Two-dimensional bionic nanostructure assembled based on receptor-ligand acting force and preparation method thereof

Abstract

The invention discloses a two-dimensional bionic nanostructure assembled based on receptor-ligand acting force and a preparation method of the two-dimensional bionic nanostructure. The core characteristic of the two-dimensional bionic nanostructure is that the ultrafine particles modified by ligand molecules are anchored on the surface of a cell membrane fragment with a corresponding receptor expressed on the surface through a receptor-ligand effect, so that uniform compounding of the ultrafine particles and the cell membrane is realized; the bottlenecks of low compounding efficiency, functional site masking and the like caused by high surface curvature of small-size particles in the traditional cell membrane complete wrapping technology are broken through. According to the preparation method disclosed by the invention, the binding force between the cell membrane and the ultramicro particles is stronger, the off-target phenomenon of the ultramicro particles in the in-vivo circulation process can be avoided, the response release of the ultramicro particles under the competition effect in a lesion region expressed by a high-abundance receptor can be realized, and the preparation method has a good application prospect in the aspect of precise drug delivery. Meanwhile, the in-vivo circulation time of the ultrafine particles is prolonged by membrane loading, the biocompatibility of the ultrafine particles is improved, and the effectiveness and low toxicity of a drug delivery system are ensured.

Description

Technical Field

[0001] The present invention belongs to the field of nanomaterial preparation and biomedical application, and specifically relates to a two-dimensional bionic nanostructure assembled based on receptor-ligand interaction force and a preparation method thereof. Background Art

[0002] In recent years, ultrafine nanoparticles have shown great potential in biomedical applications due to their advantages, such as small size, large surface area, and easy surface modification, especially their ability to efficiently penetrate the blood-brain barrier. For example, studies have found that yellow-emitting carbon dots (Y-CDs) can cross the BBB, enter neuronal cells, and effectively inhibit the overexpression of amyloid precursor protein Aβ. However, while the small size of ultrafine nanoparticles facilitates their penetration of the blood-brain barrier, it also presents some challenges. On the one hand, they are easily metabolized and cleared by organs such as the kidneys, resulting in insufficient accumulation of drugs in the lesion area. On the other hand, ultrafine nanoparticles may be recognized as foreign bodies by the immune system and then phagocytosed and degraded by immune cells such as macrophages, further reducing their delivery efficiency. These factors have greatly limited the practical application of ultrafine nanoparticles in the biomedical field. To overcome these problems, researchers have attempted to encapsulate ultrafine nanoparticles with cell membranes to increase their biocompatibility and immune evasion, but this encapsulation method still faces many challenges: (1) When the size of nanoparticles is less than 22 nm, their high surface curvature makes the nanoparticles resist complete encapsulation by the phospholipid layer, making it difficult to achieve complete encapsulation by the cell membrane. (2) The encapsulation process may cause the active sites of the drug to be shielded, affecting its effective binding to the target or catalytic activity; (3) During the functionalization process of modifying the target molecule on the cell membrane surface, high salt or changes in acidity and alkalinity can easily induce conformational changes in cell membrane proteins and lose some of their biological activity. (4) Nanoparticles are affected by various factors during circulation in the body, which can cause the nanoparticles in the complex synthesized based on the electrostatic adsorption strategy to fall off pre-lying. Therefore, developing a strategy that can efficiently combine ultrafine particles with cell membranes without affecting the respective characteristics of the two is of great research significance in drug development. Summary of the Invention

[0003] In response to the problem of the complex existence of cell membranes and ultrafine particles, the present invention aims to provide a two-dimensional bionic nanostructure assembled based on receptor-ligand interaction and a preparation method thereof, which belongs to the field of nanomaterial preparation and biomedicine.

[0004] A large number of studies have shown that different specific receptors are expressed on the surface of the cell membrane, which can bind to specific ligands, and their binding force is much stronger than electrostatic attraction. Therefore, the preparation method provided by the present invention is to modify specific ligands on the surface of ultrafine particles. The ligands have a certain binding force with the receptors expressed on the surface of the target cell membrane, and the ligand-receptor interaction between the two is used to achieve effective compounding of ultrafine particles and cell membranes. This composite strategy innovatively solves the problem that small-sized nanodots cannot be compounded with cell membranes using traditional full-encapsulation methods due to their high surface curvature. At the same time, the defect of the active ingredients in traditional methods being masked by the cell membrane and delaying the efficacy of the drug can be overcome by exposing the active sites of ultrafine particles. Secondly, this design directly sets the functional modification process of various target molecules in the preparation process of nanoparticles, rather than modifying them on the cell membrane. This design can avoid the risk of membrane protein denaturation and inactivation caused by conventional functional modifications, and can retain the natural biological functions of the cell membrane to the greatest extent. Finally, the receptor-ligand binding interaction strengthens the binding force between the ultrafine particles and the cell membrane, effectively preventing off-target effects during drug circulation in the body while simultaneously enabling competitive response release in the lesion area. Therefore, this biomimetic nanostructure has great application prospects in achieving precise drug delivery.

[0005] To achieve the above object, the present invention adopts the following technical solutions: A method for preparing a two-dimensional biomimetic nanostructure assembled based on receptor-ligand interaction forces, which uses ligand-receptor specific binding to anchor ligand-modified ultrafine particles on cell membrane fragments with corresponding receptors expressed on the surface, so that the ultrafine particles are distributed on the surface of the cell membrane fragments in a discrete and uniform manner, thereby obtaining a two-dimensional biomimetic nanostructure.

[0006] Furthermore, the preparation method includes the following steps: modifying a specific ligand on the surface of the ultrafine particles, and then mixing and incubating with cell membrane fragments of a certain size expressing corresponding receptors on the surface in a certain mass ratio to obtain a two-dimensional biomimetic nanostructure.

[0007] Furthermore, the ligand-receptor binding types for preparing the above-mentioned two-dimensional biomimetic nanostructure include but are not limited to mannose-mannose receptor, baicalein-Toll-like receptor (TLR4), and human brain-derived nerve growth factor-tyrosine kinase receptor.

[0008] Furthermore, the cell membranes include but are not limited to the cell membranes of microglia, macrophages, neurons, red blood cells, platelets, vascular endothelial cells, and tumor cells.

[0009] Furthermore, the above-mentioned ultrafine particles include solid solid ultrafine particles and self-assembled ultrafine particles; solid solid ultrafine particles include but are not limited to quercetin quantum dots, EGCG quantum dots, rutin quantum dots, and carbon quantum dots; self-assembled ultrafine particles include but are not limited to flavonoid compound molecular self-assembled particles and polyphenol compound self-assembled particles.

[0010] Furthermore, the particle size of the ultrafine particles is less than 20 nm, and the size of the cell membrane fragments is 80 to 200 nm.

[0011] Furthermore, the mass ratio of the ultrafine particles to the cell membrane fragments is 1:5-40; and the mixed incubation time is 12 hours.

[0012] A two-dimensional bionic nanostructure is prepared by the above preparation method.

[0013] Furthermore, the above-mentioned two-dimensional biomimetic nanostructure has the following characteristics: there is a strong binding force between the cell membrane and the ultrafine particles, which can avoid the off-target phenomenon of ultrafine particles during circulation in the body; it can achieve the release of drugs in response to competitive effects in the lesion area with high abundance receptor expression; and it can achieve the long circulation effect of ultrafine particles in the living body.

[0014] The application of the above-mentioned two-dimensional bionic nanostructure in the preparation of precise drug delivery system.

[0015] The significant advantages of the present invention are: (1) Efficient binding and reduced drug pre-leakage: A method for effectively combining ultrafine particles with cell membranes based on ligand-receptor interaction has been developed. This innovative solution addresses the problem of small-sized nanodots being unable to be combined with cell membranes using traditional full-encapsulation methods due to their high surface curvature. Furthermore, because the receptor-ligand binding energy is significantly higher than the electrostatic adsorption force, the stronger binding force can reduce the risk of off-target effects during systemic circulation and reduce drug pre-leakage during circulation.

[0016] (2) Simple technical operation: The present invention concentrates the modification of various target molecules in the nanoparticle preparation process. The composite of ultrafine particles and membranes only requires a simple mixing and incubation process, which will not destroy the protein recognition ability and other functionalities of the cell membrane surface.

[0017] (3) The present invention can overcome the defect of traditional methods in which the active ingredients are masked by the cell membrane and the efficacy is delayed by exposing the active sites of ultrafine particles. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : a is the TEM image of QMNDs; b is the TEM image of QNDs; c is the TEM image of QMCM; d is the TEM image of QCM.

[0019] Figure 2 : a is the particle size statistics diagram of QNDs; b is the stability characterization diagram of QNDs; c is the particle size statistics diagram of QMNDs; d is the stability characterization diagram of QMNDs.

[0020] Figure 3 : a is the release curve of QMCM and QCM at pH = 5; b is the drug competition response release curve of QMCM under different stimulation conditions; c is the TEM image of QMCM co-incubated with mannose.

[0021] Figure 4 : a is a characterization diagram of the biocompatibility of QMCM for PC12 cells; b is a characterization diagram of the biocompatibility of QMCM for BV2 cells.

[0022] Figure 5 : a, b are the BBB targeting characterizations of QMCM; c, d are the homologous targeting characterizations of QMCM cells.

[0023] Figure 6 : a is a diagram showing the circulation of QMNDs in zebrafish over time; b is a diagram showing the circulation of QMCM in zebrafish over time.

[0024] Figure 7 : a is the TEM image of LIBF nanodots; b is the TEM image of LTBF nanodots.

[0025] Figure 8 : a is the TEM image of LIBFMM; b is the TEM image of LIBFNM; c is the TEM image of LTBFNM.

[0026] Figure 9 : a is the drug release curve of LIBFMM under different conditions; b is the drug release curve of LTBFNM and LIBFNM at pH = 5.

[0027] Figure 10 : Characterization diagram of cell targeting of LIBFMM.

[0028] Figure 11 : Characterization of cell targeting of LTBFNM. DETAILED DESCRIPTION

[0029] To make the technical contents of the present invention easier to understand, the present invention is further described below using QMCM, LTBFMM, and LIBFNM as examples with accompanying drawings and embodiments, but the present invention is not limited thereto.

[0030] Example 1: Preparation of two-dimensional biomimetic nanostructures based on mannose-mannose receptor binding force (1) Preparation of mannose-modified quercetin quantum dots (QMNDs): 0.08 g of quercetin (Que) was weighed and dissolved in 10 ml of 2.5 mg / ml NaOH aqueous solution. 0.016 g of mannose was added and evenly dispersed by ultrasonication. The mixture was reacted in an 80°C oil bath for 1 h. After the reaction, the reaction solution was dialyzed in deionized water using a dialysis membrane with a molecular weight cutoff of 3500 kDa for 48 h. The permeate was collected to obtain a QMNDs solution.

[0031] (2) Preparation of quercetin quantum dots (QNDs): 0.08 g of Que was weighed and dissolved in 10 ml of 2.5 mg / ml NaOH aqueous solution. The mixture was evenly dispersed by ultrasonication and reacted in an 80°C oil bath for 1 h. After the reaction, the reaction solution was dialyzed in deionized water using a dialysis membrane with a molecular weight cutoff of 3500 kDa for 48 h. The permeate was collected to obtain a QNDs solution.

[0032] (3) Preparation of BV2 cell membrane fragments (CM): BV2 cells were added to a hypotonic lysis buffer (20 mM Tris-HCl, 10 mM KCl, 2 mM MgCl2, 1% PMSF; pH 7.4), incubated at 4°C for 1 h, and then washed twice with PBS buffer (pH 7.4). Finally, the lysed BV2 cells were ultrasonically disrupted using a cell disruptor (ultrasonication conditions: 650 W, 30% power, 3 s working / 7 s rest, for 6 min). After high-speed centrifugation (high-speed centrifugation conditions: 4°C, 16,000 rpm, centrifugation for 10 min), the precipitate was collected to obtain CM with a size of approximately 100 nm.

[0033] (4) Preparation of QMCM: The QMNDs solution and CM were mixed and incubated in a shaker at 37°C at a mass ratio of QMNDs:CM = 1:10 for 12 h. After the incubation, the mixture was centrifuged at 13,000 rpm for 30 min, and the precipitate was collected to obtain QMCM.

[0034] (5) Preparation of QCM: The QNDs solution and CM were mixed and incubated in a shaker at 37°C at a mass ratio of QNDs:CM = 1:10 for 12 h. After the incubation, the mixture was centrifuged at 13,000 rpm for 30 min, and the precipitate was collected to obtain QCM.

[0035] Example 2: Preparation of two-dimensional biomimetic nanostructures based on baicalein-TLR4 receptor binding ability (1) Preparation of Fmoc-modified tryptophan (Fmoc-Trp) stock solution: 0.0043 g of Fmoc-Trp was weighed and dissolved in 100 μL of Tris aqueous solution (1 M), and then 900 μL of deionized water was added and ultrasonicated to disperse it evenly to obtain a Fmoc-Trp stock solution with a concentration of 0.01 M.

[0036] (2) Preparation of FeSO4 stock solution: Weigh 0.0055 g of FeSO4, add 100 μL of HCl aqueous solution (0.1 M) to dissolve it, and then add 900 μL of pure water to obtain a FeSO4 stock solution with a concentration of 0.02 M.

[0037] (3) Preparation of baicalin (Bai) stock solution: Weigh 0.0058 g of Bai and dissolve it in DMSO to prepare a Bai stock solution with a concentration of 0.02 M.

[0038] (4) Preparation of lactoferrin (Lf) stock solution: Weigh 10 mg of Lf and dissolve it in pure water to prepare an Lf stock solution with a concentration of 10 mg / ml.

[0039] (5) Preparation of Lf-Trp-Bai-Fe nanodots (LTBF nanodots): Add 200 μL of Fmoc-Trp stock solution to the reaction flask, then add 50 μL of 0.1 M Tris aqueous solution, 500 μL of Lf stock solution and 50 μL of FeSO4 stock solution, react at room temperature for 5 minutes, then add 50 μL of Bai stock solution, react at room temperature for 15 minutes, centrifuge at 10,000 rpm for 20 minutes, collect the supernatant to obtain the LTBF nanodot solution.

[0040] (6) Preparation of macrophage membrane fragments (MM): Macrophages were collected and centrifuged at 2000 rpm for 5 min at 4°C. The obtained cell pellet was washed twice with pre-chilled PBS buffer (pH 7.4), then added to pre-chilled hypotonic lysis buffer (20 mM Tris-HCl, 10 mM KCl, 2 mM MgCl2, 1% PMSF; pH 7.4), and incubated at 4°C for 1 h for lysis. The cells were then washed twice with PBS buffer (pH 7.4). Finally, the lysed macrophages were ultrasonically disrupted using a cell disruptor (ultrasonication conditions: 650 W, 30% power, 3 s working / 7 s rest, for 6 min). 1 M sucrose solution was added to a final sucrose concentration of 0.25 M, and the cells were centrifuged at 4000 rpm for 6 min at 4°C. The supernatant was collected and further centrifuged at 5000 rpm for 15 min. The supernatant obtained was the MM solution, which was aliquoted and stored in a -20°C refrigerator for later use.

[0041] (7) Preparation of MM-loaded LTBF biomimetic nanostructures (LTBFMM): The LTBF nanodot solution and the MM solution were mixed and incubated in a shaker at 37°C at a mass ratio of LTBF nanodots:MM = 1:10 for 12 hours. After the incubation, the mixture was centrifuged at 13000 rpm for 30 minutes, and the precipitate was collected to obtain LTBFMM.

[0042] Example 3: Preparation of two-dimensional biomimetic nanostructures based on BDNF-TrkB receptor binding ability (1) Preparation of BDNF mimetic peptide stock solution: 0.0069 g of human brain-derived growth factor (BDNF) mimetic peptide was weighed, dissolved in pure water, and evenly dispersed by ultrasound to prepare a BDNF mimetic peptide stock solution with a concentration of 0.01 M.

[0043] (2) Preparation of FeSO4 stock solution: Weigh 0.0055 g of FeSO4, add 100 μL of HCl aqueous solution (0.1 M) to dissolve it, and then add 900 μL of pure water to obtain a FeSO4 stock solution with a concentration of 0.02 M.

[0044] (3) Preparation of Bai stock solution: Weigh 0.0058 g of Bai and dissolve it in DMSO to prepare a Bai stock solution with a concentration of 0.02 M.

[0045] (4) Preparation of Lf stock solution: Weigh 10 mg of Lf and dissolve it in pure water to prepare an Lf stock solution with a concentration of 10 mg / ml.

[0046] (5) Preparation of Lf-IKRG-Bai-Fe nanodots (LIBF nanodots) solution: Add 200 μL of BDNF mimetic peptide stock solution to the reaction flask, then add 50 μL of 0.1 M Tris solution, 500 μL of Lf stock solution and 50 μL of FeSO4 stock solution, react at room temperature for 5 minutes, add 50 μL of Bai stock solution, react at room temperature for 15 minutes, centrifuge at 10,000 rpm for 20 minutes, collect the supernatant to obtain the LIBF nanodot solution.

[0047] (6) Preparation of PC12 cell membrane fragments (NM): PC12 cells were collected and centrifuged at 2000 rpm for 5 min at 4°C. The obtained cell pellet was washed twice with pre-chilled PBS buffer (pH 7.4), then added to pre-chilled hypotonic lysis buffer (20 mM Tris-HCl, 10 mM KCl, 2 mM MgCl2, 1% PMSF; pH 7.4), and allowed to stand at 4°C for 1 h for lysis. The cells were then washed twice with PBS buffer (pH 7.4). Finally, the lysed macrophages were ultrasonically disrupted using a cell disruptor (ultrasonication conditions: 650 W, 30% power, 3 s working / 7 s rest, for 5 min), centrifuged at 4000 rpm for 6 min at 4°C, the supernatant was collected and further centrifuged at 5000 rpm for 15 min, and the supernatant was collected and further centrifuged at 9000 rpm for 15 min. The supernatant obtained was the NM solution, which was aliquoted and stored in a -20°C refrigerator for later use.

[0048] (7) Preparation of NM-loaded LIBF two-dimensional biomimetic nanostructures (LIBFNM): The LIBF nanodot solution and the NM solution were mixed and incubated in a shaker at 37°C at a mass ratio of LIBF nanodots:NM=1:20 for 12 h. After the incubation, the mixture was centrifuged at 13000 rpm for 30 min, and the precipitate was collected to obtain LIBFNM.

[0049] (8) Preparation of MM-loaded LIBF two-dimensional biomimetic nanostructures (LIBFMM): The LIBF nanodot solution and the MM solution obtained in Example 2 were mixed and incubated in a shaker at 37° C. at a mass ratio of LIBF nanodots:MM=1:10 for 12 h. After the incubation, the mixture was centrifuged at 13,000 rpm for 30 min, and the precipitate was collected to obtain LIBFMM.

[0050] (9) Preparation of NM-loaded LTBF two-dimensional biomimetic nanostructures (LTBFNM): The LTBF nanodot solution obtained in Example 2 and the NM solution were mixed and incubated in a shaker at 37° C. for 12 h at a mass ratio of LTBF nanodots:NM=1:20. After the incubation, the mixture was centrifuged at 13,000 rpm for 30 min, and the precipitate was collected to obtain LTBFNM.

[0051] 1. QMNDs, QNDs, QMCM and QCM were prepared into a solution with a concentration of 10 μg / mL, and then dropped onto the copper grid. After drying, the solution was characterized by electron microscopy. The results were as follows: Figure 1 As shown. Figure 1 a. Figure 1 b As can be seen, QMNDs and QNDs are small-sized nanoparticles with uniform size, less than 10 nm; Figure 1 c. Figure 1d It can be seen that based on the ligand interaction between QMNDs and CD206 on the surface of BV2 cells, the loading rate of QMCM on the MM membrane surface is higher than that of QCM.

[0052] 2. According to Figure 1 a. Figure 1 b Statistical analysis of particle size of QNDs and QMNDs was performed. The results are shown in Figure 2. Figure 2 As shown. Figure 2 a. Figure 2 As shown in Figure c, QNDs and QMNDs are small-sized nanoparticles with sizes of about 6 nm and 7 nm, respectively. The freshly prepared QNDs and QMNDs stock solutions were sealed and stored. Samples were taken at 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and 7 days to prepare solutions with a concentration of 10 μg / mL. The changes in particle size were measured. The results are shown in Figure 4. Figure 2 As shown. Figure 2 b. Figure 2 d As can be seen, the particle size of the nanoparticles did not change significantly within 7 days, indicating that the prepared nanoparticles had good stability.

[0053] 3. 1 mmol of mannose was incubated with cell membrane fragments for 2 hours in advance, and then QMNDs were added and incubated for 12 hours. After the incubation, the mixture was centrifuged at 13,000 rpm for 30 minutes, and the content of nanoparticles in the supernatant was measured. 1 mmol of BSA and Glu were incubated with QMCM for 12 hours. After the incubation, the mixture was centrifuged at 13,000 rpm for 30 minutes, and the content of nanoparticles in the supernatant was measured. Freshly prepared QMCM was dispersed in PBS buffer at pH 5, left for 12 hours, and centrifuged at 13,000 rpm for 30 minutes. The content of nanoparticles in the supernatant was measured. The results are as follows: Figure 3 As shown. As can be seen from the figure, the QMNDs content in the supernatant of the group with mannose added was significantly higher than that of the other groups. This is because the mannose added in advance occupied the CD206 site on the surface of the BV2 cell membrane, making it impossible for QMNDs to attach to the surface of the macrophage membrane; at pH = 5, a certain amount of QMNDs also fell off due to the acidic environment; while BSA and Glu had little effect on it, indicating that certain sugars and proteins in the blood would not cause the shedding of nanoparticles. Freshly prepared QCM and QMCM were dispersed in PBS buffer at pH = 5, left for 12 hours, centrifuged at 13000 rpm for 30 minutes, and the nanoparticle content in the supernatant was measured. The results are shown as follows. Figure 3 As shown in the figure, the amount of nanoparticles shedding in QCM is higher than that in QMCM, indicating that the binding force between nanoparticles in QMCM and cell membrane is stronger than that in QCM.

[0054] 4. In order to investigate the biocompatibility of different products, the toxicity test of QMCM on PC12 cells and BV2 cells was used as the basis.

[0055] When PC12 cells grow to about 90%, cell passaging and inoculation are performed as follows: first, remove the old culture medium, then add 2 mL of PBS solution to gently wash the cells, then add 1 mL of trypsin to digest the cells for about 1 minute, remove the trypsin, and add 2 mL of RPMI-1640 culture medium to stop the digestion; gently pipette the cells to prepare a cell stock solution, take 1 / 4 of the cell stock solution and transfer it to a new culture flask, and place it in a 37°C incubator for further culture.

[0056] When BV2 cells grow to about 90%, cell passaging and inoculation are performed as follows: first, remove the old culture medium, then add 2 mL of PBS solution to gently wash, then add 1 mL of trypsin to digest the cells for about 1 minute, remove the trypsin, and add 2 mL of MEM culture medium to stop digestion; gently pipette the cells to prepare a cell stock solution, take 1 / 4 of the cell stock solution and transfer it to a new culture flask, and place it in a 37°C incubator for further culture.

[0057] The PC12 and BV2 stock solutions were diluted with culture medium and dispersed into corresponding cell suspensions with a density of 10 5 / mL, and 10 4 Cells were seeded into a 96-well plate and cultured in a 37°C, 5% CO2 incubator for 24 hours. The culture medium was then removed and QMCM was added at concentrations of 0, 20, 40, 60, and 100 μg / mL, with four replicate wells set for each concentration. After 6 hours of culture, the plates were washed twice and 100 μL of fresh culture medium was added. After a further 19 hours of culture, 10 μL of 5 mg / mL MTT was added to each well and incubated for 4 to 6 hours. The culture medium was carefully removed and 150 μL of DMSO was added. The plates were then cultured at 37°C for 15 minutes. The absorbance at 490 nm was measured and the survival rate was calculated to evaluate the biocompatibility. The results are shown in the figure below. Figure 4 As shown in the figure, when the concentration of the product is 100μg / mL, the cell survival rate is also above 90%, indicating that the product has good biocompatibility.

[0058] 5. To investigate the targeting of the product, targeting experiments on PC12 cells, BV2 cells and bEnd.3 cells were conducted respectively.

[0059] Take the density as 10 5bEnd.3 cells were seeded into the upper chamber of the Transwell plate at a concentration of 100 / mL and cultured for 10 to 14 days. DMEM culture medium was added to the lower chamber to maintain nutrient balance. During this culture process, fresh culture medium was carefully replaced periodically, and the transendothelial cell membrane electrical resistance (TEER) in the upper chamber was measured using a cell resistance meter, Millicell ERS (Millipore, USA). When the TEER reached 170 Ω·cm 2 The BBB model was considered to be successfully established when QMCM and QCM solutions (50 μg / ml) were added to the upper layer, the old culture medium in the lower chamber was discarded, and PC12 cells were inoculated and cultured for 24 hours. The treated cells were analyzed using a confocal laser scanning microscope to evaluate their ability to cross the BBB. The results are shown in Figure 2. Figure 5 As shown in the figure, QMCM ( Figure 5 a) Its ability to cross the BBB is stronger than that of QCM ( Figure 5 b), indicating that mannose also has a certain BBB targeting ability.

[0060] PC12 cells and BV2 cells were seeded into confocal microscopy. After 24 hours, QMCM labeled with RhB (25 μg / ml) was added, incubated for 2 hours, and then rinsed twice with PBS buffer to eliminate free products. The treated cells were analyzed using confocal fluorescence microscopy to evaluate their uptake. The results are shown in Figure 2. Figure 5 As shown in the figure, under the same conditions, BV2 cells ( Figure 5 c) The uptake of the product is higher than that of PC12 cells ( Figure 5 d), indicating that the BV2 cell membrane fragments contained in the product have homologous targeting to BV2 cells.

[0061] 6. To investigate the in vivo circulation time of QMCM and QMNDs, experiments were conducted using zebrafish as a model.

[0062] 3dpf zebrafish fry were raised in an aqueous solution of RhB-labeled NPs (the Que concentration in the particles was 50μg / mL). Two days later, the zebrafish fry were transferred to clean water for immersion. The fluorescence of the kidneys and livers of the zebrafish fry after immersion for different times (0, 3, 6, 12, 24, 48, and 72h) was observed using a confocal fluorescence microscope. The results were taken as examples at 0, 24, and 72h. Figure 6 As shown. Figure 6It can be seen that at the beginning, the fluorescence intensity of QMNDs and QMCM in the zebrafish was basically the same. After 24 hours, most of the QMNDs in group a had been metabolized in the zebrafish, but there was still a large amount of QMCM in group b remaining in the zebrafish. After 72 hours, the QMNDs in group a had been completely metabolized in the zebrafish, but there was still a certain amount of QMCM in group b remaining in the zebrafish, indicating that loading the cell membrane can indeed prolong the drug's circulation time in the body.

[0063] 7. Prepare LIBF and LTBF into a solution with a concentration of 10 μg / mL, then drop it onto the copper grid. After it dries, perform electron microscopy characterization. The results are as follows: Figure 7 As shown in the figure, LIBF and LTBF are small-sized nanoparticles with uniform size, and the particle size is about 9 nm.

[0064] 8. Prepare LIBFMM, LIBFNM and LTBFNM into a solution with a concentration of 10 μg / mL, then drop it onto the copper grid. After drying, perform electron microscopy characterization. The results are as follows: Figure 8 As shown. Figure 8 a. Figure 8 b It can be seen that the ligand interaction between LIBF and TrkB on the surface of PC12 cells is stronger than the interaction between LIBF and TLR4 on the surface of macrophages, while there is no ligand interaction between LTBF and the surface of PC12 cells and it is only adsorbed by electrostatic interaction. Therefore, the loading rate of LIBFNM is higher than that of LIBFMM and LTBFNM, and the loading rate of LIBFMM is higher than that of LTBFNM.

[0065] 9. 100 μg / ml lipopolysaccharide (LPS) was incubated with cell membrane fragments for 2 hours in advance. The control group did not add LPS. Then, LIBF was added and incubated with cell membrane fragments for 12 hours. The mixture was centrifuged at 13,000 rpm for 30 minutes, and the nanoparticle content in the supernatant was measured. 1 mmol BSA and Glu were incubated with LIBFMM for 12 hours, centrifuged at 13,000 rpm for 30 minutes, and the nanoparticle content in the supernatant was measured. Freshly prepared LIBFMM was dispersed in PBS buffer at pH 5, allowed to stand for 12 hours, centrifuged at 13,000 rpm for 30 minutes, and the nanoparticle content in the supernatant was measured. The results are as follows: Figure 9 As shown. Figure 9As can be seen in a, the LIBF content in the supernatant of the group with LPS added was significantly higher than that of the other groups. This is because the LPS added in advance occupied the TLR4 site on the macrophage membrane surface, preventing LIBF from attaching to the macrophage membrane surface. At pH = 5, a certain amount of LIBF also shed due to the acidic environment. However, BSA and Glu had little effect on this, indicating that certain sugars and proteins in the blood do not cause the shedding of nanoparticles. Freshly prepared LIBFNM and LTBFNM were dispersed in PBS buffer at pH = 5, allowed to stand, and centrifuged at 13,000 rpm for 30 minutes. The nanoparticle content in the supernatant was measured. The results are shown in the figure. Figure 9 As shown. Figure 9 b As can be seen, the shedding amount of nanoparticles in LTBFNM is higher than that in LIBFNM, indicating that the binding force between nanoparticles in LIBFNM and cell membrane is stronger than that in LTBFNM.

[0066] 10. To investigate the inflammatory tendency of the product, an inflammation-targeted experiment was conducted on LIBFMM using BV2 cells.

[0067] BV2 cells were seeded into confocal microscopy. After 24 hours, one group was induced with 100 μg / ml LPS for 2 hours, while the other group was cultured normally. Then, LIBFMM (25 μg / ml) labeled with RhB was added and incubated for 2 hours. The cells were then rinsed twice with PBS buffer to eliminate free products. The treated cells were analyzed using a confocal fluorescence microscope to evaluate their uptake. The results are shown in Figure 2. Figure 10 As shown in the figure, Figure 10 b The uptake of the product by BV2 cells induced by LPS was significantly stronger than that of Figure 10 aNormal BV2 cells, indicating that the macrophage membrane contained in the product has the function of targeting the inflammatory environment.

[0068] 11. To investigate the homologous targeting of the product, cell targeting experiments were conducted on LTBFNM using PC12 and BV2 cells.

[0069] PC12 cells and BV2 cells were seeded into confocal microscopy. After 24 hours, RhB-labeled LTBFNM (25 μg / ml) was added, incubated for 2 hours, and then rinsed twice with PBS buffer to eliminate free products. The treated cells were analyzed using a confocal fluorescence microscope to evaluate their uptake. The results are shown in Figure 2. Figure 11 As shown in the figure, under the same conditions Figure 11 The uptake of the product by PC12 cells in b was higher than that in Figure 11 a) BV2 cells, indicating that the PC12 cell membrane fragments contained in the product have homologous cell targeting to PC12 cells.

[0070] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A method for preparing a two-dimensional biomimetic nanostructure assembled based on receptor-ligand interaction, characterized in that: Through the specific binding effect of ligand-receptor, the ligand-modified ultrafine particles are anchored on the cell membrane fragments expressing the corresponding receptors on the surface, so that the ultrafine particles are distributed on the surface of the cell membrane fragments in a discrete and uniform manner, thereby obtaining a two-dimensional biomimetic nanostructure.

2. The preparation method according to claim 1, wherein: The following steps are involved: Specific ligands are modified on the surface of ultrafine particles, and then mixed and incubated with cell membrane fragments of a certain size with corresponding receptors expressed on the surface at a certain mass ratio to obtain a two-dimensional biomimetic nanostructure.

3. The preparation method according to claim 2, wherein: The ligand-receptor binding types for preparing the two-dimensional biomimetic nanostructure include but are not limited to mannose-mannose receptor, baicalein-TLR4, and human brain-derived nerve growth factor-tyrosine kinase receptor.

4. The preparation method according to claim 2, wherein: The cell membranes include but are not limited to the cell membranes of microglia, macrophages, neurons, red blood cells, platelets, vascular endothelial cells, and tumor cells.

5. The preparation method according to claim 2, wherein: The ultrafine particles include solid ultrafine particles and self-assembled ultrafine particles; the solid ultrafine particles include but are not limited to quercetin quantum dots, EGCG quantum dots, rutin quantum dots, and carbon quantum dots; the self-assembled ultrafine particles include but are not limited to flavonoid compound molecular self-assembled particles and polyphenol compound self-assembled particles.

6. The preparation method according to claim 2, wherein: The particle size of the ultrafine particles is less than 20 nm, and the size of the cell membrane fragments is 80-200 nm.

7. The preparation method according to claim 2, characterized in that: The mass ratio of the ultrafine particles to the cell membrane fragments is 1:5-40; and the mixed incubation time is 12 hours.

8. A two-dimensional biomimetic nanostructure, characterized by: Prepared by the preparation method according to any one of claims 1 to 7.

9. The two-dimensional biomimetic nanostructure according to claim 8, characterized in that: The two-dimensional biomimetic nanostructure has the following characteristics: strong binding force between the cell membrane and the ultrafine particles, which can avoid the off-target phenomenon of ultrafine particles during circulation in the body; can achieve the release of drugs in response to competitive effects in lesions with high-abundance receptor expression; and can achieve the long-circulation effect of ultrafine particles in living bodies.

10. Use of the two-dimensional biomimetic nanostructure according to claim 8 in preparing a precision drug delivery system.

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