection of the present invention.

Claims

1. A microfluidic device for preparing composite biofilm-coated drug-loaded nanoparticles, characterized in that: The device comprises more than two cell membrane channels (1), the cell membrane channels (1) converge into a cell membrane mixing channel (3); more than two PBS channels (5), a polymer channel (4), and a nanoparticle mixing channel (2) formed by the convergence of the more than two PBS channels (5) and the polymer channel (4); and a cell membrane coating channel (6) formed by the convergence of the cell membrane mixing channel (3) and the nanoparticle mixing channel (2); the cell membrane mixing channel (3) and the nanoparticle mixing channel (2) are in the shape of any one of a single helix, a double helix, and a wave, and the cell membrane coating channel (6) is in the shape of any one of a single helix and a double helix.

2. The microfluidic device for preparing composite biofilm-coated drug-loaded nanoparticles according to claim 1, characterized in that: Specifically, two cell membrane channels (1) that pass natural biological cell membrane solutions perform cell membrane fusion in a cell membrane mixing channel (3); two PBS channels (5) that pass PBS solutions and a polymer channel that passes an organic solution containing a polymer for encapsulating drugs and a specific drug perform nanoprecipitation in a nanoparticle mixing channel (2); the cell membrane solution fused in the cell membrane mixing channel (3) and the nanoparticles precipitated in the nanoparticle mixing channel (2) intersect in front of a cell membrane coating channel (6) and are mixed in the cell membrane coating channel (6) to form fused cell membrane-coated nanoparticles.

3. The microfluidic device for preparing composite biofilm-coated drug-loaded nanoparticles according to claim 1 or 2, characterized in that: The cell membrane channel (1), polymer channel and PBS channel (5) have independent inlets respectively, and the cell membrane coating channel (6) is provided with an outlet. An injection device is provided at each inlet, which can be used to accurately control the input of each solution at a specified flow rate.

4. A method for preparing composite biofilm-coated drug-loaded nanoparticles using a microfluidic device, characterized in that: The steps include: Step 1: introducing PBS solutions of two natural biological cell membranes into two cell membrane channels (1) respectively, so that the membranes are fused in the cell membrane mixing channel (3); Step 2: introducing an organic solution containing a polymer for encapsulating drugs and a specific drug into the polymer channel, and introducing a PBS solution into the PBS channels (5) on both sides of the polymer channel, so that the solutions in the three channels are mixed in the nanoparticle mixing channel (2); Step 3: The cell membrane solution mixed and fused through the cell membrane mixing channel (3) and the drug-loaded polymer nanoparticle solution mixed and precipitated through the nanoparticle mixing channel (2) are intersected in front of the cell membrane coating channel (6) and mixed in the cell membrane coating channel (6) to coat the fused cell membrane nanoparticles; Step 4: After the preparation is completed, the mixed solution containing the composite biofilm-coated drug-loaded nanoparticles is output from the last channel port to complete the preparation of the composite biofilm-coated drug-loaded nanoparticles; The shapes of the cell membrane mixing channel (3) and the nanoparticle mixing channel (2) are any one of a single helix, a double helix, and a wave shape; the shape of the cell membrane coating channel (6) is any one of a single helix and a double helix shape; and the polymer is any one of a polylactic acid-glycolic acid copolymer, polylactic acid, and polyglycolic acid.

5. The method for preparing composite biofilm-coated drug-loaded nanoparticles using a microfluidic device according to claim 4, characterized in that: The organic solvent is any one of dimethyl sulfoxide, dimethylformamide, methanol, ethanol and acetonitrile.

6. The method for preparing composite biofilm-coated drug-loaded nanoparticles using a microfluidic device according to claim 5, characterized in that: The specific drug is any one of fat-soluble anticancer drugs such as doxorubicin, taxane, camptothecin, curcumin and daunorubicin.

7. The method for preparing composite biofilm-coated drug-loaded nanoparticles using a microfluidic device according to claim 4, 5 or 6, characterized in that: The natural biological cell membrane is any one of a blood cell membrane, a tumor cell membrane, a macrophage membrane, an exosome, and a bacterial membrane.

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