One-step cell membrane bionic lipid nanoparticles as well as preparation method, device and application thereof

Abstract

The invention discloses a one-step cell membrane bionic lipid nanoparticle as well as a preparation method, a device and application thereof, and belongs to the field of microfluidics preparation and bionic preparation. The one-step preparation chip is constructed by combining microfluidic and alternating current stimulation technologies, the preparation method is simple and convenient, and conditions are controllable. The fusion efficiency on cell membranes and lipid nanoparticles is better, the protection capability on protein components is better, and the clinical transformation possibility is good. The cell membrane bionic lipid nanoparticles can effectively carry various drugs, achieve accurate targeting of the recognition ability of pulmonary alveolar damaged pulmonary epithelial cells, achieve more efficient drug delivery ability, can achieve effective intervention in the early stage of pulmonary fibrosis, and have broad application prospects.

Description

Technical Field

[0001] The invention belongs to the field of microfluidic preparation and bionic preparation, and particularly relates to a one-step cell membrane bionic lipid nanoparticle using fluid microfluidic and alternating current technology, and a preparation method, device and application thereof. Background Art

[0002] Cell membrane bionic nano delivery carrier is a nano-sized delivery carrier obtained by coating or fusing the cell membrane with the nano carrier after the cell is broken. The cell membrane is the basis of cell physiological activities, and it retains the required part of the ligand of the cell physiological function. Therefore, after the cell membrane is organically combined with the nano carrier, the cell membrane surface ligand is also grafted into the cell membrane bionic nano delivery carrier, giving it natural biological multifunctionality. The bionic nano delivery carrier integrates the dual advantages of cell membrane and nano delivery carrier, and has unique advantages in the field of drug delivery. For example, the patent application with publication number CN117243920A discloses a five-layer nested core-shell structure bionic nano preparation modified by macrophage membrane, the invention helps the bionic nano preparation to escape the recognition of the immune system and improve its retention time in the body by modifying the macrophage membrane, and the antibody targeting endothelial cell factor is used to ensure that the nano preparation acts on the lesion blood vessel in a targeted manner, and the multiple anti-angiogenic drugs and targeted antibodies used, as well as the use of macrophage membrane, increase the therapeutic effect and targeting of the nano preparation, and effectively prevent the generation of synovial blood vessels. In addition, the patent application with publication number CN117159499A shows that liposomes modified with platelet membranes and 4T1 tumor cell membranes can effectively cross the blood-brain barrier of rats, selectively accumulate in the ischemic area of ​​the brain, and deliver drugs to damaged cells in a targeted manner.

[0003] At present, the commonly used preparation method of cell membrane bionic nano delivery system is a two-step method, that is, lipid nanoparticles are prepared first, and then cell membrane bionic modification is performed. Mechanical co-extrusion or ultrasonic fusion is the main method of cell membrane bionic modification. However, the above two bionic modification methods have the problem of wide particle size distribution of prepared nanoparticles, large drug loading and batch differences. At the same time, it is difficult to achieve precise control of cell membrane and lipid nanoparticle components in two-step preparation. And the traditional bionic modification method uses mechanical force to break up the lipid nanoparticle phospholipids and cell membrane and then reorganize. Cell membrane protein is the basis of the biological multifunctionality of cell membrane bionic carriers, but the traditional bionic preparation method relies on large mechanical force, and the large mechanical force in the process is easy to cause cell membrane protein damage, thereby affecting the targeting efficiency. At the same time, the large mechanical force reorganization process will cause the encapsulated drug to leak and reduce the encapsulation rate of the drug. Therefore, it is urgent to construct a one-step preparation method to accurately control the preparation process, improve the encapsulation rate of the drug, and reduce the damage to the cell membrane protein.

[0004] In recent years, microfluidics has been gradually applied to the preparation of lipid nanoparticles. Microfluidics technology is used to design microfluidic chips, which can realize the preparation and process control of lipid nanoparticles. For example, the patent application with publication number CN117123160A designs a microfluidic nanoparticle preparation chip, which simplifies the nano preparation steps. At the same time, microfluidics can be used to achieve effective mixing and precise control between different components, which helps to reduce batch differences. However, simple microfluidics chips can only realize the preparation of lipid nanoparticles, and can not further realize cell membrane bionic modification. Therefore, how to use microfluidics to prepare lipid nanoparticles and realize cell membrane bionic modification, while accurately controlling the ratio of cell membrane to nanocarrier in the preparation of bionic carrier, controlling the cell membrane bionic preparation process, reducing batch differences, and effectively reducing the structural damage of cell membrane proteins in the preparation process, is a technical problem that needs to be solved in this field. Summary of the invention

[0005] The object of the present invention is to provide a one-step cell membrane biomimetic lipid nanoparticle based on microfluidics and alternating current stimulation and a preparation method, device and application thereof. The device is a one-step chip, which can be used for the one-step preparation and biomimetic modification of different types of cell membranes and lipid nanoparticles with different electrical properties. The chip can effectively control the ratio of cell membrane to lipid nanoparticles, significantly improve the fusion ratio of cell membrane to lipid nanoparticles, stably carry fat-soluble, water-soluble chemicals and nucleic acid drugs, and reduce batch differences. At the same time, compared with mechanical co-extrusion and ultrasonic methods, the cell membrane biomimetic lipid nanoparticles prepared by the one-step chip have a better size control range, cell membrane and lipid nanoparticle fusion efficiency, and the cell membrane protein structure is more intact, with better cell affinity and targeting ability.

[0006] A one-step cell membrane bionic nanocarrier preparation chip based on microfluidics and electrical stimulation and its application, wherein the one-step cell membrane bionic nanocarrier preparation chip comprises a lipid nanoparticle preparation area and an alternating current stimulation lipid nanoparticle-cell membrane fusion area. Compared with the traditional two-step preparation method, the cell membrane bionic lipid nanoparticles prepared by the one-step method have a more controllable particle size range, better cell membrane and lipid nanoparticle fusion efficiency, and a more complete cell membrane protein structure, with better cell affinity and targeting ability. In addition, the one-step method improves the yield of cell membrane bionic lipid nanoparticles.

[0007] The specific technical solutions of the present invention are as follows:

[0008] In one aspect, the present invention provides a device for preparing cell membrane biomimetic lipid nanoparticles in one step, comprising a microfluidic chip, wherein the microfluidic chip comprises a flow channel, and the flow channel is divided into a first mixing flow channel, a second mixing flow channel and an electrical stimulation flow channel from upstream to downstream.

[0009] The first mixing channel has a lipid nanoparticle inlet and a drug inlet, and the lipid nanoparticles and the drug are mixed in the first mixing channel to form lipid nanoparticles carrying the drug;

[0010] A cell membrane inlet is provided at the junction of the first mixing channel and the second mixing channel, and the drug-carrying lipid nanoparticles and the cell membrane are mixed in the second mixing channel to form drug-carrying cell membrane-lipid nanoparticles;

[0011] The drug-carrying cell membrane-lipid nanoparticles are fused in the electrical stimulation channel through alternating current stimulation to obtain the cell membrane bionic lipid nanoparticles.

[0012] Preferably, the lipid nanoparticle inlet comprises two oppositely disposed inlets, the drug inlet and the two lipid nanoparticle inlets are arranged in a cross shape, and the cross-shaped nozzle can realize overall fluid acceleration, and prepare a phospholipid-drug mixed system by shearing on both sides.

[0013] Preferably, both the first mixing channel and the second mixing channel are provided with a Tesla valve structure for improving the mixing effect, which can provide turbulence and achieve particle size adjustment of the lipid nanoparticles.

[0014] Preferably, the microfluidic one-step chip channel positive mold is prepared by SU-8 photolithography.

[0015] Preferably, the microfluidic one-step chip flow channel is prepared by curing silicone, and the silicone used is Dow Corning Sylgard 184, and the curing mass ratio is 1:1-10.

[0016] The microfluidic chip is provided with electrodes for providing alternating current electrical stimulation at the electrical stimulation channel.

[0017] Preferably, the electrodes may be gold electrodes, and the interval between adjacent electrodes is 10 μm to 200 μm.

[0018] Preferably, the AC electrode bonding substrate is selected from one of a nano silicon wafer, a silicon dioxide wafer and a quartz glass.

[0019] On the other hand, the present invention also provides a one-step method for preparing cell membrane biomimetic lipid nanoparticles, which is prepared using the above-mentioned device for realizing one-step preparation of cell membrane biomimetic lipid nanoparticles, comprising the following steps:

[0020] (1) dissolving lipid nanoparticles in an organic solvent, dissolving the drug in a buffer, and dispersing the cell membrane in the buffer, thereby obtaining a lipid nanoparticle organic solvent, a drug buffer, and a cell membrane buffer, respectively;

[0021] (2) injecting the lipid nanoparticle organic solvent, drug buffer and cell membrane buffer from the lipid nanoparticle inlet, drug inlet and cell membrane inlet respectively,

[0022] The lipid nanoparticles in the lipid nanoparticle organic solvent and the drugs in the drug buffer are mixed in the first mixing channel to form drug-carrying lipid nanoparticles, and the drug-carrying lipid nanoparticles and the cell membranes in the cell membrane buffer are mixed in the second mixing channel to form drug-carrying cell membrane-lipid nanoparticles;

[0023] (3) The cell membrane-lipid nanoparticles carrying the drug are fused in the electric stimulation channel through alternating current stimulation to obtain the cell membrane biomimetic lipid nanoparticles.

[0024] Specifically, in step (1), the lipid nanoparticles are selected from one or more of egg yolk lecithin, soybean lecithin, dipalmitoylphosphatidylcholine, dioleoylphosphatidylglycerol, distearoylphosphatidylcholine, 1,2-distearoylphosphatidylethanolamine, heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecanyloxy)hexyl)amino)octanoate), ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), ((3-hydroxypropyl)azadiyl)bis(nonane-9,1-diyl)bis(dibutyl octanoate),

[0025] The drugs include fat-soluble drugs and water-soluble drugs, the fat-soluble drugs are curcumin or baicalein, the water-soluble drugs are metformin, artemisinin, cytarabine, doxorubicin, one or more of siRNA, mRNA and sgRNA,

[0026] The cell membrane is selected from one or more of a mesenchymal stem cell membrane, a bone marrow-derived macrophage membrane, a peripheral blood neutrophil membrane, an erythrocyte membrane, a tracheal epithelial cell membrane, a vascular epithelial cell membrane, and a lung cancer tumor cell membrane.

[0027] The organic solvent is selected from one of methanol, ethanol, ether and acetone, and the buffer is selected from one of sodium citrate solution, phosphate buffer and RNAse-free and ion-free sterilized water.

[0028] Preferably, in step (2), the flow rate ratio of the lipid nanoparticle organic solvent, the drug buffer and the cell membrane buffer is 1:1-10:0.5;

[0029] The flow rate of the drug buffer or cell membrane buffer is 0.1-3.0 ml / min;

[0030] The mass ratio of the phospholipid in the lipid nanoparticles to the protein of the cell membrane in the cell membrane buffer is 1:0.1-4.

[0031] Different flow rates and organic solution / buffered aqueous solution flow rate ratios will affect the particle size and particle size range of the prepared cell membrane biomimetic lipid nanoparticles. Further preferably, the organic solvent flow rate is adjusted to 93.3 μl / min and the buffer solution flow rate is adjusted to 700 μl / min. Figure 1 The outlet 5 shown in A can receive the cell membrane biomimetic lipid nanoparticle solution containing an organic solvent.

[0032] The particle size of the cell membrane bionic lipid nanoparticles is 50-500nm, and the polydispersity coefficient is 0.01-0.2.

[0033] Preferably, in step (3), the AC electrode voltage is 5-25V, the frequency is 0.25-25kHz, and the waveform is one of 25% pulse wave, 50% pulse wave, 75% pulse wave, sine wave and irregular wave;

[0034] Different voltages, frequencies and waveforms will affect the fusion efficiency of cell membrane and lipid nanoparticles and the integrity of cell membrane proteins, and further affect the targeting efficiency of the carrier. Preferably, the AC voltage is 10V, the frequency is 12.5kHz, and the waveform is 50% pulse wave.

[0035] The one-step preparation method of cell membrane biomimetic lipid nanoparticles of the present invention further comprises removing the organic solvent from the cell membrane biomimetic lipid nanoparticles obtained in step (3) by dialysis or negative pressure vacuum method.

[0036] Specific process: Place the cell membrane biomimetic lipid nanoparticle solution containing organic solvent in a vacuum drying oven, adjust the air pressure to 0.01Mpa, and the temperature to 20°C. After vacuuming for 30 minutes, a cell membrane biomimetic lipid nanoparticle solution without organic solvent is obtained.

[0037] The invention also provides cell membrane bionic lipid nanoparticles prepared by the preparation method.

[0038] The present invention also provides the use of the cell membrane bionic lipid nanoparticles in preparing drugs for early intervention treatment of pulmonary fibrosis.

[0039] The drug encapsulation rate of the microfluidic one-step method reached more than 90%, which is much higher than the encapsulation rates of the co-extrusion method or the ultrasonic method, indicating that the microfluidic one-step method has better nucleic acid carrying capacity.

[0040] In particular, because the one-step microfluidic preparation method can retain a better cell membrane structure, the prepared cell membrane biomimetic lipid nanoparticles have better recognition ability for damaged alveolar lung epithelial cells, and can deliver more therapeutic drugs to damaged lung epithelial cells, thereby achieving early and effective intervention treatment.

[0041] Preferably, the delivery method of the above-mentioned cell membrane biomimetic lipid nanoparticles in the early intervention treatment of pulmonary fibrosis includes one of intravenous injection, airway instillation and nebulized inhalation.

[0042] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0043] (1) The present invention combines microfluidics and alternating current stimulation technology to construct a one-step preparation chip, which has a simple preparation method and controllable conditions. It has better fusion efficiency for cell membranes and lipid nanoparticles, better protection ability for protein components, and has good clinical transformation potential.

[0044] (2) The cell membrane biomimetic lipid nanoparticles of the present invention can effectively carry various drugs, achieve precise targeting of the recognition ability of damaged alveolar lung epithelial cells, and achieve more efficient drug delivery capabilities, which can achieve effective early intervention in pulmonary fibrosis and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a design diagram of the microfluidic one-step microfluidic chip in Example 1, wherein 1 and 3 are drug inlets, 2 is a lipid nanoparticle inlet, 4 is a cell membrane inlet, and 5 is a product collection port.

[0046] Figure 2 The figure is a process flow chart of the microfluidic one-step microfluidic chip of the present invention.

[0047] Figure 3 This is a characterization result diagram of the microfluidic one-step microfluidic chip in Example 1.

[0048] Figure 4 This is a diagram of the flow rate condition screening experiment for preparing cell membrane biomimetic lipid nanoparticles by the microfluidic one-step method in Example 2.

[0049] Figure 5 This is a graph showing the results of screening flow rate conditions for preparing cell membrane biomimetic lipid nanoparticles by the one-step microfluidic method in Example 2.

[0050] Figure 6 This is a graph showing the screening results of AC voltage and frequency for the AC electrical stimulation conditions for preparing cell membrane biomimetic lipid nanoparticles by the one-step microfluidic method in Example 3.

[0051] Figure 7 This is a graph showing the results of screening the alternating current waveform for the alternating current stimulation conditions for preparing cell membrane biomimetic lipid nanoparticles by the one-step microfluidic method in Example 3.

[0052] Figure 8 This is the confocal observation result of different cell membrane universality of cell membrane biomimetic lipid nanoparticles prepared by microfluidic one-step method in Application Example 1.

[0053] Fig. 9 This is a graph showing the size, polydispersity coefficient and colocalization Pearson coefficient results of different cell membrane universality of cell membrane biomimetic lipid nanoparticles prepared by the one-step microfluidic method in Application Example 1.

[0054] Fig.10 The transmission electron microscopy observation results and size and polydispersity coefficient results of the cell membrane biomimetic lipid nanoparticles prepared by the microfluidic one-step method and the traditional method in Application Example 2.

[0055] Fig.11 This is a fusion efficiency result diagram comparing the cell membrane phospholipid fusion efficiency and protein protection of cell membrane biomimetic lipid nanoparticles prepared by the microfluidic one-step method and the traditional method in Application Example 3.

[0056] Fig.12 This is a graph showing the cell membrane protein protection results comparing the cell membrane phospholipid fusion efficiency and protein protection of cell membrane biomimetic lipid nanoparticles prepared by the microfluidic one-step method and the traditional method in Application Example 3.

[0057] Fig.13 This is a graph comparing the nucleic acid carrying capacity of cell membrane biomimetic lipid nanoparticles prepared by the microfluidic one-step method and the traditional method in Application Example 4.

[0058] Fig.14 This is a graph showing the results of the nucleic acid delivery ability of cell membrane biomimetic lipid nanoparticles prepared by the microfluidic one-step method and the traditional method in Application Example 4.

[0059] Fig.15 This is a graph showing the in vivo distribution comparison and lung accumulation statistics of cell membrane biomimetic lipid nanoparticles prepared by the microfluidic one-step method and the traditional method in the pulmonary fibrosis model in Application Example 5.

[0060] Fig.16 This is a graph comparing the therapeutic effects of the pulmonary fibrosis model using the microfluidic one-step method and the traditional method to prepare cell membrane biomimetic lipid nanoparticles in Application Example 6. DETAILED DESCRIPTION

[0061] Example 1: Preparation and characterization of microfluidic one-step chip

[0062] The process flow chart of the microfluidic one-step chip preparation process of the present invention is as follows: Figure 2 shown.

[0063] 1) Preparation of SU-8 positive mold: Spin coating of SU-82050, pour about 4 mL of SU-82050 on the center of the silicon wafer, spin coating parameters are 1500 rpm, 30 seconds. Program drying, 65 ° C, drying for 10 minutes, then heating to 95 ° C, drying for 30 minutes. UV lithography, dose 500 mJ / cm 2, the photolithography time is 0.01s. Transfer the photolithographic silicon wafer to the heating plate, 65℃, dry for 5min, then heat to 95℃ and dry for 20min. Take the dried silicon wafer, put it into a container containing SU-8 Developer, develop under yellow light for 15min, rinse with isopropanol and deionized water, and blow dry. Harden and bake the developed silicon wafer, start heating at 65℃, rise to 180℃ in 30min, and keep constant at 180℃ for 60min. After cooling, the SU-8 positive mold is obtained.

[0064] 2) Microfluidic preparation: Take Corning Sylgard 184, prepare silicone and curing liquid in a mass ratio of 10:1, take 20g and pour it on the surface of SU-8 cation film. After the silicone is self-leveling, remove the bubbles from the base added with silicone. In a vacuum oven, evacuate at room temperature until there are no bubbles in the colloid. Transfer the silicon wafer containing silicone to the heating table and bake at 100℃ for 40 minutes to solidify the silicone. After the silicone cools down, demold it to obtain a microfluidic chip. The results are as follows Figure 1 As shown, ports 1 and 3 are the drug (drug-buffer solution) inlets, port 2 is the lipid nanoparticle (lipid nanoparticle organic solvent) inlet, port 4 is the cell membrane (cell membrane-buffer) inlet, and port 5 is the product collection port.

[0065] 3) Preparation of gold electrodes: Ultrasonic clean the glass slides with acetone, ethanol, and deionized water for 5 minutes respectively, and blow dry with an air gun. Spin-coat AZ5214E ​​on the clean substrate, and set the speed to pre-spin 500rpm, acceleration 100rpm / s, time 10s, post-spin 2000rpm, acceleration 300rpm / s, time 40s. Transfer the substrate to a hot plate and bake at 110°C for 3min. Use ultraviolet lithography to expose the mask with an electrode pattern for 10s. Develop in SU-8Developer developer for 45s to form an electrode pattern. Vacuum evaporate Cr / Au (5 / 80nm) by thermal evaporation. After evaporation, lift-off the photoresist in acetone, clean the electrode substrate with acetone, blow dry with an air gun, and seal in a clean container for later use.

[0066] 4) Plasma bonding: Place the microfluidic chip and the electrode base in anhydrous ethanol for 5 minutes and blow dry. Then, transfer both to a plasma cleaner for cleaning at 100W for 30 seconds. Align the microfluidic chip and the electrode base, press them tightly, transfer them to a heating table, and heat and bake them at 65℃ for 30 minutes. Press and let stand overnight to obtain a one-step microfluidic chip.

[0067] 5) Characterization of microfluidic one-step preparation chip: The above-mentioned silica gel microchannel was placed under a scanning electron microscope and the morphology of the silica gel channel was observed at a voltage of 3.0 kV. Figure 3As shown, the cross-shaped nozzle can realize overall fluid acceleration, prepare the phospholipid-drug mixture system by shearing on both sides, and the Tesla valve structure can provide turbulence to achieve particle size adjustment of lipid nanoparticles.

[0068] 6) Place the gold electrode under a phase contrast microscope to observe the electrode morphology. Figure 3 As shown, the gold electrode matrix is ​​arranged uniformly with a gap of 50 μm.

[0069] Example 2: Screening of flow rate conditions for preparing cell membrane biomimetic lipid nanoparticles by one-step microfluidic method

[0070] Weigh 10 mg of each of cationic (heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecanyloxy)hexyl)amino)octanoate)), anionic (dioleoylphosphatidylglycerol) and electrically neutral (distearoylphosphatidylcholine) phospholipids, respectively, and dissolve them in 10 ml of ethanol to prepare a phospholipid ethanol solution. Quantify the mesenchymal stem cell membrane-sodium phosphate solution using a protein quantification kit and adjust the concentration to 0.5 mg / ml (with total cell membrane protein as the quantitative standard). Dissolve 5OD NC-siRNA in 0.1M sodium citrate buffer (NC-siRNA sequence: 5'-3': UUCUCCGAACGUGUCACGUTT, 3'-5': ACGUGACACGUUCGGAGA ATT) to prepare a nucleic acid-citric acid solution.

[0071] Transfer the above phospholipid ethanol solution, nucleic acid-sodium citrate buffer solution and cell membrane-sodium phosphate buffer solution to a microinjection pump. Figure 1 Port 2 in A enters the first flow channel, and the nucleic acid-sodium citrate buffer solution flows from Figure 1 Ports 1 and 3 in A enter the first flow channel, and the cell membrane-sodium phosphate buffer flows from Figure 1 The second flow channel was entered from port 4 in A. The flow rates of the nucleic acid-sodium citrate aqueous solution were adjusted to 250, 400, 550, 700, and 850 μl / min, respectively. Under each flow rate condition, the flow rate ratio of the nucleic acid-sodium citrate aqueous solution (aqueous phase) to the phospholipid ethanol solution (organic phase) was adjusted to 1:1, 5:1, 7.5:1, and 10:1, respectively (see Figure 5 ), and the specific flow rate conditions are shown in Table 1. At the same time, the flow rate of the 0.5 mg / ml mesenchymal cell membrane phosphate solution was adjusted to be 1 / 2 of the phospholipid ethanol solution under each group of conditions. The solution containing cell membrane biomimetic lipid nanoparticles was collected at outlet 5, and the cell membrane biomimetic lipid nanoparticle solution was obtained after removing the organic solvent in a vacuum drying oven.

[0072] Table 1

[0073]

[0074]

[0075] The cell membrane biomimetic lipid nanoparticles prepared under the above different flow rate conditions were taken, and the particle size and polydispersity coefficient of the biomimetic lipid nanoparticles under different flow rate conditions were detected by dynamic light scattering instrument. The optimal flow rate condition was screened with a particle size of 100 nm and a polydispersity coefficient of less than 0.1 as the screening conditions. The screening experiment process is as follows Figure 4 The results are shown in Figure 5 As shown, for phospholipid materials with different electrical properties, the present invention can achieve fusion with cell membranes to prepare cell membrane biomimetic lipid nanoparticles. When the water phase flow rate is 700 μl / min and the water phase / organic phase flow rate ratio is 7.5:1, the particle size of the constructed lipid nanoparticles is about 100 nm, and the polydispersity coefficient is less than 0.1, so the water phase flow rate is 700 μl / min and the water phase / organic phase flow rate ratio is 7.5:1 as the optimal flow rate conditions.

[0076] Example 3: Screening of alternating current stimulation conditions for preparing cell membrane biomimetic lipid nanoparticles by one-step microfluidic method

[0077] 1) Screening of voltage and frequency conditions: Use the green cell membrane probe DiO to stain the stem cell membrane (10μM), and use the red cell membrane probe DiD to stain the phospholipid material (10μM). Fix the optimal flow rate conditions, adjust the voltage to 5V, 10V, 25V, and the frequency to 0.25kHz, 5kHz, 10kHz, 12.5kHz and 25kHz. Take the cell membrane bionic lipid nanoparticles prepared under the above different electrical stimulation conditions, and observe the red and green fluorescence signals under a confocal microscope. The Pearson coefficient is a parameter used to indicate the degree of overlap of two fluorescence signals. The Pearson coefficient obtained under each condition is calculated and statistically analyzed. The results are as follows Figure 6 As shown, when the voltage is 10V and the frequency is 12.5kHz, the Pearson coefficient is the highest at 0.806, so this condition is selected as the optimal voltage and frequency condition.

[0078] 2) Waveform condition screening: Use the green cell membrane probe DiO to stain the stem cell membrane (10μM), and use the red cell membrane probe DiD to stain the phospholipid material (10μM). The voltage is fixed at 10V, the frequency is 12.5kHz, and the AC waveform is adjusted to 25% pulse wave, 50% pulse wave, 75% pulse wave, sine wave and irregular wave. Take the cell membrane bionic lipid nanoparticles prepared under the above-mentioned different waveform electrical stimulation conditions, place them under a confocal microscope to observe the red and green fluorescence signals, calculate and count the Pearson coefficients obtained under each condition, and use a dynamic light scattering instrument to determine the particle size and polydispersity coefficient of the cell membrane lipid nanoparticles under different waveforms. The results are as follows Figure 7As shown, when the waveform is 50% pulse wave, the highest Pearson coefficient is 0.92, and there is no significant effect on the particle size and polydispersity coefficient. Therefore, the 50% pulse wave is selected as the optimal electrical stimulation waveform.

[0079] Application Example 1: Universality of one-step microfluidic method for preparing cell membrane biomimetic lipid nanoparticles

[0080] 1) Stem cell acquisition: Stem cells were obtained from the femur and tibia of 4-week-old SD rats, and the in vitro culture form was adherent culture. The specific method is as follows: 4-week-old SD rats were killed, and the bilateral femurs and tibias were separated in a sterile environment. The culture medium was drawn with a 1mL syringe to repeatedly rinse the femur and tibia, and the obtained bone marrow was passed through a 200-mesh cell sieve, centrifuged at 1200rpm for 5min, the supernatant was discarded, and the precipitate was resuspended in the culture medium to obtain MSCs for in vitro culture. DMEM culture medium containing 1% penicillin-streptomycin (100U / mL) double antibody solution, 1% glutamine, and 10% fetal bovine serum was used as a complete culture medium for in vitro adherent culture of MSCs.

[0081] 2) Obtaining bone marrow-derived macrophage membranes: Mouse bone marrow-derived macrophages were obtained from 4-week-old C57BL / 6 mice. The tibia and femur of the mouse were separated and removed under sterile conditions. Cut open both ends of the tibia and femur, and then use a 1 ml syringe to absorb complete culture medium. Wash the bone marrow cells from one end of the bone into a 50 ml sterile centrifuge tube. Repeat twice until the bone turns white. Add 5 times the volume of red blood cell lysis buffer to the 50 mL centrifuge tube, pipette repeatedly with a Pasteur pipette, and let it stand for 15 minutes. After standing, centrifuge at 1000 rpm for 10 minutes and discard the supernatant. Add an appropriate amount of 1640 cell culture medium to resuspend the cells, and then filter the cells with a 200 mesh filter. Centrifuge at 1000 rpm for 10 minutes, discard the supernatant, and repeat twice. Add 1640 complete culture medium containing 10 ng / mL-1M-CSF to the test tube to resuspend the cells. After one week of in vitro culture, bone marrow cells were induced to differentiate into macrophages.

[0082] 3) Obtaining bone marrow-derived neutrophils: Mouse bone marrow-derived neutrophils were obtained from 4-week-old C57BL / 6 mice and completely disinfected with 75% alcohol. The tibia and femur of the mouse were separated and removed under sterile conditions. Cut both ends of the tibia and femur, and then use a 1ml syringe to absorb the complete culture medium. Wash the bone marrow cells from one end of the bone into a 50ml sterile centrifuge tube. Repeat twice until the bone turns white. Pass the bone marrow cell suspension through a 200-mesh cell sieve, transfer it to a 15mL centrifuge tube, and centrifuge it at 1700rpm for 5 minutes at 4°C. Discard the supernatant, add 3-5 times the red blood cell lysis buffer to the cell pellet, gently pipette until mixed, lyse for 1-2 minutes, centrifuge at 1700rpm for 5 minutes at 4°C, and collect the cell pellet. Use discontinuous density gradient centrifugation to separate and purify neutrophils from bone marrow cells. Add an appropriate amount of serum to a 15mL centrifuge tube, rinse the tube wall and discard it. Add 2 ml of 78% Percoll solution. Use a sterile syringe to add 2mL of 65% and 55% Percoll solutions to 78% Percoll solution in sequence. Above the liquid level. Remove the cell suspension with a pipette, draw up the cell suspension with a sterile syringe, slowly add the top of the 55% Percoll solution along the wall of the centrifuge tube, and centrifuge at 1600g for 30 minutes at 25°C. A layer of white material will appear at the interface between 78% and 65%. Pipette this layer and transfer it to a centrifuge tube, add an equal volume of cold PBS to wash, and then centrifuge at 1700rpm for 3 minutes at 4°C to remove excess percoll. Finally, add an appropriate amount of RPMI 1640 and resuspend to obtain neutrophils.

[0083] 4) Red blood cell acquisition: Take 4 to 8 week old C57BL / 6 mice and collect blood from the eye sockets to obtain whole blood. Transfer 10 mL of whole blood into a 50 mL centrifuge tube, add 10 mL of PBS solution to dilute, and gently mix. Take two 15 mL centrifuge tubes and first add 5 mL of Ficoll solution. Then gently add the diluted blood to the upper layer of Ficoll in the two centrifuge tubes, centrifuge at 2000 rpm for 20 minutes, and the precipitate is the red blood cells.

[0084] 5) Acquisition of alveolar epithelial cells: TC-1 cells of mouse alveolar epithelial cell line in the logarithmic growth phase were obtained, digested with trypsin, and adjusted to a concentration of 1x10 5 / mL, centrifuge at 1200rpm for 5min, and the precipitate is the alveolar epithelial cells.

[0085] 6) Obtaining lung cancer tumor cells: Mouse lung cancer cell line A549 cells in the logarithmic growth phase were obtained, digested with trypsin, and the concentration was adjusted to 1x10 5 / mL, centrifuge at 1200rpm for 5min, and the precipitate is lung cancer tumor cells.

[0086] 7) Take the above-mentioned cells, stain the cells with the green cell membrane probe DiO (10 μM), and then freeze and thaw repeatedly in liquid nitrogen and 37°C water bath for 3 times, transfer to a glass homogenizer and homogenize on ice for 20 times. Subsequently, centrifuge the homogenate (20,000×g, 25min, 4°C), discard the precipitate, centrifuge the supernatant again (100,000×g, 35min, 4°C), and collect the precipitate to obtain the cell membrane.

[0087] 8) 10 mg of each of cationic (heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)amino)octanoate)), anionic (dioleoylphosphatidylglycerol) and electrically neutral (distearoylphosphatidylcholine) phospholipids were accurately weighed and dissolved in 10 ml of ethanol. The above phospholipid materials were stained with the red cell membrane probe DiD (10 μM), and various cell membranes were respectively injected into the microfluidic one-step preparation chip with the above phospholipid-ethanol solution through a microinjection pump, with an aqueous phase flow rate of 700 μl / min, an aqueous phase / organic phase flow rate ratio of 7.5:1, a voltage of 10 V, a frequency of 12.5 kHz, and a waveform of 50% pulse wave for preparation. The cell membrane biomimetic lipid nanoparticles prepared with different electrical properties and different cell membranes were placed under a confocal microscope to observe the red and green fluorescence signals, and the Pearson coefficients obtained under each condition were calculated and statistically analyzed. The particle size, potential and polydispersity coefficient of cell membrane biomimetic lipid nanoparticles under different waveforms were measured using a dynamic light scattering instrument. The results are shown in Figure 2. Figure 8 and 9 As shown in the figure, different types of cell membranes can achieve good fusion with phospholipids, and the Pearson coefficient is above 0.9. Phospholipids with different electrical properties can fuse with different types of cell membranes, and the particle size range is about 140nm, and the potential is negative and negative.

[0088] Application Example 2: Characterization of Cell Membrane Biomimetic Lipid Nanoparticles Prepared by Microfluidic One-Step Method and Traditional Method

[0089] 1) Weigh 10 mg of lecithin with different electrical properties, add 20 mL of anhydrous ethanol to completely dissolve, and then perform rotary evaporation at 30°C for 2 hours. Then, add a certain volume of phosphate buffer. After a short water bath ultrasound, adjust the mixing ratio of stem cell membrane (based on protein amount) and soybean lecithin (500 μg / ml) and mix the two at a ratio of 1:0.5 (soybean lecithin and cell membrane protein mass ratio).

[0090] 2) The co-extrusion method is to use a handheld liposome extrusion instrument to pass through a 400nm and 200nm polycarbonate membrane respectively, and repeatedly extrude 20 times to obtain cell membrane biomimetic lipid nanoparticles.

[0091] 3) The ultrasonic method is to subject the mixed solution to probe ultrasonic particle size adjustment in an ice bath, with an ultrasonic power of 100 W, working for 3 seconds and resting for 2 seconds, and a total working time of 3 minutes, to obtain cell membrane biomimetic lipid nanoparticles.

[0092] 4) The microfluidic one-step method uses a microfluidic one-step chip to transfer 2 mg / mL soybean lecithin solution and 1 mg / mL stem cell membrane solution to a microinjection pump, with an aqueous phase flow rate of 700 μl / min, an aqueous phase / organic phase flow rate ratio of 7.5:1, a voltage of 10 V, a frequency of 12.5 kHz, and a waveform of 50% pulse wave to obtain cell membrane biomimetic lipid nanoparticles.

[0093] 5) The morphology of the cell membrane biomimetic lipid nanoparticles prepared by the above three preparation methods was observed by projection electron microscopy, and the particle size and polydispersity coefficient of the cell membrane biomimetic lipid nanoparticles prepared by the above three preparation methods were detected by dynamic light scattering. Fig.10 As shown. For phospholipids with different electrical properties, the cell membrane biomimetic lipid nanoparticles prepared by the microfluidic one-step chip have good dispersibility. The cell membrane biomimetic lipid nanoparticles prepared by the co-extrusion method are easy to fuse and show a nested result. The biomimetic lipid nanoparticles prepared by the ultrasound method are not uniform and are prone to aggregation. The results of particle size and polydispersity coefficient also show that the microfluidic one-step method has a better particle size control range. Application Example 3: Comparison of cell membrane phospholipid fusion efficiency and protein protection of cell membrane biomimetic lipid nanoparticles prepared by microfluidic one-step method and traditional method

[0094] 1) The stem cell membrane was stained with green cell membrane probe DiO (10 μM), and the phospholipid material was stained with red cell membrane probe DiD (10 μM). Cell membrane biomimetic lipid nanoparticles were prepared by microfluidic one-step chip, co-extrusion and ultrasound respectively. The double positive ratio of the two fluorescences was detected by nanoflow cytometry. The results are shown in Figure 2. Fig.11 As shown, the cell membrane biomimetic lipid nanoparticles prepared by the microfluidic one-step method have a higher double-positive ratio, and the double-positive ratios of the three phospholipids, cationic, anionic and electrically neutral, are higher than those of the traditional method, indicating that the microfluidic one-step method has better cell membrane and lipid nanoparticle fusion efficiency.

[0095] 2) The cell membrane was dispersed in RNase-free and ion-free sterile water, and cell membrane biomimetic lipid nanoparticles were prepared by microfluidic one-step chip, co-extrusion and ultrasound. The tertiary structure of the protein was detected by circular dichroism, and the orientation of CD44, VLA-4, CD47 and CXCR4 proteins was detected by dotblot. The results are shown in Figure 2. Fig.12As shown in the figure, the extrusion method and the ultrasonic method are more likely to damage or make CD44, VLA-4, and CD47 face inward during the preparation process, thereby affecting the precise targeting ability of the prepared cell membrane biomimetic lipid nanoparticles to damaged lung epithelial cells. The cell membrane biomimetic lipid nanoparticles prepared by the microfluidic one-step method have better membrane protein protection ability. Among the CD44, VLA-4, CD47 and CXCR4 proteins tested, most of the proteins are facing outward, indicating that the microfluidic one-step method can make more targeting-related proteins face outward through electrical induction, retaining better precise targeting ability.

[0096] Application Example 4: Comparison of nucleic acid carrying and delivery efficiency of cell membrane biomimetic lipid nanoparticles prepared by microfluidic one-step method and traditional method

[0097] 1) Microfluidic one-step nucleic acid / phospholipid ratio screening. Weigh 5 mg of heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)amino)octanoate), 5 mg of soybean lecithin and 2 mg of cholesterol, dissolve in 6 mL of ethanol, and inject into the chip through the organic phase inlet (inlet 1). Dissolve 5 OD of TGF β1 siRNA in ion-free DNA / RNA enzyme-free sterile water (TGF β1 siRNA sequence is 5'-3': GAACCAAGGAGACGGAAUATT, 3'-5': UAUUCCGUCUCCUUGGUUCTT) and inject into the chip through the aqueous phase inlet (inlets 1 and 3). Inject 1 mg / mL of stem cell membrane solution into the chip through inlet 4. The aqueous phase flow rate was 700 μl / min, and the aqueous phase / organic phase flow rate ratio was adjusted so that the mass ratio of siRNA / cell membrane biomimetic lipid nanoparticles was 1:1, 1:2, 1:2.5, 1:5, 1:10, 1:20, 1:30 and 1:40. The voltage was 10 V, the frequency was 12.5 kHz, and the waveform was 50% pulse wave. The product was collected at outlet 5, and after removing the ethanol, it was centrifuged at 15000 g to remove free siRNA. The precipitate was resuspended in 2 mL of phosphate buffer to obtain cell membrane biomimetic lipid nanoparticles. 1% agarose gel electrophoresis was used for nucleic acid encapsulation detection, and the results are shown in the figure. Fig.13 As shown in A, when the mass ratio of siRNA / cell membrane biomimetic lipid nanoparticles is 1:10, all siRNA is encapsulated.

[0098] 2) Preparation by one-step microfluidic method. Weigh 5 mg of heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)amino)octanoate), 5 mg of soybean lecithin and 2 mg of cholesterol, dissolve in 6 mL of ethanol and inject into the chip through the organic phase inlet (inlet 1). Dissolve 5 OD of TGF β1 siRNA in ion-free, DNA / RNA enzyme-free sterilized water and inject into the chip through the aqueous phase inlet (inlets 1 and 3). Inject 1 mg / mL of stem cell membrane solution into the chip through inlet 4. The aqueous phase flow rate was 700 μl / min, and the aqueous / organic phase flow rate ratio was adjusted to 7.5:1, the voltage was 10 V, the frequency was 12.5 kHz, and the waveform was 50% pulse wave. Collect the product at outlet 5, remove the ethanol and centrifuge at 15000 g to remove free siRNA. Resuspend the precipitate in 2 mL of phosphate buffer to obtain cell membrane biomimetic lipid nanoparticles.

[0099] 3) Preparation by traditional method. Weigh 5 mg of heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)amino)octanoate), 5 mg of soybean lecithin and 2 mg of cholesterol and dissolve them in 20 mL of ethanol. Rotary evaporation was performed at 30°C for 2 h to prepare a thin film. 1.2 OD of TGF β1 siRNA was dissolved in ion-free, DNA / RNA enzyme-free sterile water. The siRNA solution was added to the film, and after a short sonication in a water bath, free siRNA was removed by centrifugation at 15000 g. The precipitate was resuspended in 2 mL of phosphate buffer, and the mixing ratio of stem cell membrane (in terms of protein amount) and phospholipid (500 μg / ml) was adjusted respectively, and the two were mixed at a ratio of 1:0.5 (phospholipid and cell membrane protein mass ratio). The cell membrane was fused with lipid nanoparticles by co-extrusion or ultrasound to obtain cell membrane biomimetic lipid nanoparticles carrying siRNA.

[0100] 4) Use 1% agarose gel electrophoresis to test the nucleic acid loading of different preparations. Fig.13 As shown in B and C, when the mass ratio of siRNA / cell membrane biomimetic lipid nanoparticles is 1:10, both the coextrusion method and the ultrasound method will cause the encapsulated nucleic acid to leak, while the cell membrane and lipid nanoparticle fusion process of the microfluidic one-step method does not rely on mechanical force, so the drug will not leak. The siRNA encapsulation rate of the microfluidic one-step method reaches more than 90%, while the encapsulation rates of the other two methods are only 40%-50%, indicating that the microfluidic one-step method has a better nucleic acid carrying capacity.

[0101] 5) Treat mouse lung epithelial cell TC-1 cell line with 10 ng / mL bleomycin solution and incubate at 37°C overnight. Incubate TC-1 cells with FAM siRNA-cell membrane biomimetic lipid nanoparticles prepared by the above different methods at 37°C for 6 hours, discard the culture medium, digest with trypsin to form a single cell suspension, and detect the proportion of positive cells by flow cytometry. The results are as follows: Fig.14 As shown in A, the positive rate of cell membrane biomimetic lipid nanoparticles prepared by the microfluidic one-step method reached about 80%, while the positive rates of cell membrane biomimetic lipid nanoparticles prepared by the two traditional methods were both lower than 40%, indicating that the microfluidic one-step method has better nucleic acid delivery ability.

[0102] 6) Treat mouse lung epithelial cell TC-1 cell line with 10 ng / mL bleomycin solution and incubate at 37°C overnight. Cell membrane biomimetic lipid nanoparticles carrying TGF β1 siRNA prepared by the above different methods were incubated with TC-1 cells. After incubation at 37°C for 6 hours, the culture medium was replaced and the expression of TGF β1 in the cells was detected after 24 hours. The results are shown in Figure 2. Fig.14 As shown in B, the cell membrane biomimetic lipid nanoparticles prepared by the microfluidic one-step method have a better ability to inhibit the expression of TGF β1. Fig.14 As shown in Figure C, the siRNA-cell membrane biomimetic lipid nanoparticles prepared by the co-extrusion method and the ultrasound method are more likely to enter the lysosomal pathway in the damaged lung epithelial cells, while the siRNA-cell membrane biomimetic lipid nanoparticles prepared by the microfluidic one-step method have siRNA rapidly released after entering the cells, thereby achieving a better therapeutic effect.

[0103] Application Example 5: Comparison of the in vivo distribution of cell membrane biomimetic lipid nanoparticles prepared by microfluidic one-step method and traditional method in a pulmonary fibrosis model

[0104] The 20 g C57BL / 6 mice were instilled with bleomycin (0.8 mg / kg) through the trachea to establish the pulmonary fibrosis model. After 48 h, the drug was administered by a small animal handheld nebulizer, with each mouse inhaling 50 μl of the drug (the DiD dosage was 400 μg kg -1 ) were used to prepare cell membrane biomimetic lipid nanoparticles by a one-step microfluidic method and a traditional method. After 2, 4, 8, 12, and 24 hours of drug administration, the mouse organs were separated to observe the fluorescence content in the mouse heart, liver, spleen, lung, kidney and other organs, and semi-quantitative statistics of the fluorescence in the lung were performed. The lung tissue was fixed and sliced, and lung epithelial cells were stained to observe the co-localization results of cell membrane biomimetic lipid nanoparticles prepared by different preparation methods and damaged epithelial cells. The results are shown in Fig.15As shown in the figure, the biomimetic lipid nanoparticles prepared by the one-step microfluidic chip accumulate in the lungs for a longer time and have a stronger fluorescence signal. The slice results also show that the biomimetic lipid nanoparticles prepared by the one-step microfluidic chip have better co-localization results with damaged lung epithelial cells, while the cell membrane biomimetic lipid nanoparticles prepared by the two traditional methods are less co-localized with epithelial cells, indicating that their ability to target damaged epithelial cells is weaker.

[0105] Application Example 6: Comparison of the efficacy of cell membrane biomimetic lipid nanoparticles prepared by microfluidic one-step method and traditional method in pulmonary fibrosis model

[0106] 20g C57BL / 6 mice were given bleomycin (0.8mg / kg) by tracheal instillation to establish pulmonary fibrosis model. On the 1st, 4th and 7th day after modeling, TGF β1 siRNAs-cell membrane biomimetic lipid nanoparticles prepared by the above different methods were administered by handheld atomization. 21 days after modeling, the mouse lungs were isolated, the lung tissues were ground to obtain tissue homogenates, and the contents of TGF β1, MMP9 and hydroxyproline in the lungs were detected. The lung tissues were fixed and sliced, and hematoxylin-eosin staining and Masson's III staining were performed. The results are shown in Figure 2. Fig.16 As shown, compared with TGF β1 siRNAs-cell membrane biomimetic lipid nanoparticles obtained by traditional preparation methods, TGF β1 siRNAs-cell membrane biomimetic lipid nanoparticles prepared by microfluidic one-step method have better therapeutic effects, indicating that microfluidic one-step method of preparing chips has better application and transformation prospects.

Claims

1. A device for preparing cell membrane biomimetic lipid nanoparticles in one step, characterized in that: The microfluidic chip includes a flow channel, which is divided into a first mixing flow channel, a second mixing flow channel and an electrical stimulation flow channel from upstream to downstream. The first mixing channel has a lipid nanoparticle inlet and a drug inlet, and the lipid nanoparticles and the drug are mixed in the first mixing channel to form lipid nanoparticles carrying the drug; A cell membrane inlet is provided at the junction of the first mixing channel and the second mixing channel, and the drug-carrying lipid nanoparticles and the cell membrane are mixed in the second mixing channel to form drug-carrying cell membrane-lipid nanoparticles; The drug-carrying cell membrane-lipid nanoparticles are fused in the electrical stimulation channel through alternating current stimulation to obtain the cell membrane bionic lipid nanoparticles.

2. The device for preparing cell membrane biomimetic lipid nanoparticles in one step according to claim 1, characterized in that: The lipid nanoparticle inlet comprises two oppositely disposed inlets, and the drug inlet and the two lipid nanoparticle inlets are arranged in a cross shape.

3. The device for preparing cell membrane biomimetic lipid nanoparticles in one step according to claim 1, characterized in that: The first mixing flow channel and the second mixing flow channel are both provided with Tesla valve structures for improving the mixing effect.

4. The device for preparing cell membrane biomimetic lipid nanoparticles in one step according to claim 1, characterized in that: The microfluidic chip is provided with electrodes for providing alternating current electrical stimulation at the electrical stimulation channel.

5. A one-step method for preparing cell membrane biomimetic lipid nanoparticles, characterized in that: The preparation is carried out using the device for realizing one-step preparation of cell membrane biomimetic lipid nanoparticles according to any one of claims 1 to 3, comprising the following steps: (1) dissolving lipid nanoparticles in an organic solvent, dissolving the drug in a buffer, and dispersing the cell membrane in the buffer, thereby obtaining a lipid nanoparticle organic solvent, a drug buffer, and a cell membrane buffer, respectively; (2) injecting the lipid nanoparticle organic solvent, drug buffer and cell membrane buffer from the lipid nanoparticle inlet, drug inlet and cell membrane inlet respectively, The lipid nanoparticles in the lipid nanoparticle organic solvent and the drugs in the drug buffer are mixed in the first mixing channel to form drug-carrying lipid nanoparticles, and the drug-carrying lipid nanoparticles and the cell membranes in the cell membrane buffer are mixed in the second mixing channel to form drug-carrying cell membrane-lipid nanoparticles; (3) The cell membrane-lipid nanoparticles carrying the drug are fused in the electric stimulation channel through alternating current stimulation to obtain the cell membrane biomimetic lipid nanoparticles.

6. The method for preparing cell membrane biomimetic lipid nanoparticles in one step according to claim 5, characterized in that: In step (1), the lipid nanoparticles are selected from one or more of egg yolk lecithin, soybean lecithin, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecanyloxy)hexyl)amino)octanoate), ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), ((3-hydroxypropyl)azadiyl)bis(nonane-9,1-diyl)bis(dibutyl octanoate), The drugs include fat-soluble drugs and water-soluble drugs, the fat-soluble drugs are curcumin or baicalein, the water-soluble drugs are metformin, artemisinin, cytarabine, doxorubicin, one or more of siRNA, mRNA and sgRNA, The cell membrane is selected from one or more of a mesenchymal stem cell membrane, a bone marrow-derived macrophage membrane, a peripheral blood neutrophil membrane, an erythrocyte membrane, a tracheal epithelial cell membrane, a vascular epithelial cell membrane, and a lung cancer tumor cell membrane. The organic solvent is selected from one of methanol, ethanol, ether and acetone, and the buffer is selected from one of sodium citrate solution, phosphate buffer and RNAse-free and ion-free sterilized water.

7. The method for preparing cell membrane biomimetic lipid nanoparticles in one step according to claim 5, characterized in that: In step (2), the flow rate ratio of the lipid nanoparticle organic solvent, the drug buffer and the cell membrane buffer is 1:1-10:0.5; The flow rate of the drug buffer or cell membrane buffer is 0.1-3.0 ml / min; The mass ratio of the phospholipid in the lipid nanoparticles to the protein of the cell membrane in the cell membrane buffer is 1:0.1-4.

8. The method for preparing cell membrane biomimetic lipid nanoparticles in one step according to claim 5, characterized in that: In step (3), the AC electrode voltage is 5-25V, the frequency is 0.25-25kHz, and the waveform is one of 25% pulse wave, 50% pulse wave, 75% pulse wave, sine wave and irregular wave; The particle size of the cell membrane bionic lipid nanoparticles is 50-500nm, and the polydispersity coefficient is 0.01-0.

2.

9. Cell membrane biomimetic lipid nanoparticles prepared by any preparation method described in claims 5-8.

10. Use of the cell membrane biomimetic lipid nanoparticles according to claim 9 in the preparation of drugs for early intervention treatment of pulmonary fibrosis.

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