Method for constructing biofilm bionic drug-loaded nano system and micro-fluidic chip

Abstract

The invention belongs to the technical field of bionic drug-loaded nano-particle preparation, and discloses a method for constructing a biofilm bionic drug-loaded nano-system and a micro-fluidic chip, the method comprises the following steps: S1, preparing a nano-particle solution and a biofilm solution; s2, respectively pumping the biological membrane solution and the nano-particle solution into an inlet of the micro-fluidic chip, mixing the biological membrane solution and the nano-particle solution in a solution mixing area to obtain a mixed solution, converging the mixed solution, sequentially introducing the converged mixed solution into at least two entrapment runners of an entrapment area, enabling the mixed solution to penetrate through a membrane with the aperture not greater than 2 mu m for multiple times in each entrapment runner, and forming the micro-fluidic chip. Fusing the biological membrane in the mixed solution with the medicine or the medicine-carrying nano-particles; and S3, collecting a mixed solution containing the biofilm bionic drug-loaded nano system to obtain the biofilm bionic drug-loaded nano system. According to the preparation method, accurate control over preparation of the biofilm bionic drug-loaded nano system is achieved, the biofilm bionic drug-loaded nano system which is uniform and controllable in particle size is obtained, and the protein structure of a cell membrane is reserved.

Description

Technical Field

[0001] The present invention relates to the technical field of the preparation of bionic drug-loaded nanoparticles, and particularly relates to a method for constructing a bionic drug-loaded nano-system of a biological membrane, a microfluidic chip, and specifically relates to a method for constructing a bionic drug-loaded nano-system of a biological membrane by a microfluidic chip and the microfluidic chip. Background Art

[0002] Nano-drug carriers are advantageous technologies for solving existing tumor drugs. Nano-drug carriers are prepared by encapsulating drugs with nanoparticles. Nano-drug carriers have characteristics such as reducing the toxic and side effects of drugs, improving the hydrophobicity of drugs, enhancing the uptake of tumor cells, and enhancing the drug efficacy. However, current targeted nano-drug carriers still have problems such as ligand inactivation and immune clearance. Therefore, constructing a new type of targeted nano-system is the key to current research. In recent years, bionic drug-loaded nano-systems of biological membranes have received extensive attention. A bionic drug-loaded nano-system of a biological membrane is a nano-sized delivery carrier obtained by disrupting cells to obtain cell membranes and then coating or fusing them with drug-loaded nanoparticles.

[0003] A bionic drug-loaded nano-system of a biological membrane utilizes the homing property of cell membranes, which is affected by adhesion proteins on the cell membrane surface. The same type of cell membrane surface has the same adhesion protein molecules, which can recognize and aggregate with each other. At the same time, due to the presence of immune escape proteins on the cell membrane surface, immune clearance is avoided, realizing the targeted distribution of drugs in the body and enhancing the therapeutic effect.

[0004] The construction of a bionic drug-loaded nano-system of a biological membrane currently uses mechanical co-extrusion method, ultrasonic fusion method or electrostimulation fusion method. The mechanical co-extrusion method is to use mechanical force to disperse cell membranes and then recombine them with nanoparticles. However, the relatively large mechanical force in the preparation process is likely to cause damage to cell membrane proteins, thereby affecting the targeting efficiency.

[0005] The ultrasonic fusion method and the electrostimulation fusion method are prepared using microfluidic technology. The ultrasonic fusion method, such as the method for synthesizing biofilm nanoparticles using a microfluidic chip and the microfluidic chip in Chinese Patent CN 110560186B, includes the following steps: Step 1: Place the microfluidic chip in an ultrasonic bath and introduce a biofilm solution into the biofilm channel; Step 2: Introduce an organic solution containing a polymer into the polymer channel to mix the organic solution with the biofilm solution in the biofilm channel. The mixed solution flows through the first mixing channel and is split at the end of the first mixing channel to enter the first mixing channel respectively; Step 3: Introduce a PBS solution into the PBS channel to mix the PBS solution with the mixed solution output from the first mixing channel; Step 4: The PBS channel outputs the mixed solution to the second mixing channel. When the solution enters the second mixing channel, the polymer in the mixed solution is squeezed into the biofilm under the action of pressure and ultrasonic waves; Step 5: After the preparation is completed, the mixed solution containing biofilm nanoparticles is output from the second mixing channel to complete the preparation of biofilm nanoparticles. The electrostimulation fusion method, such as the method for preparing a one-step cell membrane biomimetic lipid nanoparticle disclosed in Chinese Patent CN 119098121A, is as follows: (1) Prepare lipid nanoparticle organic solvent, drug buffer solution and cell membrane buffer solution respectively; (2) Inject the lipid nanoparticle organic solvent, drug buffer solution and cell membrane buffer solution from the lipid nanoparticle inlet, drug inlet and cell membrane inlet respectively. The lipid nanoparticles in the lipid nanoparticle organic solvent and the drug in the drug buffer solution are mixed in the first mixing channel to form drug-loaded lipid nanoparticles, and the drug-loaded lipid nanoparticles are mixed with the cell membrane in the cell membrane buffer solution in the second mixing channel to form drug-loaded cell membrane-lipid nanoparticles; (3) The drug-loaded cell membrane-lipid nanoparticles are fused by alternating current stimulation in the electrostimulation channel to obtain the cell membrane biomimetic lipid nanoparticles.

[0006] Using microfluidic technology can achieve effective mixing and precise control between different components. However, the ultrasonic fusion method still has the problem of a wide particle size distribution of the prepared nanoparticles. The electrostimulation fusion method requires strict control of the alternating current electrode parameters. For different cell membranes, the alternating current electrode parameters used are likely to cause damage to the structure of cell membrane proteins and cannot achieve cell membrane biomimetic modification. Therefore, the existing methods for preparing biofilm biomimetic drug-loaded nanosystems have problems such as a wide particle size distribution of the nanoparticles, difficult parameter control, easy damage to the structure of cell membrane proteins, and low applicability, and cannot further achieve cell membrane biomimetic modification. Summary of the Invention

[0007] The purpose of the present invention is to solve the problems in the existing methods for preparing biofilm biomimetic drug-loaded nanosystems, such as a wide particle size distribution of the nanoparticles, difficult parameter control, easy damage to the structure of cell membrane proteins in the middle, and low applicability, and to provide a method for constructing a biofilm biomimetic drug-loaded nanosystem and a microfluidic chip.

[0008] To achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0009] A method for constructing a biomimetic drug-loaded nanosystem of a biofilm, comprising the following steps:

[0010] S1. Prepare a nanoparticle solution and a biofilm solution. The nanoparticle solution includes a drug solution or a drug-loaded nanoparticle solution;

[0011] S2. Based on microfluidic technology, pump the biofilm solution and the nanoparticle solution prepared in step S1 into the inlets of a microfluidic chip respectively. In the solution mixing area of the microfluidic chip, mix the biofilm solution and the nanoparticle solution to obtain a mixed solution. After the mixed solution converges, it is successively introduced into at least two loading channels in the loading area of the microfluidic chip. In each loading channel, the mixed solution passes through a membrane with a pore size not greater than 2 μm multiple times, so that the biofilm and the drug or the drug-loaded nanoparticles in the mixed solution are fused into a biomimetic drug-loaded nanosystem of the biofilm;

[0012] S3. After the preparation is completed, collect the mixed solution containing the biomimetic drug-loaded nanosystem of the biofilm to obtain the biomimetic drug-loaded nanosystem of the biofilm.

[0013] In the above technical solution, the present application first prepares a nanoparticle solution and a biofilm solution. The drug solution is prepared by mixing a drug with an organic solvent or a drug with a buffer solution. The drug-loaded nanoparticle solution is prepared by mixing a drug, polymer nanoparticles, an organic solvent or a drug, polymer nanoparticles, and a buffer solution. The fusion of the biofilm and the drug or the biofilm and the drug-loaded nanoparticles is realized through a microfluidic chip. The inlets and outlets of the microfluidic chip are used to connect external pipelines to respectively input and output solutions. The solution mixing area is used to form a mixed solution of the input nanoparticle solution and the biofilm solution and input it into the loading area, improving the subsequent extrusion efficiency after mixing; the loading area is the key area for realizing the loading and fusion of the biofilm and the drug or the biofilm and the drug-loaded nanoparticles. In each loading channel, the mixed solution passes through a membrane with a pore size not greater than 2 μm multiple times. The biofilm in the mixed solution deforms when passing through the micropores on the membrane, wrapping the drug or the drug-loaded nanoparticles. Through the multiple actions of at least two loading channels, the full fusion of the biofilm and the drug or the biofilm and the drug-loaded nanoparticles is ensured to form a biomimetic drug-loaded nanosystem of the biofilm.

[0014] In the technical solution of the present invention, a microfluidic chip is used to construct a biomimetic drug-loaded nanosystem. After mixing the biomembrane solution and the nanoparticle solution, the mixture flows into the encapsulation area. By controlling the number of encapsulation channels, the number of times the mixed solution passes through the membrane, and the pore size of the membrane, the fusion time, fusion pressure, and encapsulation process between the biomembrane and the drug or drug-loaded nanoparticles are adjusted to achieve precise control of the preparation of the biomimetic drug-loaded nanosystem, thereby obtaining a uniform biomimetic drug-loaded nanosystem with controllable particle size and retaining the protein structure of the biomembrane, solving the problems in the prior art that the particle size distribution of nanoparticles in the preparation method is wide, the parameters are not easy to control, it is easy to cause damage to the structure of cell membrane proteins, and the applicability is not high. In addition, the concentration of the biomembrane solution, the concentration of the drug-loaded nanoparticles, and the solution flow rate in the microfluidic chip channels can be adjusted to achieve fine control of the particle size, drug loading amount, and membrane structure.

[0015] As a preferred embodiment of the present invention, in step S2, the pumping speed of the nanoparticle solution is 20-80 μL / h; the pumping speed of the biomembrane solution is 40-200 μL / h, and the flow rate ratio of the nanoparticle solution to the biomembrane solution is 1:1.5-4.

[0016] As a preferred embodiment of the present invention, in each of the encapsulation channels, the number of times the mixed solution passes through the membrane is 6-30, preferably 10-18.

[0017] As a preferred embodiment of the present invention, when the mixed solution flows into different encapsulation channels, from the upstream to the downstream of the encapsulation channel, the pore size of the membrane through which the mixed solution passes in different encapsulation channels shows a decreasing trend. In the above technical solution, the pore size of the micro-holes of the membrane in different encapsulation channels shows a decreasing trend, resulting in inconsistent pressures between different encapsulation channels. Due to the inconsistent pressures, the biomembrane in the mixed solution will be squeezed to different degrees in different encapsulation channels. In the channels with smaller pore sizes, the pressure is higher, and the biomembrane is more obviously squeezed. By designing at least two encapsulation channels and the pore size of the membrane through which the mixed solution passes in different encapsulation channels shows a decreasing trend, it can fully ensure the structural deformation or rupture of the biomembrane in the mixed solution to fuse with the drug-loaded nanoparticles and improve the fusion efficiency.

[0018] As a preferred embodiment of the present invention, three of the encapsulation channels are provided in the encapsulation area, namely the first-stage encapsulation channel, the second-stage encapsulation channel, and the third-stage encapsulation channel. A diaphragm is provided in each of the encapsulation channels, namely the first-stage diaphragm, the second-stage diaphragm, and the third-stage diaphragm. The aperture of the first-stage diaphragm is 0.5 to 2 μm, the aperture of the second-stage diaphragm is 0.2 to 1.2 μm, and the aperture of the third-stage diaphragm is 0.05 to 0.8 μm. The apertures of the first-stage diaphragm, the second-stage diaphragm, and the third-stage diaphragm show a decreasing trend. In the above technical solution, the apertures of the first-stage diaphragm, the second-stage diaphragm, and the third-stage diaphragm decrease. As the aperture of the diaphragm decreases, the resistance of the biofilm passing through the diaphragm increases, and it is not easy to pass through the diaphragm. Moreover, in the channel with a smaller aperture, the pressure is higher, and the extrusion of the biofilm is more obvious. However, excessive extrusion may affect the activity and function of the biofilm. For example, the protein structure in the biofilm may be damaged due to extrusion, affecting its metabolic activity or biological function. Therefore, by controlling the aperture of the first-stage diaphragm to be 0.5 to 2 μm, the aperture of the second-stage diaphragm to be 0.2 to 1.2 μm, and the aperture of the third-stage diaphragm to be 0.05 to 0.8 μm, it can not only ensure that the biofilm can pass through the diaphragm but also retain the protein structure of the biofilm, improving the fusion efficiency.

[0019] As a more preferred embodiment of the present invention, the aperture of the first-stage diaphragm is 0.6 to 1 μm, the aperture of the second-stage diaphragm is 0.3 to 0.8 μm, and the aperture of the third-stage diaphragm is 0.1 to 0.4 μm.

[0020] As a preferred embodiment of the present invention, the biofilm solution is prepared from cell membranes, and the cell membranes include at least one of exosomes, tumor cell membranes, bone marrow stromal cell membranes, macrophage membranes, red blood cell membranes, white blood cell membranes, platelet membranes, mesenchymal stem cell membranes, bacterial membranes, and fungal membranes.

[0021] As a more preferred embodiment of the present invention, the cell membranes are extracted from cells, the cell density is 10 6 ~10 8 CFU / mL, and the concentration of the cell membranes in the biofilm solution is expressed as the membrane protein concentration. After extracting the cell membranes, the membrane protein concentration is 0.1 to 10 mg / mL.

[0022] As a preferred embodiment of the present invention, the drugs used in the preparation of the drug solution or the drug-loaded nanoparticle solution include at least one of clinically common drugs such as bortezomib, doxorubicin, taxane, camptothecin, curcumin, and daunorubicin.

[0023] As a preferred embodiment of the present invention, the drug-loaded nanoparticle solution is prepared by mixing a drug, polymer nanoparticles, an organic solvent or a drug, polymer nanoparticles, and a buffer solution. The organic solvent used in the preparation of the drug-loaded nanoparticle solution includes at least one of dimethyl sulfoxide, dimethylformamide, methanol, ethanol, acetonitrile, ether, and acetone; the buffer solution includes at least one of sodium citrate solution, phosphate buffer solution, RNase-free and ion-free sterile water, etc.

[0024] As a more preferred embodiment of the present invention, the concentration of the drug-loaded nanoparticle solution is 0.05 to 10 mg / mL.

[0025] As a more preferred embodiment of the present invention, the polymer nanoparticles are at least one of poly(lactic-co-glycolic acid), poly(D,L-lactic acid) (PDLLA), poly(lactide-co-glycolide) (PLGA), polycaprolactone (PCL), polyglycolic acid, and polycaprolactone-polycaprolactone block copolymer. More preferably, the polymer nanoparticles are PCL-PEG-PCL block copolymer, which is an amphiphilic BAB triblock copolymer composed of a hydrophilic block of polyethylene glycol (PEG) and a hydrophobic block of polycaprolactone (PCL), having good biocompatibility and degradation properties, and its nano-system can effectively load hydrophobic drugs, antigens, etc., and has good biological properties.

[0026] As a preferred embodiment of the present invention, the drug solution is prepared by mixing a drug with an organic solvent or a drug with a buffer solution. The organic solvent includes at least one of dimethyl sulfoxide, dimethylformamide, methanol, ethanol, acetonitrile, ether, and acetone; the buffer solution includes at least one of sodium citrate solution, phosphate buffer solution, RNase-free and ion-free sterile water, etc.

[0027] As a preferred embodiment of the present invention, the method is used to prepare a biomimetic drug-loaded nanosystem with fluorescent molecules. When preparing the nanoparticle solution, it is prepared by mixing fluorescent nanoparticles, a drug, an organic solvent or fluorescent nanoparticles, a drug, and a buffer solution.

[0028] The present invention also provides the application of the biomimetic drug-loaded nanosystem obtained by the method for constructing the biomimetic drug-loaded nanosystem as described above in the preparation of drugs for tumor treatment.

[0029] The present invention also provides a microfluidic chip for implementing the method for constructing a biomimetic drug-loaded nanosystem as described above. The microfluidic chip is provided with a closed microchannel cavity, and the microchannel cavity is provided with an inlet and an outlet communicating with the cavity;

[0030] From upstream to downstream, the microchannel cavity includes a connected solution mixing area and a loading area. The loading area includes at least two connected loading channels. Each loading channel includes at least two sub-loading channels arranged in parallel. A diaphragm is provided in each loading channel. Each sub-loading channel is distributed on both sides of the diaphragm and communicated through the diaphragm for the mixed liquid to pass through the diaphragm multiple times. Micro-holes are provided on the diaphragm, and the aperture of the micro-holes is not greater than 2 μm. From the upstream to the downstream of the loading channel, the aperture of the micro-holes of the diaphragm in different loading channels shows a decreasing trend.

[0031] In the above technical solution, a novel microfluidic chip structure is provided, which can realize the reaction control of the biofilm solution and the nanoparticle solution in a tiny reaction space. Through the microchannel design inside the chip, the formation of the biofilm biomimetic drug-loaded nano-system can be effectively controlled, and the particle size, drug loading amount and membrane structure of the biofilm biomimetic drug-loaded nano-system can be finely regulated by controlling the loading channels and the aperture of the diaphragm of the microfluidic chip.

[0032] As a preferred solution of the present invention, at least two inlets are provided.

[0033] As a preferred solution of the present invention, the number of the sub-loading channels is 10 - 35.

[0034] As a preferred solution of the present invention, a first detection port is provided between the solution mixing area and the loading area, and / or a second detection port is provided between different loading channels, and / or a third detection port is provided between the end of the last loading channel and the outlet. The first detection port, the second detection port and the third detection port are used for detecting the physical and chemical parameters in the flow channel.

[0035] As a preferred solution of the present invention, the microfluidic chip adopts a multi-layer substrate structure, including a cover plate, a bottom plate and a microchannel plate. The microchannel plate is arranged between the cover plate and the bottom plate to form a closed microchannel cavity.

[0036] As a preferred solution of the present invention, the inlet is arranged on the cover plate or the bottom plate, and the outlet is arranged on the cover plate or the bottom plate.

[0037] As a preferred solution of the present invention, an injection device is provided at each inlet, which can be used to accurately regulate the input of each solution at a specified flow rate.

[0038] As a preferred embodiment of the present invention, the microchannel plate includes an upper plate and a lower plate. The sub-carrier channels include a plurality of first through-hole structures and second through-hole structures. A plurality of the first through-hole structures are provided on the upper plate, and a plurality of the second through-hole structures are provided on the lower plate. The first through-hole structures and the second through-hole structures are equal in number and arranged in a staggered manner. In each sub-carrier channel, the diaphragm is disposed between the upper plate and the lower plate. The first through-hole structures and the second through-hole structures are distributed on both sides of the diaphragm and are connected through the diaphragm to form the sub-carrier channels.

[0039] As a preferred embodiment of the present invention, the solution mixing area is a mixing channel provided on the upper plate. The upstream of the mixing channel is communicated with the inlet, and the downstream of the mixing channel converges the mixed liquid and is communicated with the carrier area. The mixing channel is at least one of a spiral shape, an S shape, a dendritic shape, a comb shape, and a circular shape. The cross-section of the mixing channel is a circular, semi-circular or square structure, and the inner diameter of the mixing channel is 0.01 - 2 mm.

[0040] As a preferred embodiment of the present invention, three sub-carrier channels are provided in the carrier area, namely a first-stage sub-carrier channel, a second-stage sub-carrier channel, and a third-stage sub-carrier channel. A diaphragm is provided in each sub-carrier channel, namely a first-stage diaphragm, a second-stage diaphragm, and a third-stage diaphragm. The pore diameter of the first-stage diaphragm is 0.5 - 2 μm, the pore diameter of the second-stage diaphragm is 0.2 - 1.2 μm, and the pore diameter of the third-stage diaphragm is 0.05 - 0.8 μm. The pore diameters of the first-stage diaphragm, the second-stage diaphragm, and the third-stage diaphragm show a decreasing trend.

[0041] As a more preferred embodiment of the present invention, the pore diameter of the first-stage diaphragm is 0.6 - 1 μm, the pore diameter of the second-stage diaphragm is 0.3 - 0.8 μm, and the pore diameter of the third-stage diaphragm is 0.1 - 0.4 μm.

[0042] As a more preferred embodiment of the present invention, the pore diameter of the first-stage diaphragm is 0.8 μm, the pore diameter of the second-stage diaphragm is 0.4 μm, and the pore diameter of the third-stage diaphragm is 0.2 μm.

[0043] As a more preferred embodiment of the present invention, the cross-section of the sub-carrier channel is a circular, semi-circular or square structure, and the inner diameter of the sub-carrier channel is 0.01 - 2 mm. From the upstream to the downstream of the sub-carrier channel, the cross-sectional areas of the sub-carrier channels in different sub-carrier channels show an increasing trend.

[0044] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0045] 1. The present invention provides a method for constructing a biomimetic drug-loaded nanosystem using a microfluidic chip to construct the biomimetic drug-loaded nanosystem. After mixing the biomembrane solution and the nanoparticle solution, they flow into the encapsulation area. By controlling the number of encapsulation channels, the number of times the mixed solution passes through the membrane, and the pore size of the membrane, the fusion time, fusion pressure, and encapsulation process between the biomembrane and the drug or drug-loaded nanoparticles are adjusted to achieve precise control over the preparation of the biomimetic drug-loaded nanosystem, thereby obtaining a uniform and controllable particle size biomimetic drug-loaded nanosystem that retains the protein structure of the cell membrane, solving the problems in the prior art that the particle size distribution of nanoparticles is wide, the parameters are difficult to control, the structure of cell membrane proteins is easily damaged, and the applicability is not high. In addition, the concentration of the biomembrane solution, the concentration of the drug-loaded nanoparticles, and the solution flow rate in the microfluidic chip channels can be adjusted to achieve fine control over the particle size, drug loading amount, and membrane structure.

[0046] 2. During the preparation of the biomimetic drug-loaded nanosystem of the present invention, the pore size of the microholes in the membrane in different encapsulation channels shows a decreasing trend. In the channels with a smaller pore size, the pressure is higher, and the extrusion of the biomembrane is more obvious. By designing at least two encapsulation channels with a decreasing trend in the pore size of the membrane through which the mixed solution passes in different encapsulation channels, it can fully ensure the structural deformation or rupture of the biomembrane in the mixed solution to fuse with the drug-loaded nanoparticles and improve the fusion efficiency.

[0047] 3. The present invention provides a microfluidic chip. Through the design of the microchannels inside the chip, the formation of the biomimetic drug-loaded nanosystem can be effectively controlled, and the particle size, drug loading amount, and membrane structure of the biomimetic drug-loaded nanosystem can be finely regulated by controlling the encapsulation channels and the pore size of the membrane of the microfluidic chip.

[0048] 4. When using the method of the present invention to prepare the biomimetic drug-loaded nanosystem, when the particle size of the drug-loaded nanoparticles with a theoretical drug loading of 10% is 75 nm, the particle size of the biomimetic drug-loaded nanosystem obtained after being encapsulated by the cell membrane is 105 nm, the dispersion coefficient is less than 0.15, and the particle size distribution is stable; preparing the biomimetic drug-loaded nanosystem improves the targeting and uptake rate of the drug to tumor cells, enhances the tumor suppression effect, and reduces the toxic and side effects of the drug. Description of the Drawings

[0049] Figure 1 is a flowchart of a method for constructing a biomimetic drug-loaded nanosystem of the present invention;

[0050] Figure 2 is a three-dimensional view of the microfluidic chip in Example 2;

[0051] Figure 3 is a three-dimensional view of the cover plate of the microfluidic chip in Example 2;

[0052] Figure 4 It is the top view of the upper plate of the microfluidic chip in Example 2;

[0053] Figure 5 It is Figure 4 the enlarged view of part A in

[0054] Figure 6 It is the three-dimensional view of the back side of the upper plate of the microfluidic chip in Example 2;

[0055] Figure 7 It is the three-dimensional view of the front side of the lower plate of the microfluidic chip in Example 2;

[0056] Figure 8 It is the three-dimensional view of the back side of the lower plate of the microfluidic chip in Example 2;

[0057] Figure 9 It is the cross-sectional view of the microfluidic chip in Example 2;

[0058] Figure 10 It is Figure 9 the enlarged view of part B in

[0059] Figure 11 It is the three-dimensional view of the first-stage diaphragm of the microfluidic chip in Example 2;

[0060] Figure 12 It is Figure 11 the enlarged view of part C in

[0061] Figure 13 It is the schematic diagram of preparing the biomembrane biomimetic drug-loaded nanosystem (PCEC / mem) by wrapping the extracted multiple myeloma cell membrane around the outer layer of Bort / PCEC nanoparticles in Example 5;

[0062] Figure 14 It is the TEM photo of the biomembrane biomimetic drug-loaded nanosystem in Example 5;

[0063] Markings in the figure: 1 - cover plate, 2 - bottom plate, 3 - upper plate, 31 - mixing flow channel, 32 - first through-hole structure, 4 - lower plate, 41 - groove, 42 - second through-hole structure, 51 - first-stage diaphragm, 52 - second-stage diaphragm, 53 - third-stage diaphragm, 6 - inlet, 7 - outlet. Detailed implementation manner

[0064] In order to more clearly describe the invention purpose, technical solution and technical effect advantages in the specific embodiments of the present invention, the following will elaborate on the solutions in the specific embodiments in combination with the accompanying drawings of the present invention. The specific technical solutions involved in the following specific embodiments are only for clearly and completely describing the innovative technical solutions of the present invention. They are only part of the specific implementation solutions that the present invention can adopt, not all embodiments, and should not be construed as a limitation on the innovative solutions of the present invention. Any solution adopting the same inventive concept of the present invention should be included in the protection scope of the present invention.

[0065] Secondly, the relevant descriptions of the accompanying drawings in the specific embodiments of the present invention are only for facilitating those skilled in the art to understand the present invention's solution. The partial details shown in the drawings are for clearly presenting the technical solution. It should not be considered that all the technical features in the drawings must be incorporated into the specific embodiments, nor can the detailed features in the drawings be regarded as additional limitations on the innovative technical solutions of the present invention. The components in each embodiment described and shown in the drawings can be combined and arranged in different configurations. These variations in combination and arrangement should all be regarded as part of all the embodiments of the innovative solutions of the present invention and be included in the scope to be protected by the present invention.

[0066] It should be noted that, without special instructions, in the description of the specific embodiments of the present invention, the expression terms indicating the orientation or positional relationship such as "upper", "lower", "left", "right", "center", "inner", "outer", etc. are all based on the orientation or positional relationship shown in the drawings, or the orientation or positional relationship when the invention product / device / apparatus is normally used. These terms of orientation or positional relationship are only for facilitating the description of the present invention's solution or simplifying the description in the specific embodiments, to help those skilled in the art quickly understand the solution, rather than indicating or implying that a specific device / component / element must have a specific orientation or be constructed and operated in a specific positional relationship. Therefore, it cannot be construed as a limitation on the present invention.

[0067] In addition, when terms such as "horizontal", "vertical", "hanging", etc. appear, it does not mean that the corresponding device / component / element is required to be absolutely horizontal, vertical or hanging, but it can be slightly inclined. For example, "horizontal" only means that its direction is more horizontal relative to "vertical", and it does not mean that the structure must be completely horizontal, but it can be slightly inclined. Or, it can be simply understood that the corresponding device / component / element is arranged in a specific direction such as "horizontal", "vertical", "hanging", etc., and can have an error / deviation of ±10% relative to the corresponding direction setting, more preferably an error / deviation within ±8%, more preferably an error / deviation within ±6%, more preferably an error / deviation within ±5%, more preferably an error / deviation within ±4%. As long as the corresponding device / component / element is within the error / deviation range, it can still achieve its function in the solution of the present invention.

[0068] In addition, when expressions such as "first", "second", "third", etc. appear in the terms, they are only used to distinguish the description of the same or similar components, and should not be understood as emphasizing or implying the relative importance of specific components.

[0069] In addition, in the description of the embodiments of the present invention, "several", "multiple", "a number of" represent at least 2. It can be any situation such as 3, 4, 5, 6, 7, 8, 9, etc., or even more than 9.

[0070] In addition, in the description of the technical solution of the present invention, unless otherwise clearly specified / defined / restricted, when terms such as "set", "installed", "connected", "linked" appear, they should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection. It can be connection means commonly used in the art such as welding, riveting, bolting, threaded connection, etc. Such a connection can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the communication inside two components.

[0071] Embodiment 1

[0072] Multiple myeloma is a currently incurable blood tumor. Bortezomib is the main drug for the clinical treatment of multiple myeloma, but there are problems such as non-specific distribution, short half-life, and low stability. Nano-drugs are a favorable technology to improve the above problems. However, the current targeted nano-systems still have problems such as ligand inactivation and immune clearance. Therefore, constructing a new type of targeted nano-system is the key to current research. Since tumor cells have homologous targeting, the same adhesion molecules exist on the surface of the same type of tumor cells, which can recognize and aggregate with each other. Multiple myeloma has the property of bone marrow homing, which is affected by the adhesion proteins on the surface of myeloma cell membranes. The importance of biomimetic drug-loaded nano-systems in drug delivery has become increasingly prominent. By mimicking the membrane structure, nano-carriers can imitate the functions of cell membranes and have better biocompatibility, targeting, and stability. Therefore, how to efficiently construct such biomimetic drug-loaded nano-systems has become an important research direction in the field of nano-drug delivery. Microfluidic chips are technical tools with the characteristics of high precision, controllability, and high throughput. Through microfluidic technology, reaction conditions such as flow rate, temperature, and concentration of chemical components can be precisely controlled. Based on the above problems, this embodiment provides a method for constructing a biomimetic drug-loaded nano-system based on microfluidic technology, as Figure 1 shown, including the following steps:

[0073] S1. Prepare a nano-particle solution and a biomembrane solution. The nano-particle solution includes a drug solution or a drug-loaded nano-particle solution;

[0074] S2. Based on microfluidic technology, pump the biomembrane solution and the nano-particle solution prepared in step S1 into the inlets of the microfluidic chip respectively. In the solution mixing area of the microfluidic chip, mix the biomembrane solution and the nano-particle solution to obtain a mixed solution. After the mixed solution converges, it is successively introduced into at least two encapsulation channels in the encapsulation area. In each encapsulation channel, the mixed solution passes through a membrane with a pore size not greater than 2 μm multiple times, so that the biomembrane and the drug or the drug-loaded nano-particle in the mixed solution are fused into a biomimetic drug-loaded nano-system;

[0075] S3. After preparation, collect the mixed solution containing the biomimetic drug-loaded nano-system to obtain the biomimetic drug-loaded nano-system.

[0076] The drug solution is prepared by mixing a drug with a buffer solution or a drug with an organic solvent. The drug-loaded nano-particle solution is prepared by mixing a drug, polymer nano-particles, an organic solvent or a drug, polymer nano-particles, a buffer solution. The fusion of the biomembrane and the drug or the biomembrane and the drug-loaded nano-particle is achieved through the microfluidic chip.

[0077] Example 2

[0078] This embodiment provides a microfluidic chip. The microfluidic chip is provided with a closed microchannel cavity, and the microchannel cavity is provided with an inlet 6 and an outlet 7 communicating with the cavity; from upstream to downstream, the microchannel cavity includes a connected solution mixing area and a loading area, and the loading area includes at least two connected loading channels. Each loading channel includes at least two sub-loading channels arranged in parallel. A diaphragm is provided in each loading channel. Each sub-loading channel is distributed on both sides of the diaphragm and communicated through the diaphragm for allowing the mixed liquid to pass through the diaphragm multiple times; micro-holes are provided on the diaphragm, and the aperture of the micro-holes is not greater than 2 μm; from the upstream to the downstream of the loading channel, the aperture of the micro-holes on the diaphragm in different loading channels shows a decreasing trend.

[0079] As Figures 2 - 12 shown, the microfluidic chip adopts a multi-layer substrate structure, including a cover plate 1, a bottom plate 2, and a microchannel plate. The microchannel plate is arranged between the cover plate 1 and the bottom plate 2 to form a closed microchannel cavity. In this embodiment, both the inlet 6 and the outlet 7 are arranged on the cover plate 1. As Figure 3 shown, there are at least two inlets 6, and injection devices are arranged at the inlets 6, which can be used to precisely control each solution to be input at a specified flow rate and control the pumping speed of the solution.

[0080] In some embodiments, the microchannel plate includes an upper plate 3 and a lower plate 4. The solution mixing area is a mixing channel 31 arranged on the upper plate 3. As Figure 4 , Figure 5 , Figure 6 shown, the upstream of the mixing channel 31 is communicated with the inlet 6, and the downstream of the mixing channel 31 converges the mixed liquid and is communicated with the loading area; the mixing channel 31 is at least one of a spiral shape, an S shape, a dendritic shape, a comb shape, and a ring shape. In this embodiment, the mixing channel 31 is in a dendritic shape, and the cross-section of the mixing channel 31 is a circular, semi-circular or square structure, and the inner diameter is 0.01 - 2 mm.

[0081] The loading area includes at least two connected loading channels, specifically three loading channels. Each loading channel includes at least two sub-loading channels arranged in parallel. Preferably, the number of sub-loading channels is 10 - 35, and the number of sub-loading channels in different loading areas can be different. A single loading channel shares the same diaphragm. A groove 41 is provided on the upper surface of the lower plate 4 for placing the diaphragm. The sub-loading channel includes a plurality of first through-hole structures 32 and second through-hole structures 42. A plurality of first through-hole structures 32 are arranged on the upper plate 3, and a plurality of second through-hole structures 42 are arranged on the lower plate 4. Figure 7 , Figure 8As shown, the number of the first through-hole structures 32 matches that of the second through-hole structures 42 and they are arranged in a staggered manner. In each encapsulation flow channel, the diaphragm is arranged between the upper plate 3 and the lower plate 4. The first through-hole structures 32 and the second through-hole structures 42 are distributed on both sides of the diaphragm and are connected through the diaphragm to form sub-encapsulation flow channels, constituting a three-dimensional micro-channel. The number of the first through-hole structures 32 and the second through-hole structures 42 is set to ensure that in each encapsulation flow channel, the mixed liquid passes through the diaphragm 6 to 30 times, preferably 10 to 18 times. In the above technical solution, there are many parallel sub-encapsulation flow channels in each encapsulation flow channel. Each sub-encapsulation flow channel is composed of the first through-hole structure 32, the second through-hole structure 42 and the diaphragm clamped in the middle. The number of the first through-hole structures 32 and the second through-hole structures 42 matches and they are arranged in a staggered manner. When the mixed liquid flows in the micro-hole encapsulation flow channel arranged on the sub-diaphragm, it reciprocally passes through the micro-holes from the first through-hole structure 32 to the second through-hole structure 42 and from the second through-hole structure 42 to the first through-hole structure 32, so as to realize the deformation of the biofilm in the mixed liquid when passing through the micro-holes on the diaphragm and fuse to wrap the drug or drug-loaded nanoparticles. More specifically, as Figure 9 , Figure 10 , the first through-hole structure 32 includes a first vertical hole, a first horizontal hole and a second vertical hole which are sequentially connected. The first vertical hole and the second vertical hole are vertically arranged on the upper plate 3, and the first horizontal hole is horizontally arranged on the upper surface of the upper plate 3. The second through-hole structure 42 includes a third vertical hole, a second horizontal hole and a fourth vertical hole which are sequentially connected. The third vertical hole and the fourth vertical hole are vertically arranged on the lower plate 4, and the third horizontal hole is horizontally arranged on the lower surface of the lower plate 4. The second vertical hole is connected to the third vertical hole through the micro-holes of the diaphragm, and the fourth vertical hole is connected to the first vertical hole through the micro-holes of the diaphragm.

[0082] In this embodiment, three encapsulation flow channels are arranged in the encapsulation area, namely a first-stage encapsulation flow channel, a second-stage encapsulation flow channel and a third-stage encapsulation flow channel. The front end of the first-stage encapsulation flow channel is connected to the end of the mixing flow channel 31, and the end of the third-stage encapsulation flow channel is connected to the outlet 7; diaphragms are arranged in each encapsulation flow channel, namely a first-stage diaphragm 51, a second-stage diaphragm 52 and a third-stage diaphragm 53. The pore diameter of the first-stage diaphragm 51 is 0.5 to 2 μm, the pore diameter of the second-stage diaphragm 52 is 0.2 to 1.2 μm, and the pore diameter of the third-stage diaphragm 53 is 0.05 to 0.8 μm, as Figure 11 , Figure 12, micro holes are provided on the first-stage diaphragm 51, the second-stage diaphragm 52, and the third-stage diaphragm 53. The micro holes are through holes. The pore diameters of the first-stage diaphragm 51, the second-stage diaphragm 52, and the third-stage diaphragm 53 show a decreasing trend to achieve the purpose of finally constructing a stable and uniform system. More preferably, the pore diameter of the first-stage diaphragm 51 is 0.6 - 1 μm, the pore diameter of the second-stage diaphragm 52 is 0.3 - 0.8 μm, and the pore diameter of the third-stage diaphragm 53 is 0.1 - 0.4 μm. Specifically, in this embodiment, the diaphragm is a polycarbonate membrane, the pore diameter of the first-stage diaphragm 51 is 800 nm, the pore diameter of the second-stage diaphragm 52 is 400 nm, and the pore diameter of the third-stage diaphragm 53 is 200 nm.

[0083] The cross-section of the sub-carrier channel is a circular, semi-circular or square structure, and the inner diameter is 0.01 - 2 mm. From the upstream to the downstream of the sub-carrier channel, the cross-sectional areas of the sub-carrier channels in different carrier channels show an increasing trend. In the above technical solution, the pore diameters of the micro holes of the diaphragm in different carrier channels show a decreasing trend. Such a setting makes the pressure of the mixed liquid increase as the pore diameter becomes smaller when the mixed liquid is extruded through diaphragms with different pore diameters. Therefore, the cross-sectional areas of different carrier channels gradually increase according to the user's needs to improve the extrusion filtration efficiency.

[0084] The cover plate 1 and the bottom plate 2 are made of conductive glass with an indium tin oxide coating. The indium tin oxide coatings on the inner sides of the cover plate 1 and the bottom plate 2 form a conductive layer for connecting an external power supply. The material of the microchannel plate is selected from any one or more of polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), cycloolefin copolymer (COC), polycarbonate (PC), quartz, and glass. Based on soft lithography technology, a microchannel plate with a microchannel pattern is prepared and irreversibly sealed with the cover plate 1 and the bottom plate 2 to obtain a microfluidic chip.

[0085] Example 3

[0086] This embodiment provides a microfluidic chip, which is similar to Example 2, except that a microchannel liquid one-way valve is provided at the connection between the solution mixing area and the carrier area in this embodiment to control the introduction of the solution.

[0087] Example 4

[0088] This embodiment provides a microfluidic chip, which is similar to Embodiment 2. The difference is that in this embodiment, a first detection port is provided between the solution mixing area and the encapsulation area, and / or a second detection port is provided between different encapsulation channels, and / or a third detection port is provided between the end of the last encapsulation channel and the outlet. The first detection port, the second detection port, and the third detection port are used to detect the physical and chemical parameters in the channels. In the above technical solution, the pore diameters of the membrane micropores in different encapsulation channels show a decreasing trend, resulting in inconsistent pressures between different encapsulation channels. The first detection port, the second detection port, and the third detection port can be set at different positions of the microfluidic chip according to requirements, and an external sensor is connected to detect the physical and chemical parameters such as pressure and temperature in the channels, and the pressure in the channels is adjusted in real time according to the pressure in each area of the microfluidic chip to ensure the construction efficiency.

[0089] Embodiment 5

[0090] This embodiment is similar to Embodiment 1. Based on the microfluidic chip of Embodiment 2, a biofilm biomimetic drug-loaded nanosystem is constructed. PCEC (PCL-PEG-PCL) drug-loaded nanoparticles are selected as the core. The hydrophobic end inside the PCEC nanoparticles effectively loads the bortezomib (Bort) drug. The extracted multiple myeloma cell membrane is wrapped around the outer layer of the drug-loaded nanoparticles to prepare a tumor cell biomimetic nanosystem for the treatment of multiple myeloma, such as Figure 13 . The construction method includes the following steps:

[0091] S1. Prepare a drug-loaded nanoparticle solution and a biofilm solution. The nanoparticle solution is a drug-loaded nanoparticle solution.

[0092] The PCEC block copolymer is synthesized by ring-opening polymerization, and then anion PCEC nanoparticles are prepared by the emulsion solvent evaporation method. Specifically, a certain amount of PCEC powder and SDS emulsifier are respectively dissolved in an organic solvent and an aqueous solution, and are emulsified and dispersed to form an oil-in-water state (O / W). The organic solvent in the emulsion is evaporated by a rotary evaporator, and the droplets shrink and the polymer solidifies to form PCEC nanoparticles. According to the drug loading amount, a certain amount of Bort is weighed and dissolved in an organic solvent, and the PCEC nanoparticles are dissolved in an organic solvent. Then the two solutions are mixed and stirred, and the organic solvent in the emulsion is evaporated by a rotary evaporator to obtain drug-loaded nanoparticles. Then the drug-loaded nanoparticles are dissolved in dimethyl sulfoxide to obtain a drug-loaded nanoparticle solution with a concentration of 0.2 mg / mL.

[0093] Multiple myeloma cells (5-TGM1) are extracted by the hypotonic technique and gradient centrifugation method. A certain amount of multiple myeloma cells are cultured, and the cell density is 5*10 6 ~5*10 7CFU / mL. After collection, it was washed three times with PBS, dispersed in the prepared hypotonic solution, and homogenized with a Dounce homogenizer. The homogenized liquid was centrifuged at 3200 g for 5 minutes, and the supernatant was taken. The precipitate was repeated the above homogenization and centrifugation operations, and the supernatants of the two times were combined and centrifuged at 20000 g for 20 minutes. 100000 g of the supernatant was centrifuged for 1.5 hours, and the lower transparent liquid was taken. Then it was washed once with a solution with a pH of 7.5 (10 mM Tris-HCl and 1 mM EDTA) to obtain a colorless and transparent liquid as the extracted cell membrane. The prepared cell membrane was dispersed into phosphate buffer to obtain a biomembrane solution, and the membrane protein concentration was 0.5 - 1 mg / mL.

[0094] S2. The biomembrane solution and the drug-loaded nanoparticle solution prepared in step S1 were respectively pumped into two inlets of the microfluidic chip. The biomembrane solution and the drug-loaded nanoparticle solution were mixed in the solution mixing area of the microfluidic chip to obtain a mixed solution. After the mixed solution converged, it was successively introduced into the encapsulation channels in the encapsulation area. In each encapsulation channel, the mixed solution passed through a membrane with a pore size not greater than 2 μm multiple times, so that the biomembrane and the drug-loaded nanoparticles in the mixed solution were fused into a biomembrane biomimetic drug-loaded nanoparticle system. During the preparation process, the pumping speed of the nanoparticle solution was 20 - 80 μL / h; the pumping speed of the biomembrane solution was 40 - 200 μL / h, and the flow rate ratio of the nanoparticle solution to the biomembrane solution was 1:1.5 - 4.

[0095] S3. After the preparation was completed, the mixed solution containing the biomembrane biomimetic drug-loaded nanoparticle system was collected at the outlet, and the solution was removed by evaporation or centrifugation to obtain the biomembrane biomimetic drug-loaded nanoparticle system.

[0096] When preparing the drug-loaded nanoparticles, drug-loaded nanoparticles with a theoretical drug loading of 10% were prepared. The concentration of drug Bort was detected by high performance liquid chromatography, and the result was the actual drug loading. The particle size distributions of the biomembrane biomimetic drug-loaded nanoparticle systems prepared from drug-loaded nanoparticles with different theoretical drug loadings were respectively tested. The particle size distribution and surface characteristics of the synthesized nanoparticles were analyzed by techniques such as TEM (transmission electron microscopy) and DLS (dynamic light scattering), and the results are shown in Table 1.

[0097] Table 1 Performance of drug-loaded nanoparticles and biomembrane biomimetic drug-loaded nanoparticle systems

[0098]

[0099] Among them, the particle size of the drug-loaded nanoparticles with a theoretical drug loading of 10% was 75 nm. The drug-loaded nanoparticles loaded with drugs had a stable particle size distribution. The particle size of the biomembrane biomimetic drug-loaded nanoparticle system obtained after being wrapped by the cell membrane was 105 nm, and the dispersion coefficient was less than 0.15, as Figure 14 , and the cell membrane was successfully wrapped on the surface of the drug-loaded nanoparticles.

[0100] In this embodiment, the biomimetic drug-loaded nanosystem selects PCEC drug-loaded nanoparticles as the core, and the extracted multiple myeloma cell membrane is wrapped around the outer layer of the drug-loaded nanoparticles to prepare a tumor cell biomimetic nanosystem for the treatment of multiple myeloma. This biomimetic drug-loaded nanosystem migrates into the bone marrow by mimicking bone marrow homing through the regulation of proteins on the cell membrane surface; targets tumor cells using the homologous targeting of surface molecules of tumor cells; and at the same time, escapes phagocytosis by the immune system due to immune escape proteins on the cell membrane surface, avoiding immune clearance.

[0101] Example 6

[0102] In this example, the method of Example 5 was used to test the flow rate conditions of the solution in the preparation of the biomimetic drug-loaded nanosystem. The drug-loaded nanoparticle solution with a theoretical drug loading of 10% and the biomembrane solution prepared by the method of Example 5 were respectively pumped into the inlets of the microfluidic chip of Example 2. The pumping speeds of the nanoparticle solution were adjusted to 40 and 80 μL / h respectively. When the pumping speed was 40 μL / h, the flow rate ratios of the drug-loaded nanoparticle solution to the biomembrane solution were adjusted to 1:1.25, 1:2.5, and 1:5 respectively. When the pumping speed was 40 μL / h, the flow rate ratios of the drug-loaded nanoparticle solution to the biomembrane solution were adjusted to 1:1.25, 1:1.5, and 1:1.5 respectively. The specific flow rate conditions are shown in Table 2. The biomimetic drug-loaded nanosystem was prepared by the construction method of Example 5. The biomimetic drug-loaded nanosystems prepared under the above different flow rate conditions were taken, and the particle size and dispersion coefficient of the biomimetic drug-loaded nanosystem under different flow rate conditions were detected by a dynamic light scattering instrument. The results are shown in Table 2.

[0103] Table 2 Flow rate conditions of the solution in the preparation of the biomimetic drug-loaded nanosystem and test results

[0104]

[0105] As can be seen from the above results, when the flow rate ratio of the nanoparticle solution to the biofilm solution is 1:1.5 - 4, the particle size of the biofilm biomimetic drug-loaded nanosystem is relatively stable, with the particle size in the range of 104 - 108 nm. When the flow rate ratio of the nanoparticle solution to the biofilm solution is less than 1:1.5, the particle size of the biofilm biomimetic drug-loaded nanosystem is larger, greater than 118 nm, and the dispersion coefficient is greater than 0.15, indicating that the particle size is uneven after the biofilm encapsulates the drug-loaded nanoparticles. When the flow rate ratio of the nanoparticle solution to the biofilm solution is greater than 1:4, the particle size of the biofilm biomimetic drug-loaded nanosystem is smaller. The possible reason is that the flow rate of the biofilm is too fast, and the drug-loaded nanoparticles are not fully incorporated into the cell membrane, resulting in the biofilm biomimetic drug-loaded nanosystem obtained by testing being closer to the particle size of the PCEC drug-loaded nanoparticles. Therefore, during the preparation process, the pumping speed of the nanoparticle solution is 20 - 80 μL / h; the pumping speed of the biofilm solution is 40 - 200 μL / h, and the flow rate ratio of the nanoparticle solution to the biofilm solution is 1:1.5 - 4.

[0106] For those skilled in the art, when understanding the solutions described in the specific embodiments of the present invention, they can refer to the conventional technical manuals in the art. At the same time, for the places where the above terms appear, they can make appropriate understandings or adjustments referentially. Without creative labor, the same or similar technical solution implementation situations can be deduced.

[0107] The above embodiments only describe the basic principles, main features, and / or advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and the description in the invention content part of the specification are only the principles or specific cases of the present invention. Without departing from the essence of the innovative idea of the present invention, there are various changes and improvements to the innovative solutions of the present invention, and these changes and improvements all fall within the scope of protection required by the present invention.

Claims

1. A method for constructing a biomimetic drug-loaded nanosystem of biofilm, characterized in that, It includes the following steps: S1. Prepare a nanoparticle solution and a biofilm solution. The nanoparticle solution includes a drug solution or a drug-loaded nanoparticle solution; S2. Based on microfluidic technology, pump the biofilm solution and the nanoparticle solution prepared in step S1 into the inlets of a microfluidic chip respectively. Mix the biofilm solution and the nanoparticle solution in the solution mixing area of the microfluidic chip to obtain a mixture. After the mixture converges, it is sequentially introduced into at least two loading channels in the loading area of the microfluidic chip. In each loading channel, the mixture passes through a diaphragm with a pore size not greater than 2 μm multiple times, so that the biofilm and the drug or drug-loaded nanoparticles in the mixture are fused into a biofilm biomimetic drug-loaded nano-system; S3. After preparation, collect the mixture containing the biofilm biomimetic drug-loaded nano-system to obtain the biofilm biomimetic drug-loaded nano-system.

2. The method for constructing a biomimetic drug-loaded nanosystem according to claim 1, wherein In step S2, the pumping speed of the nanoparticle solution is 20-80 μL / h; the pumping speed of the biofilm solution is 40-200 μL / h, and the flow rate ratio of the nanoparticle solution to the biofilm solution is 1:1.5-4.

3. A method for constructing a biomimetic drug-loaded nanosystem according to claim 1, characterized in that, When the mixture flows into different loading channels, from the upstream to the downstream of the loading channel, the pore size of the diaphragm through which the mixture passes in different loading channels shows a decreasing trend.

4. The method for constructing a biomimetic drug-loaded nanosystem according to claim 3, characterized in that, There are three loading channels in the loading area, namely the first-stage loading channel, the second-stage loading channel, and the third-stage loading channel. A diaphragm is provided in each loading channel, namely the first-stage diaphragm, the second-stage diaphragm, and the third-stage diaphragm. The pore size of the first-stage diaphragm is 0.5-2 μm, the pore size of the second-stage diaphragm is 0.2-1.2 μm, and the pore size of the third-stage diaphragm is 0.05-0.8 μm. The pore sizes of the first-stage diaphragm, the second-stage diaphragm, and the third-stage diaphragm show a decreasing trend.

5. A method for constructing a biomimetic drug-loaded nanosystem according to claim 1, characterized in that, The biofilm solution is prepared using cell membranes, and the cell membranes include at least one of exosomes, tumor cell membranes, bone marrow stromal cell membranes, macrophage membranes, red blood cell membranes, white blood cell membranes, platelet membranes, mesenchymal stem cell membranes, bacterial membranes, and fungal membranes; the drugs used in the preparation of the drug solution or the drug-loaded nanoparticle solution include at least one of clinical drugs such as bortezomib, doxorubicin, taxane, camptothecin, curcumin, and daunorubicin.

6. Application of the biofilm biomimetic drug-loaded nano-system obtained by the method for constructing a biofilm biomimetic drug-loaded nano-system according to any one of claims 1-5 in the preparation of drugs for tumor treatment.

7. A microfluidic chip, characterized in that, For implementing the method for constructing a biofilm biomimetic drug-loaded nano-system according to any one of claims 1-5, the microfluidic chip is provided with a closed microchannel cavity, and the microchannel cavity is provided with an inlet and an outlet; From upstream to downstream, the microchannel cavity includes a connected solution mixing area and a loading area. The loading area includes at least two connected loading channels. Each loading channel includes at least two sub-loading channels arranged in parallel. A diaphragm is provided in each loading channel. Each sub-loading channel is distributed on both sides of the diaphragm and is connected through the diaphragm to enable the mixed liquid to pass through the diaphragm multiple times. Micro-holes are provided on the diaphragm, and the aperture of the micro-holes is not greater than 2 μm. From the upstream to the downstream of the loading channel, the aperture of the micro-holes on the diaphragms in different loading channels shows a decreasing trend.

8. The microfluidic chip according to claim 7, wherein The microfluidic chip includes a cover plate, a bottom plate, and a microchannel plate. The microchannel plate is arranged between the cover plate and the bottom plate to form a closed microchannel cavity.

9. The microfluidic chip according to claim 8, characterized in that, The microchannel plate includes an upper plate and a lower plate. The solution mixing area is a mixing channel provided on the upper plate. The upstream of the mixing channel is connected to the inlet, and the downstream of the mixing channel converges the mixed liquid and is connected to the loading area. The sub-loading channel includes a plurality of first through-hole structures and second through-hole structures. The plurality of first through-hole structures are provided on the upper plate, and the plurality of second through-hole structures are provided on the lower plate. The number of the first through-hole structures matches that of the second through-hole structures and they are arranged in a staggered manner. In each loading channel, the diaphragm is arranged between the upper plate and the lower plate. The first through-hole structures and the second through-hole structures are distributed on both sides of the diaphragm and are connected through the diaphragm.

10. The microfluidic chip according to any one of claims 7-9, characterized in that, A first detection port is provided between the solution mixing area and the loading area, and / or a second detection port is provided between different loading channels, and / or a third detection port is provided between the end of the last loading channel and the outlet. The first detection port, the second detection port, and the third detection port are used for detecting the physical and chemical parameters in the flow channel.

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